G-PROTEIN-COUPLED RECEPTOR INTERNAL SENSORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application

No. 62/426,173, filed on November 23, 2016 and U.S. Provisional Patent Application No. 62/513,991, filed on June 1, 2017, which are hereby incorporated herein by reference in their entireties for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under Grant Nos.

DP2MH019427 and U01NS090604, awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

[0003] G-protein coupled receptors compose the largest family of membrane receptors in eukaryotes with more than 800 members and are involved in a variety of physiological processes. They are composed of 7-trasmembrane domains and relay an extracellular signal (i.e. light, hormone, neurotransmitter, peptide, small molecule ligand etc.) to intracellular signaling through a conformational change that triggers G-protein binding and affects a second messenger cascade. Recent crystallographic efforts have yielded important structural information for both the active and inactive state of ligand- activated GPCRs, the first of which was the Beta2AR, greatly improving our understanding of their activation mechanism [1-3]. Biopharmaceutical research has long focused on developing drugs targeting GPCRs, however these efforts have been doomed by a high failure rate, in part due to lack of tools to study drug-receptor interaction in intact living systems.

[0004] Molecular biosensors may be used in cultured cells or brain slice, or expressed in animals [4]. However, an ideal biosensor is genetically encoded and can be expressed in situ from a transgene. This allows targeting to defined cell populations by promoters and enhancers [5], conditional expression [6], and subcellular targeting with signal peptides and retention sequences [7]. Genetically encoded sensors typically employ either a single fluorescent protein or a FRET pair of donor and acceptor FPs as a reporter element. Currently available systems to study GPCR-drug interactions primarily rely on the principles of Forster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET), between a donor and an acceptor inserted in two conformationally sensitive sites of a GPCR [8-10]. This system is bimolecular, as it necessitates a couple of fluorescence emitting molecules with partially overlapping excitation/emission spectrum to be genetically inserted into the GPCR (usually a combination of two fluorescent proteins (FPs), or one FP combined with either a peptide motif for specific dye labeling or luciferase), and therefore it consumes a large portion of the available spectrum for optical readouts. As yet another critical limitation of this system, FRET-based sensors typically afford very low signal-to-noise ratio (SNR) and dynamic range, and thus cannot be easily applied in living systems. The herein described sensors provide an improved method for single-wavelength fluorescent sensor development that allows easy generation of GPCR sensors with large SNR and dynamic range.

[0005] Fluorescent sensors based on circularly permuted single FPs (either green fluorescent protein or related FPs) have been engineered to sense small molecules so that these can be visualized directly with a change in fluorescence intensity of the chromophore

[11]. Single-FP based indicators offer several appealing advantages, such as superior sensitivity, enhanced photostability, broader dynamic range and faster kinetics compared to FRET-based indicators. They are relatively small, thus relatively easier to be targeted to sub-cellular locations, such as spines and axonal terminals. The preserved spectrum bandwidth of single-FP indicators can allow for multiplex imaging or use alongside optogenetic effectors such as channel rhodopsin.

[0006] In systems employing fluorescent sensors based on circularly permuted single FPs, the conformation of the sensor controls the protonation state of its chromophore, and therefore its fluorescence intensity. The engineering process for these types of sensors can be streamlined and relies mostly on randomization of the linking regions between the conformational sensor (i.e. a bacterial Periplasmic Binding Protein) and a circularly permuted FP. This sensor design strategy has been able to yield sensors with 5-6 fold increases in GFP fluorescence in response to the stimulus and has encountered great applicability in living systems [12, 13]. Therefore, we demonstrate the design and creation of single-FP based sensors using GPCRs as a scaffold. SUMMARY

[0007] In one aspect, provided is a G protein-coupled receptor (GPCR) comprising a sensor, e.g., a fluorescent sensor, integrated into the third intracellular loop of the G protein- coupled receptor. In varying embodiments, the sensor comprises the following polypeptide structure: LI -cpFP-L2, wherein:

(1) LI comprises a peptide linker having LSS at the N-terminus and from 5 to 13 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid;

(2) cpFP comprises a circularly permuted fluorescent protein, wherein the circularly permuted N-terminus is positioned within beta strand seven of a non-permuted fluorescent protein; and

(3) L2 comprises a peptide linker having DQL at the C-terminus and from 5 to 6 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid. In some embodiments, LI comprises LSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, LI comprises QLQKIDLSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, X1X2 is selected from the group consisting of leucine-isoleucine (LI), alanine-valine (AV), isoleucine-lysine (IK), serine- argjnine (SR), lysine-valine (KV), leucine-alanine (LA), cysteine-proline (CP), glycine- methionine (GM), valine-arginine (VR), asparagjne-valine (NV), arginine-valine (RV), argjnine-glycine (RG), leucine-glutamate (LE), serine-glycine (SG), valine-aspartate (VD), alanine-phenylalanine (AF), threonine-aspartate (TD), methionine-arginine (MR), leucine- glycine (LG), arginine-glutamine (RQ), serine-tryptophan (SW), serine-glycine (SG), valine-aspartate (VD), leucine-glutamate (LE), alanine-phenylalanine (AF), serine- tryptophan (SW), argjnine-glycine (RG), threonine-aspartate (TD), leucine-glycine (LG), arginine-glutamine (RQ), threonine-tyrosine (TY), leucine-leucine (LL), valine-leucine (VL), threoni ne-glutami ne (TQ), valine-phenylalanine (VF), threonine-threonine (TT), leucine-valine (LV), valine-isoleucine (VI), valine-valine (W), proline-valine (PV), glycine-valine (GV), serine-valine (SV), phenylalanine-valine (FV), cysteine-valine (CV), glutamate- valine (EV), glutamine-valine (QV), and lysine-valine (KV), arginine-tryptophan (RW), glycine-aspartate (GD), alanine-leucine (AL), proline-methionine (PM), glycine- argjnine (GR), glycine-tyrosine (GY), isoleucine-cysteine (IC), and glycine-leucine (GL). In some embodiments, X3X4 is selected from the group consisting of asparagine-histidine (NH), threonine-arginine (TR), isoleucine-isoleucine (Π), proline-proline (PP), leucine- phenylalanine (LF), valine-threonine (VT), glutamine-glycine (QG), alanine-leucine (AL), proline-arginine (PR), arginine-glycine (RG), threonine-leucine (TL), threonine-proline (TP), glycine-valine (GV), threonine-threonine (TT), cysteine-cysteine (CC), alanine- threonine (AT), leucine-proline (LP), tyrosine-proline (YP), tryptophan-proline (WP), serine-leucine (SL), glutamate-arginine (ER), methionine-cysteine (MC), methionine- histidine (MH), tryptophan-leucine (YL), leucine-serine (LS), arginine-proline (RP), lysine- proline (KP), tyrosine-proline (YP), tryptophan-proline (WP), serine-serine (SS), glycine- valine (GV), valine-serine (VS), glutamine-asparagine (QN), lysine-serine (KS), lysine- threonine (KT), lysine-histidine (KH), lysine-valine (KV), lysine-glutamine (KQ), lysine- arginine (KR), cysteine-proline (CP), alanine-proline (AP), serine-proline (SP), isoleucine- proline (IP), tyrosine-proline (YP), threonine-proline (TP), arginine-proline (RP), aspartate- histidine (DH), histidine-tyrosine (HY), glycine-glycine (GG), proline-histidine (PH), serine-threonine (ST), arginine-serine (RS), arginine-histidine (RH), and tryptophan-proline (WP). In some embodiments, X1X2 comprises alanine-valine (AV) and X3X4 comprises lysine-proline (KP); threonine-arginine (TR); aspartate-histidine (DH); threonine-threonine (TT); serine-serine (SS); glycine-valine (GV); cysteine-cysteine (CC); valine-serine (VS); glutamine-asparagine (QN); lysine-serine (KS); lysine-threonine (KT); lysine-histidine (KH); lysine-valine (KV); lysine-glutamine (KQ); lysine-arginine (KR); lysine-proline (KP); cysteine-proline (CP); alanine-proline (AP); serine-proline (SP); isoleucine-proline (IP); tyrosine-proline (YP); threonine-proline (TP); or arginine-proline (RP);

X1X2 comprises leucine-valine (LV) and X3X4 comprises threonine-arginine (TR), lysine- proline (KP) or valine-threonine (VT); X1X2 comprises arginine-valine (RV) and X3X4 comprises threonine-arginine (TR), lysine-proline (KP) or threonine-proline (TP); X1X2 comprises arginine-glycine (RG) and X3X4 comprises tyrosine-leucine (YL) or threonine- arginine (TR); X1X2 comprises serine-arginine (SR) and X3X4 comprises leucine- phenylalanine (LF) or proline-proline (PP); X1X2 comprises proline-methionine (PM) and X3X4 comprises proline-histidine (PH) or serine-serine (SS); X1X2 comprises valine-valine (W) and X3X4 comprises threonine-arginine (TR) or lysine-proline (KP); X1X2 comprises leucine-isoleucine (LI) and X3X4 comprises threonine-arginine (TR); XI X2 comprises threonine-tyrosine (TY) and X3X4 comprises threonine-arginine (TR); X1X2 comprises isoleucine-lysine (IK) and X3X4 comprises isoleucine-isoleucine (Π); XI X2 comprises cysteine-proline (CP) and X3X4 comprises alanine-leucine (AL); X1X2 comprises glycine- methionine (GM) and X3X4 comprises proline-arginine (PR); X1X2 comprises leucine- alanine (LA) and X3X4 comprises glutamine-glycine (QG); X1X2 comprises valine- argjnine (VR) and X3X4 comprises arginine-glycine (RG); X1X2 comprises serine-glycine (SG) and X3X4 comprises tyrosine-proline (YP); XI X2 comprises valine-aspartate (VD) and X3X4 comprises tryptophan-proline (WP); X1X2 comprises leucine-glutamate (LE) and X3X4 comprises leucine-proline (UP); XI X2 comprises alanine-phenylalanine (AF) and X3X4 comprises serine-leucine (SL); X1X2 comprises serine-tryptophan (SW) and X3X4 comprises arginine-proline (RP); X1X2 comprises threonine-aspartate (TD) and X3X4 comprises glutamate-arginine (ER); X1X2 comprises leucine-glycine (LG) and X3X4 comprises methionine-histidine (MH); X1X2 comprises argjnine-glutamine (RQ) and X3X4 comprises leucine-serine (LS); X1X2 comprises methionine-arginine (MR) and X3X4 comprises methionine-cysteine (MC); X1X2 comprises leucine-leucine (LL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-leucine (VL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises threonine-glutamine (TQ) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-phenylalanine (VF) and X3X4 comprises threonine-arginine (TR); X1X2 comprises threonine-threonine (TT) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-isoleucine (VI) and X3X4 comprises threonine-arginine (TR); X1X2 comprises proline-valine (PV) and X3X4 comprises lysine-proline (KP); XI X2 comprises glycine-valine (GV) and X3X4 comprises lysine-proline (KP); X1X2 comprises serine-valine (SV) and X3X4 comprises lysine- proline (KP); X1X2 comprises asparagine-valine (NV) and X3X4 comprises lysine-proline (KP); X1X2 comprises phenylalanine-valine (FV) and X3X4 comprises lysine-proline (KP); X1X2 comprises cysteine-valine (CV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glutamate-valine (EV) and X3X4 comprises lysine-proline (KP); XI X2 comprises glutamine-valine (QV) and X3X4 comprises lysine-proline (KP); X1X2 comprises lysine-valine (KV) and X3X4 comprises lysine-proline (KP); X1X2 comprises argjnine-tryptophan (RW) and X3X4 comprises histidine-tyrosine (HY); X1X2 comprises glycine-aspartate (GD) and X3X4 comprises glycine-glycine (GG); X1X2 comprises alanine-leucine (AL) and X3X4 comprises asparagine-histidine (NH); XI X2 comprises glycine-arginine (GR) and X3X4 comprises serine-threonine (ST); XI X2 comprises glycine-tyrosine (GY) and X3X4 comprises arginine-serine (RS); X1X2 comprises isoleucine-cysteine (IC) and X3X4 comprises arginine-histidine (RH); or X1X2 comprises glycine-leucine (GL) and X3X4 comprises tryptophan-proline (WP). In some

embodiments, LI comprises LSSLIX1 and L2 comprises X2NHDQL, wherein XI, X2 are independently any amino acid. In some embodiments, XI is selected from the group consisting of I, W, V, L, F, P, N, Y and D; and X2 is selected from the group consisting of G, N, M, R T, S, K, L, Y, H, F, E, I and W. In some embodiments, XI is I and X2 is N or S; XI is W and X2 is M, T, F, E or I; XI is V and X2 is R, H or T; XI is L and X2 is T; XI is F and X2 is S; XI is P and X2 is K or S; XI is Y and X2 is S, L; or XI is D and X2 is W. In some embodiments, the circularly permuted N-terminus of the cpFP is positioned within the motif YN(Y/F)(N/I)SHNV (SEQ ID NO: 19) or

WE(A/PAO(S/L/N/T)(S/E/T)E(R/M/T/K)(M/L) (SEQ ID NO:20) of a non-permuted fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 7 of the amino acid motif YN(Y/FXN/I)SHNV (SEQ ID NO: 19) of a non-permuted green fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 3, 4, 5, 6 or 7 of the amino acid motif

WE(A/P/VXS/L/NA")(S/E/T)E(R/MyT/K)(>l/L) (SEQ ID NO:20) of a non-permuted red- fluorescent protein. In some embodiments, the circularly permuted fluorescent protein is from a photo-convertible or photoactivable fluorescent protein. In some embodiments, the photo-convertible or photoactivable fluorescent protein is selected from the group consisting of paGFP, mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple, mKate2, mMaple, mCardinal, mNeptune, far-red single-domain cyanbacteriochrome WP 016871037 and far-red single-domain cyanbacteriochrome anacy 2551g3. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein. In some embodiments, the circularly permuted fluorescent protein is from a fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a non-permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, SEQ ID NO: 1, wherein the tyrosine at residue position 69 of SEQ ID NO: 1 is replaced with a tryptophan (Y 69W) to generate a cyan fluorescent protein (CFP) sensor. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, SEQ ID NO: 1, wherein the threonine at residue position 206 of SEQ ID NO: 1 is replaced with a tyrosine (T206Y) to generate a yellow fluorescent protein (YFP) sensor. In some embodiments, the circularly permuted fluorescent protein has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a circularly permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 15-18. In some embodiments, the sensor is integrated into the third intracellular loop of the GPCR. In some embodiments, the GPCR is a class A type or alpha GPCR. In some embodiments, the GPCR is a Gs, Gi or Gq-coupled receptor. In some embodiments, the GPCR is selected from the group consisting of an adrenoceptor or adrenergic receptor, an opioid receptor, a 5-Hydroxytryptamine (5 HT) receptor, a dopamine receptor, a muscarinic acetylcholine receptor, an adenosine receptor, a glutamate metabotropic receptor, a gamma-aminobutyric acid (GABA) type B receptor, corticotropin- releasing factor (CRF) receptor, a tachykinin or neurokinin (NK) receptor, an angiotensin receptor, an apelin receptor, a bile acid receptor, a bombesin receptor, a bradykinin receptor, a cannabinoid receptor, a chemokine receptor, a cholecystokinin receptor, a complement peptide receptor, an endothelin receptor, a formylpeptide receptor, a free fatty acid receptor, a galanin receptor, a ghrelin receptor, a glycoprotein hormone, a gonadotrophin-releasing hormone receptor, a G protein-coupled estrogen receptor, an histamine receptor, a leukotriene receptor, a lysophospholipid (LP A) receptor, a lysophospholipid (S 1 P) receptor, a melanocortin receptor, a melatonin receptor, a neuropeptide receptor, a neurotensin receptor, an orexin receptor, a P2Y receptor, a prostanoid or prostaglandin receptor, somatostatin receptor, a tachykinin receptor, a thyrotropin-releasing hormone receptor, a urotensin receptor, and a vasopressin/oxytocin receptor. In some embodiments, the GPCR is selected from the group consisting of an ADRBl (Bl AR) adrenoceptor beta 1, an

ADRB2 (B2AR) adrenoceptor beta 2, an adrenoceptor alpha 2A (ADRA2A), an ADORAl (A1AR) adenosine Al receptor, an ADORA2A (A2AR) adenosine A2a receptor, a mu (μ)- type opioid receptor (OPRM or MOR), a kappa (ic)-type opioid receptor (OPRK or KOR), a delta (6)-type opioid receptor (OPRD or DOR), a Dopamine Receptor type-1 (DRDl); a Dopamine Receptor type-2 (DRD2); a Dopamine Receptor type-4 (DRD4); a Serotonin Receptor type-2 A (5HT2A); a Serotonin Receptor type-2B (5HT2B), a MTNR1B (MT2) melatonin receptor IB, a CNR1 (CB1) cannabinoid receptor 1, a histamine receptor HI (HRHl), a neuropeptide Y receptor Yl (NPY1R), a cholinergic receptor muscarinic 2 (CHRM2), a hypocretin (orexin) receptor 1 (HCRTR1), a tachykinin receptor 1 (TACR1) (a.k.a. neurokinin 1 receptor (NK1R)), a corticotropin releasing hormone receptor 1

(CRHRl), a glutamate metabotropic receptor 1 (GRM1), a gamma-aminobutyric acid (GABA) type B receptor subunit 1 (GABBRl), a Metabotropic Glutamate Receptor type-3 (MGLUR3); a Metabotropic Glutamate Receptor type-5 (MGLUR5); a Gamma- aminobutyric acid Receptor type-2 (GABABl); a Gamma-aminobutyric acid Receptor type- 2 (GABAB2); a Gonadotropin-Releasing Hormone Receptor (GNRHR); a Vasopressin Receptor type-1 (VIA); an Oxytocin Receptor (OTR); an Acetylcholine Muscarinic Receptor type-2 (M2R); an Histamine Receptor type-1 (H1R); a Tachykinin Receptor type- 1 (NK1); a Tachykinin Receptor type-2 (NK2); a Tachykinin Receptor type-3 (NK3); a P2 purinoceptor type Yl (P2Y1); an Angiotensin-Π Receptor type-1 (ATI). In some embodiments, the GPCR is selected from the group consisting of ADRBl (Bl AR) adrenoceptor beta 1, ADRB2 (B2AR) adrenoceptor beta 2, ADORAl (Al AR) adenosine Al receptor, ADORA2A (A2AR) adenosine A2a receptor, a mu ^)-type opioid receptor (OPRM or MOR), a kappa (ic)-type opioid receptor (OPRK or KOR), a dopamine receptor Dl (DRDl), a dopamine receptor D2 (DRD2), a dopamine receptor D4 (DRD4), a 5 hydroxy-tryptamine receptor 2A (5-HT2A), MTNRIB (MT2) melatonin receptor IB. In some embodiments, the GPCR:

i) is a human dopamine receptor Dl (DRDl), and the N-terminus of the sensor abuts the amino acid sequence RIYRIAQK of the receptor and the C-terminus of the sensor abuts the amino acid sequence KRETKVLK of the receptor;

ii) is a human ADRB 1 (B 1 AR) adrenoceptor beta 1 receptor, and the N- terminus of the sensor abuts the amino acid sequence RVFREAQK of the receptor and the C-terminus of the sensor abuts the amino acid sequence REQKALKT of the receptor;

iii) is a human ADRB2 (B2AR) adrenoceptor beta 2 receptor, and the N- terminus of the sensor abuts the amino acid sequence RVFQEAKR of the receptor and the C-terminus of the sensor abuts the amino acid sequence KEHKALKT of the receptor;

iv) is a human dopamine receptor D2 (DRD2), and the N-terminus of the sensor abuts the amino acid sequence IVLRRRRK of the receptor and the C-terminus of the sensor abuts the amino acid sequence QKEKKATQ of the receptor;

v) is a human dopamine receptor D4 (DRD4), and the N-terminus of the sensor abuts the amino acid sequence RGLQRWEV of the receptor and the C-terminus of the sensor abuts the amino acid sequence GRERKAMR of the receptor;

vi) is a human kappa (ic)-type opioid receptor (OPRK or KOR), and the N- terminus of the sensor abuts the amino acid sequence LMILRLKS of the receptor and the C- terminus of the sensor abuts the amino acid sequence REKDRNLR of the receptor;

vii) is a human mu (μ)-ί3φβ opioid receptor (OPRM or MOR), and the N- terminus of the sensor abuts the amino acid sequence LMILRLKS of the receptor and the C- terminus of the sensor abuts the amino acid sequence KEKDRNLR of the receptor; viii) is a human ADORA2A (A2AR) adenosine A2a receptor, and the N-terminus of the sensor abuts the amino acid sequence RIYQIAKR of the receptor and the C-terminus of the sensor abuts the amino acid sequence REKRFTFV of the receptor;

ix) is a human MTNR1 B (MT2) melatonin receptor IB, and the N-terminus of the sensor abuts the amino acid sequence VLVLQARR of the receptor and the C-terminus of the sensor abuts the amino acid sequence KPSDLRSF of the receptor;

x) is a human 5 hydroxy-tryptamine receptor 2A (5-HT2A), and the N-terminus of the sensor abuts the amino acid sequence LTIKSLQK of the receptor and the C-terminus of the sensor abuts the amino acid sequence NEQKACKV of the receptor; or

xi) is a human ADORAl (Al AR) adenosine Al receptor, and the N-terminus of the sensor abuts the amino acid sequence RVYVVAKR of the receptor and the C-terminus of the sensor abuts the amino acid sequence SREKKAAK of the receptor. In some embodiments, the receptor is mutated to be signaling incompetent or incapable. In some embodiments, the receptor is substantially isolated and/or purified and/or solubilized. In some embodiments, the GPCR comprises a beta2 adrenergic receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 22 or SEQ ID NO:32. In some embodiments, one or more of amino acid residues S355 and S356 (residues 624-625 in SEQ ID NO: 22) are replaced with alanine residues. In some embodiments, X at amino acid residue 163 in SEQ ID NO: 22 or at residue 139 of SEQ ID NO:32 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a mu ^)-type opioid receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 24 or SEQ ID NO:37. In some embodiments, X at amino acid residue 199 in SEQ ID NO: 24 or at residue 175 of SEQ ID NO:37 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a dopamine receptor Dl (DRDl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 26 or SEQ ID NO:30. In some embodiments, X at amino acid residue 153 in SEQ ID NO: 26 or at residue 129 of SEQ ID NO:30 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a 5 hydroxy-tryptamine 2A (5-HT2A) receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 28 or SEQ ID NO:33. In some embodiments, X at amino acid residue 205 in SEQ ID NO: 28 or at residue 181 of SEQ ID NO:33 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises an adrenoceptor beta 1 (ADRB1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:31. In some embodiments, X at amino acid residue 164 in SEQ ID NO: 31 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises an adenosine A2a receptor (ADORA2A) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 34. In some embodiments, X at amino acid residue 110 in SEQ ID NO: 34 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises an adrenoceptor alpha 2A (ADRA2A) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 35. In some embodiments, X at amino acid residue 139 in SEQ ID NO: 35 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a kappa receptor delta 1 (OPRK1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 36. In some embodiments, X at amino acid residue 164 in SEQ ID NO: 36 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises an opioid receptor delta 1 (OPRD1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 38. In some embodiments, X at amino acid residue 154 in SEQ ID NO: 38 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a melatonin receptor IB (MTNRIB) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 39. In some embodiments, X at amino acid residue 146 in SEQ ID NO: 39 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a cannabinoid receptor type 1 (CNR1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 40. In some embodiments, X at amino acid residue 222 in SEQ ID NO: 40 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a histamine receptor HI (HRH1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 41. In some embodiments, X at amino acid residue 133 in SEQ ID NO: 41 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a neuropeptide Y receptor Yl (NPY1R) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 42. In some embodiments, the GPCR comprises a muscarinic cholinergic receptor type 2 (CHRM2) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 43. In some embodiments, X at amino acid residue 129 in SEQ ID NO: 43 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a hypocretin (orexin) receptor 1 (HCRTR1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 44. In some embodiments, X at amino acid residue 152 in SEQ ID NO: 44 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V. In some embodiments, the GPCR comprises a dopamine receptor D2 (DRD2) having at least 90% sequence identity e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 46. In some embodiments, the GPCR comprises a dopamine receptor D4 (DRD4) having at least 90% sequence identity e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 48.

[0008] In a further aspect, provided is a nanodisc comprising the GPCR having a cpFP sensor integrated into its third intracellular loop, as described above and herein, as described above and herein. In a further aspect, provided is a solid support attached to one or more GPCR, or one or more nanodiscs, the GPCRs and nanodiscs being described above and herein. In some embodiments, the solid support is a bead or a microarray.

[0009] In a further aspect, provided is a polynucleotide encoding the GPCR having a cpFP sensor integrated into its third intracellular loop, as described above and herein, as described above and herein. Further provided is an expression cassette comprising the polynucleotide encoding the GPCR having an integrated sensor, as described above and herein. Further provided is a vector comprising the polynucleotide of encoding the GPCR having an integrated sensor, as described above and herein. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the vector is a viral vector from a virus selected from the group consisting of a retrovirus, a lentivirus, an adeno virus, and an adeno-associated virus.

[0010] In another aspect, provided is cell comprising the GPCR having a cpFP sensor integrated into its third intracellular loop, as described above and herein, as described above and herein, e.g., integrated into the extracellular membrane of the cell. In another aspect, provided is cell comprising the polynucleotide encoding the GPCR, as described above and herein, e.g., integrated into the genome of the cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an astrocyte or a neuronal cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is selected from a Chinese hamster ovary (CHO) cell, an HEK 293 T cell and a HeLa cell.

[0011] In another aspect, provided is a transgenic animal comprising the GPCR having a cpFP sensor integrated into its third intracellular loop, as described above and herein. In some embodiments, the animal is selected from a mouse, a rat, a worm, a fly and a zebrafish. In some embodiments, the animal is a mouse, and the GPCR is expressed in the CNS tissues of the mouse. In some embodiments, the animal is a mouse, and the GPCR is expressed in the brain cortex of the mouse.

[0012] In a further aspect, provided is a kit comprising the GPCR having a cpFP sensor integrated into its third intracellular loop, as described above and herein, the solid support, the nanodisc, the polynucleotide, the expression cassette, the vector, the cell, and/or the transgenic animal, as described above and herein.

[0013] In another aspect, provided are methods of detecting binding of a ligand to a GPCR. In some embodiments, the methods comprise:

a) contacting the ligand with a GPCR, as described above and herein, under conditions sufficient for the ligand to bind to the GPCR; and

b) determining a change in an optics signal from the sensor integrated into the third intracellular loop of the GPCR, wherein a detectable change in fluorescence signal indicates binding of the ligand to the GPCR. In some embodiments, the optics signal is a linear optics signal. In some embodiments, the linear optics signal comprises fluorescence. In some embodiments, the change in fluorescence signal comprises a change in intensity of the fluorescence signal. In some embodiments, the change in fluorescence intensity is at least about 10% over, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, or more, over baseline, in the absence of ligand binding. In some embodiments, the change in

fluorescence signal comprises a change in color (spectrum or wavelength) of the

fluorescence signal. In some embodiments, the optics signal is a non-linear optics signal. In some embodiments, the non-linear optics signal is selected from the group consisting of fiber optics, miniature fiber optics, fiber photometry, one photon imaging, two photon imaging, and three photon imaging. In some embodiments, a binding ligand further indicates activation of intracellular signaling from the GPCR. In some embodiments, the ligand is a suspected agonist of the GPCR. In some embodiments, the ligand is a suspected inverse agonist of the GPCR. In some embodiments, the ligand is a suspected antagonist of the GPCR. In some embodiments, the GPCR is in vitro. In some embodiments, the GPCR is in vivo.

[0014] In another aspect, provided are methods of screening for binding of a ligand to a GPCR. In some embodiments, the methods comprise:

a) contacting a plurality of members from a library of ligands with a plurality of

GPCRs, as described above and herein, under conditions sufficient for the ligand members to bind to the GPCRs, wherein the plurality of GPCRs are arranged in an array of predetermined addressable locations; and

b) determining a change in one or more optics signals from the sensor integrated into the third intracellular loop of the plurality GPCRs, wherein a detectable change in the one or more fluorescence signals indicates binding of one or more members of the library of ligands to at least one of the plurality GPCR. In some embodiments, the one or more optics signals comprise a linear optics signal. In some embodiments, the linear optics signal comprises fluorescence. In some embodiments, the one or more fluorescence signals fluoresce at the same wavelength. In some embodiments, the one or more fluorescence signals fluoresce at different wavelengths. In some embodiments, the change in fluorescence signal comprises a change in intensity of the fluorescence signal. In some embodiments, the change in fluorescence intensity is at least about 10% over, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, or more, over baseline, in the absence of ligand binding. In some embodiments, the change in fluorescence signal comprises a change in color (spectrum or wavelength) of the fluorescence signal. In some embodiments, the optics signal is a non-linear optics signal. In some embodiments, the non-linear optics signal is selected from the group consisting of fiber optics, miniature fiber optics, fiber photometry, one photon imaging, two photon imaging, and three photon imaging. In some

embodiments, a binding ligand further indicates activation of intracellular signaling from the GPCR. In some embodiments, two or more members of the plurality of GPCRs are different. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different type of GPCR. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different subtype of a GPCR. In some embodiments, two or more members of the plurality of GPCRs comprise a sensor that fluoresces at a different wavelength.

[0015] In a further aspect, further provided is a fluorescent sensor. In some embodiments, sensor comprises the following polypeptide structure: Ll-cpFP-L2, wherein:

(1) LI comprises a peptide linker having LSS at the N-terminus and from 5 to 13 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid;

(2) cpFP comprises a circularly permuted fluorescent protein, wherein the circularly permuted N-terminus is positioned within beta strand seven of a non-permuted fluorescent protein; and

(3) L2 comprises a peptide linker having DQL at the C-terminus and from 5 to 6 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid. In some embodiments, LI comprises LSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, wherein LI comprises QLQKIDLSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, X1X2 is selected from the group consisting of leucine-isoleucine (LI), alanine-valine (AV), isoleucine-lysine (IK), serine- arginine (SR), lysine-valine (KV), leucine-alanine (LA), cysteine-proline (CP), glycine- methionine (GM), valine-arginine (VR), asparagine-valine (NV), arginine-valine (RV), arginine-glycine (RG), leucine-glutamate (LE), serine-glycine (SG), valine-aspartate (VD), alanine-phenylalanine (AF), threonine-aspartate (TD), methionine-arginine (MR), leucine- glycine (LG), arginine-glutamine (RQ), serine-tryptophan (SW), serine-glycine (SG), valine-aspartate (VD), leucine-glutamate (LE), alanine-phenylalanine (AF), serine- tryptophan (SW), arginine-glycine (RG), threonine-aspartate (TD), leucine-glycine (LG), arginine-glutamine (RQ), threonine-tyrosine (TY), leucine-leucine (LL), valine-leucine (VL), threonine-glutamine (TQ), valine-phenylalanine (VF), threonine-threonine (TT), leucine-valine (LV), valine-isoleucine (VI), valine-valine (W), proline-valine (PV), glycine-valine (GV), serine-valine (SV), phenylalanine-valine (FV), cysteine-valine (CV), glutamate-valine (EV), glutamine-valine (QV), and lysine-valine (KV), arginine-tryptophan (RW), glycine-aspartate (GD), alanine-leucine (AL), proline-methionine (PM), glycine- arginine (GR), glycine-tyrosine (GY), isoleucine-cysteine (IC), and glycine-leucine (GL). In some embodiments, X3X4 is selected from the group consisting of asparagjne-histidine (NH), threonine-arginine (TR), isoleucine-isoleucine (Π), proline-proline (PP), leucine- phenylalanine (LF), valine-threonine (VT), glutamine-glycine (QG), alanine-leucine (AL), proline-arginine (PR), arginine-glycine (RG), threonine-leucine (TL), threonine-proline (TP), glycine-valine (GV), threonine-threonine (TT), cysteine-cysteine (CC), alanine- threonine (AT), leucine-proline (LP), tyrosine-proline (YP), tryptophan-proline (WP), serine-leucine (SL), glutamate-arginine (ER), methionine-cysteine (MC), methionine- histidine (MH), tryptophan-leucine (YL), leucine-serine (LS), argjnine-proline (RP), lysine- proline (KP), tyrosine-proline (YP), tryptophan-proline (WP), serine-serine (SS), glycine- valine (GV), valine-serine (VS), glutamine-asparagine (QN), lysine-serine (KS), lysine- threonine (KT), lysine-histidine (KH), lysine-valine (KV), lysine-glutamine (KQ), lysine- argjnine (KR), cysteine-proline (CP), alanine-proline (AP), serine-proline (SP), isoleucine- proline (IP), tyrosine-proline (YP), threonine-proline (TP), argjnine-proline (RP), aspartate- histidine (DH), histidine-tyrosine (HY), glycine-glycine (GG), proline-histidine (PH), serine-threonine (ST), argjnine-serine (RS), arginine-histidine (RH), and tryptophan-proline (WP). In some embodiments, X1X2 comprises alanine-valine (AV) and X3X4 comprises lysine-proline (KP); threonine-arginine (TR); aspartate-histidine (DH); threonine-threonine (TT); serine-serine (SS); glycine-valine (GV); cysteine-cysteine (CC); valine-serine (VS); glutamine-asparagine (QN); lysine-serine (KS); lysine-threonine (KT); lysine-histidine (KH); lysine-valine (KV); lysine-glutamine (KQ); lysine-arginine (KR); lysine-proline (KP); cysteine-proline (CP); alanine-proline (AP); serine-proline (SP); isoleucine-proline (IP); tyrosine-proline (YP); threonine-proline (TP); or arginine-proline (RP);

X1X2 comprises leucine-valine (LV) and X3X4 comprises threonine-arginine (TR), lysine-proline (KP) or valine-threonine (VT); XI X2 comprises arginine-valine (RV) and X3X4 comprises threonine-arginine (TR), lysine-proline (KP) or threonine-proline (TP); X1X2 comprises arginine-glycine (RG) and X3X4 comprises tyrosine-leucine (YL) or threonine-arginine (TR); X1X2 comprises serine-arginine (SR) and X3X4 comprises leucine-phenylalanine (LF) or proline-proline (PP); X1X2 comprises proline-methionine (PM) and X3X4 comprises proline-histidine (PH) or serine-serine (SS); X1X2 comprises valine-valine (W) and X3X4 comprises threonine-arginine (TR) or lysine-proline (KP); X1X2 comprises leucine-isoleucine (LI) and X3X4 comprises threonine-arginine (TR); XI X2 comprises threonine-tyrosine (TY) and X3X4 comprises threonine-arginine (TR); X1X2 comprises isoleucine-lysine (IK) and X3X4 comprises isoleucine-isoleucine (Π); X1X2 comprises cysteine-proline (CP) and X3X4 comprises alanine-leucine (AL); XI X2 comprises glycine-methionine (GM) and X3X4 comprises proline-arginine (PR); X1X2 comprises leucine-alanine (LA) and X3X4 comprises glutamine-glycine (QG); X1X2 comprises valine-arginine (VR) and X3X4 comprises argjnine-glycine (RG); X1X2 comprises serine-glycine (SG) and X3X4 comprises tyrosine-proline (YP); X1X2 comprises valine-aspartate (VD) and X3X4 comprises tryptophan-proline (WP); XI X2 comprises leucine-glutamate (LE) and X3X4 comprises leucine-proline (LP); X1X2 comprises alanine-phenylalanine (AF) and X3X4 comprises serine-leucine (SL); XI X2 comprises serine-tryptophan (SW) and X3X4 comprises arginine-proline (RP); X1X2 comprises threonine-aspartate (TD) and X3X4 comprises glutamate-arginine (ER); X1X2 comprises leucine-glycine (LG) and X3X4 comprises methionine-histidine (MH); X1X2 comprises arginine-glutamine (RQ) and X3X4 comprises leucine-serine (LS); X1X2 comprises methi oni ne-arginine (MR) and X3X4 comprises methionine-cysteine (MC); X1X2 comprises leucine-leucine (LL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-leucine (VL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises threonine-glutamine (TQ) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-phenylalanine (VF) and X3X4 comprises threonine-arginine (TR); XI X2 comprises threonine-threonine (TT) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-isoleucine (VT) and X3X4 comprises threonine-arginine (TR); X1X2 comprises proline-valine (PV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glycine-valine (GV) and X3X4 comprises lysine-proline (KP); XI X2 comprises serine- valine (SV) and X3X4 comprises lysine-proline (KP); X1X2 comprises asparagine-valine (NV) and X3X4 comprises lysine-proline (KP); X1X2 comprises phenylalanine-valine (FV) and X3X4 comprises lysine-proline (KP); X1X2 comprises cysteine-valine (CV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glutamate-valine (EV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glutamine-valine (QV) and X3X4 comprises lysine-proline (KP); XI X2 comprises lysine-valine (KV) and X3X4 comprises lysine-proline (KP); X1X2 comprises arginine-tryptophan (RW) and X3X4 comprises histidine-tyrosine (HY); X1X2 comprises glycine-aspartate (GD) and X3X4 comprises glycine-glycine (GG); X1X2 comprises alanine-leucine (AL) and X3X4 comprises asparagine-histidine (NH); X1X2 comprises glycine-arginine (GR) and X3X4 comprises serine-threonine (ST); X1X2 comprises glycine-tyrosine (GY) and X3X4 comprises arginine-serine (RS); XI X2 comprises isoleucine-cysteine (IC) and X3X4 comprises argjnine-histidine (RH); or X1X2 comprises glycine-leucine (GL) and X3X4 comprises tryptophan-proline (WP). In some embodiments, LI comprises LSSLDC1 and L2 comprises X2NHDQL, wherein XI, X2 are independently any amino acid. In some embodiments, XI is selected from the group consisting of L W, V, L, F, P, N, Y and D; and X2 is selected from the group consisting of G, N, M, R T, S, K, L, Y, H, F, E, I and W. In some embodiments, XI is I and X2 is N or S; XI is W and X2 is M, T, F, E or I; XI is V and X2 is R, H or T; Xl is L and X2 is T; Xl is F and X2 is S; XI is P and X2 is K or S; XI is Y and X2 is S, L; or XI is D and X2 is W. In some embodiments, the circularly permuted N- terminus is positioned within the motif YN(Y/F)(N/I)SHNV (SEQ ID NO: 19) or

WE(A/P/VXS/L/NA")(S/E/T)E(R/MyT/K)(>l/L) (SEQ ID NO:20) of a non-permuted fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 7 of the amino acid motif YN(Y/FXN/I)SHNV (SEQ ID NO:19) ofa non-permuted green fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 3, 4, 5, 6 or 7 of the amino acid motif

WE(A/PAO(S/L/N/T)(S/E/T)E(R/M/T/K)(M/L) (SEQ ID NO:20) of a non-permuted red- fluorescent protein. In some embodiments, the circularly permuted fluorescent protein is from a photo-convertible or photoactivable fluorescent protein. In some embodiments, the photo-convertible or photoactivable fluorescent protein is selected from the group consisting of paGFP, mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple, mKate2, mMaple, mCardinal, mNeptune, far-red single-domain cyanbacteriochrome WP 016871037 and far-red single-domain cyanbacteriochrome anacy 255 lg3. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein. In some embodiments, the circularly permuted fluorescent protein is from a fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a non-permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, SEQ ID NO: 1, wherein the tyrosine at residue position 69 of SEQ ID NO: 1 is replaced with a tryptophan (Y 69W) to generate a cyan fluorescent protein (CFP) sensor. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, SEQ ID NO: 1, wherein the threonine at residue position 206 of SEQ ID NO: 1 is replaced with a tyrosine (T206Y) to generate a yellow fluorescent protein (YFP) sensor. In some embodiments, the circularly permuted fluorescent protein has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a circularly permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 15-18. Further provided is a polynucleotide encoding the fluorescent sensor, as described above and herein. DEFINITIONS

[0016] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., share at least about 80% identity, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a specified region to a reference sequence, e.g., any of SEQ ID NOs: 1-44, as described herein, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full-length of a reference sequence.

[0017] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to fluorescent proteins, circularly permuted fluorescent proteins, and GPCR nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters are used. [0018] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is

immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically

substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0019] The term "isolated," and variants thereof when applied to a protein (e.g., a population of GPCRs having an integrated cpFP sensor), denotes that the protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution, or solubilized. Purity and homogeneity are typically determined using known techniques, such as polyacryl amide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0020] The term "purified" denotes that a protein (e.g., a population of GPCRs having an integrated cpFP sensor) gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 80%, 85% or 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 illustrates computationally-guided design of cpGFP insertion site into Beta2AR. Graph indicating amino acid by amino acid changes in the torsion angle of the polypeptide chain of Beta2AR between active and inactive state. The torsion angle describes rotations of the polypeptide backbone around the bonds between nitrogen atom and the alpha-carbon. The numbering on the X axis corresponds to the amino acid numbers on the full-length human Beta2AR protein sequence (GenBank Accession Number:

AAB82150.1). [0022] Figure 2 illustrates the design of a circularly permuted green fluorescent protein (cpGFP) sensor integrated into the third intracellular loop of the beta2 adrenergic receptor (Beta2AR). A circularly-permuted GFP is inserted in the intracellular Loop3 of the Beta2AR and is connected to the GPCR via two linker regions (highlighted as Linker 1 in red and Linker 2 in blue). Agonist induced conformational activation of the receptor triggers a reversible increase in cpGFP fluorescence (excitation λ 488 nm, emission λ 510 nm).

[0023] Figures 3 A-C illustrate full-agonist titrations on a cpGFP sensor integrated into the third intracellular loop of the Beta2AR. Titrations of three different Beta2AR full- agonists (A, epinephrine, EPI; B, norepinephrine, NE; C, isoproterenol, ISO) were performed on HEK293T cells expressing Beta2AR with a cpGFP integrated into the third intracellular loop using a constant perfusion chamber with buffer exchange. Images of the cells were taken on a confocal microscope every 4 seconds and analyzed on Fiji using manually drawn ROIs. n=3 ROIs per trace.

[0024] Figures 4A-B illustrate affinity and specificity characterization of a cpGFP sensor integrated into the third intracellular loop of the Beta2AR. A. Drug/response curves for the three different full-agonists tested on Beta2AR with a cpGFP integrated into the third intracellular loop. The curves were fit with a one-site total binding curve using GraphPad Prism 6 software. B. Fluorescence trace of Beta2AR with a cpGFP integrated into the third intracellular loop expressed on HEK293T cells during bath application of non- Beta2AR ligands (Serotonin (SER) and Dopamine (DA)) followed by application of the Beta2AR agonist ISO and successive inhibitory competition using a higher concentration (50 uM) of the Beta2AR inverse agonist CGP-12177.

[0025] Figures 5A-D illustrate that Beta2ARwith a cpGFP integrated into the third intracellular loop can distinguish different classes of ligands. Fluorescence trace of

Beta2AR with a cpGFP integrated into the third intracellular loop in response to three different agonists (one full agonist: Norepinephrine, followed by inverse agonist competition, and two partial agonists: Terbutaline and Dobutamine) applied under identical conditions but in three separate experiments.

[0026] Figures 6A-D illustrates characterization of Beta2AR with a cpGFP integrated into the third intracellular loop in neuronal cultures. Representative images before (A) and after (B) drug application (ISO, 10 uM) of DIV14 primary hippocampal neurons after 5 days of infection with a Lentivirus carrying a Synapsin promoter-driven Beta2AR with a cpGFP integrated into the third intracellular loop. C. AFF image obtained using a custom-made script on MatLab. D. Fluorescent signal trace during bath application ofNE. [0027] Figures 7A-D illustrate that Beta2AR with a cpGFP integrated into the third intracellular loop is signaling incompetent. Membrane relocalization of a conformationally- sensitive nanobody (Nb80) is used as an indication of Beta2AR activation. Wild-type Beta2AR with a GFP tag on it C-terminus is used as a control. Representative images before and after 10 μΜ drug application and fluorescence profiles are shown for Beta2AR-GFP in A and B, and for Beta2AR with a cpGFP integrated into the third intracellular loop in C and D, respectively.

[0028] Figures 8A-D illustrate an opioid sensor based on the Mu-opioid receptor.

An opioid sensor was designed by inserting cpGFP into the Loop3 of the Mu-opioid receptor. A. Representative images of a HEK293T cell expressing the opioid sensor before and after addition of a specific Mu-opioid receptor agonist (DAMGO, 10 uM). B.

Fluorescent signal trace upon addition of DAMGO, 10 uM. C. Titration curve upon addition of increasing concentrations of DAMGO (1 nm to 1 μΜ in 10-fold increases) and ligand washout. D. Drug/response curve of DAMGO for the opioid sensor indicates an apparent Kd of25 nM.

[0029] Figure 9 illustrates an alignment of fluorescent proteins of use in the present sensors.

[0030] Figure 10 illustrates an over-imposition of the PBD structures of illustrative fluorescent proteins useful in the present sensors (with the exclusion of the far-red single- domain cyanbacteriochromes for which no structure is available). The arrow indicates the sites of circular permutation of the various FPs.

[0031] Figures 11 A-E illustrate an alignment of the third intracellular loop from different G protein-coupled receptors. Legend: MGLUR3: Metabotropic Glutamate Receptor type-3; MGLUR5: Metabotropic Glutamate Receptor type-5; GABAB1: Gamma- aminobutyric acid Receptor type-2; GABAB2: Gamma-aminobutyric acid Receptor type-2; CB1: Cannabinoid Receptor type- 1; GNRHR: Gonadotropin-Rel easing Hormone Receptor; VIA: Vasopressin Receptor type- 1; OTR: Oxytocin Receptor; A2A: Adenosine Receptor type-2; B2AR: Beta-2 Adrenergic Receptor; DRDl: Dopamine Receptor type- 1; M2R: Acetylcholine Muscarinic Receptor type-2; H1R: Histamine Receptor type-1; DRD2:

Dopamine Receptor type-2; 5HT2A: Serotonin Receptor type-2A; 5HT2B: Serotonin

Receptor type-2B; NK1 : Tachykinin Receptor type-1; NK3: Tachykinin Receptor type-3; NK2: Tachykinin Receptor type-2; MTNR1B: Melatonin Receptor type-IB; P2Y1: P2 purinoceptor type Yl; ATI : Angiotensin-II Receptor type-1; KOR1 : Kappa Opioid Receptor type-1; MORI : Mu Opioid Receptor type-1; and DOR1 : Delta Opioid Receptor type-1.

[0032] Figuresl2A-D illustrate an alignment of the third intracellular loop from different G protein-coupled receptors. Legend: MT2R: melatonin receptor type IB (NCBI Reference Sequence: NP_005950.1); KOR1 : Kappa Opioid Receptor type-1 (GenBank: AAC50158.1); 5HT2A: Serotonin Receptor type-2A (NCBI Reference Sequence:

NP 000612.1); A2AR: Alpha-2C Adrenergic Receptor (NCBI Reference Sequence:

NP 000674.2); B2AR: Beta-2 Adrenergic Receptor (GenBank: AAB82151.1); and DRDl: Dopamine Receptor type-1 (GenBank: AAH96837.1). [0033] Figures 13A-D illustrate proof-of-principle experiment to show that a universal sensor (e.g., LSSLI-cpGFP-NHDQL or QLQKIDLSSLI-cpGFP-NHDQL) is capable of detecting the action of pharmacological drugs. (A-B) Screening of a panel of drugs using the b2AR with universal module 1 (e.g., QLQKIDLSSLI-cpGFP-NHDQL) in 293 cells and representative time-lapse curves are shown for each individual drug application. (C) a representative image of the HEK293t cells expressing the sensor shows good membrane expression. (D) a quantification of the maximal DF/F versus drug type. Both full agonists (isoetharine, isoproterenol), partial agonists (Salbutamol, Blenbuterol, Terbutaline), inverse agonist (CGP-12177) and antagonists (Alprenolol, Timolol) were used. [0034] Figure 14 illustrates representative time-lapse curves for each of the GPCR sensors developed with universal module 1. Agonist application is indicated by an arrow in each graph.

[0035] Figures 15 A-E provide representative time-lapse curves for each of the

GPCR sensors developed with universal module 2 (e.g., LSSLI-cpGFP-NHDQL) (A, C, D, E). Agonist application is indicated by an arrow in each graph. (B) In situ titration of the dopamine DRDl -based sensor with apparent Kd of ~70nM, while other non-selective ligands (norepinephrine, epinephrine) can only trigger a similar response with ~200-fold lower affinity (-16, 14 μΜ, respectively).

[0036] Figures 16A-B illustrate measuring GPCR activation in the living brain. p2AR-sensor signals (pink traces in B corresponding to yellow ROIs in A) measured in the cortex of a mouse reported endogenous norepinephrine release triggered by running on a spherical treadmill. Running speed is indicated in the top blue trace and correlates with signal peaks.

[0037] Figure 17 illustrates Graphic description of universal module insertion sites into 11 example GPCR-sensors. Each raw contains from left to right: Abbreviated name of the GPCR used, sequence of the 8 amino acids preceding the universal module (in the direction from N-terminus to C-terminus), universal module, sequence of the 8 amino acids following the universal module.

[0038] Figure 18 illustrates graph describing the fluorescent response (fold-change, or DF/F0). The data are represented as Box and Whiskers view, with error bars being the standard error and the horizontal line inside the box being the median.

[0039] Figures 19A-B illustrate A. Alignment of the 8 amino acids comprising the

GPCR sequence abutting the N-terminus of the sensor. B. Alignment of the 8 amino acids comprising the GPCR sequence abutting the C-terminus of the sensor. Alignments were done in Jalview Conservation.

[0040] Figure 20 illustrates results from screening a library of linker variants obtained by randomly mutating the X1X2X3X4 residues all at once. The first column from the left shows the fluorescence fold-change of each variant. The second column from the left shows the amino acid sequence of the X1X2 linker residues for each variant. The second column from the left shows the amino acid sequence of the X3X4 linker residues for each variant.

[0041] Figure 21 illustrates results from screening a library of linker variants obtained by inserting a random amino acid after LI and NH parts of the universal module, to create an LIX1 - cpGFP - NHX2 library. The first column from the left shows the fluorescence fold-change of each variant. The second column from the left shows the amino acid sequence of the LIX1 linker residues for each variant. The second column from the left shows the amino acid sequence of the NHX2 linker residues for each variant.

[0042] Figure 22 illustrates graph showing the fluorescence fold-change response of

DRD1 -based dopamine sensor where the amino acid sequence of the GPCR prior to the beginning of the universal module has been sequentially deleted of 1, 2 and 3 amino acids.

[0043] Figure 23 illustrates graph showing the fluorescence fold-change response of

DRDl -based dopamine sensor where the amino acid sequence of the GPCR after the end of the universal module has been added or deleted of 2 amino acids according to the DRD1 amino acid sequence.

[0044] Figure 24 illustrates graph showing the results from screening a library of

DRDl-sensor linker variants obtained by randomly mutating the X1X2X3X4 residues replacing "LI" and "NH" all at once.

[0045] Figure 25 illustrates graph showing the florescence fold-change response of

DRD2-based dopamine sensor where after the amino acid sequence of the GPCR-sensor preceding the beginning of the universal module an insertion has been made of 1, 2, 3 and 8 amino acids, respectively, according to the DRD2 amino acid sequence.

[0046] Figure 26 illustrates graph showing the florescence fold-change response of

DRD2-based dopamine sensor where after the amino acid sequence of the GPCR-sensor following the end of the universal module an insertion has been made of 1 and 2 amino acids, respectively, according to the DRD2 amino acid sequence.

DETAILED DESCRIPTION

1. Introduction

[0047] G-protein coupled receptors (GPCRs) are widely expressed in nervous systems and respond to a wide variety of ligands including hormones, neurotransmitters and neuromodulators. Drugs targeting members of this integral membrane protein superfamily represents the core of modern medicine. Here we developed a toolbox of optogenetic sensors for visualizing GPCR activation; the conformational dynamics triggered by ligand binding to the GPCR is monitored via ligand induced changes in fluorescence. This toolbox enables high-throughput cell-based screening, mapping neuromodulation networks in the brain and in vivo validation of potential therapeutics, which is expected to accelerate the discovery process of drugs for treating neurological disorders.

[0048] Using the prototype GPCR Beta2AR as a starting point, we inserted circular permuted green fluorescent protein (cpGFP) into the third intracellular loop region of the receptor to transform the ligand-induced conformational changes of Beta2AR into changes of fluorescence intensity of the GFP chromophore. A cell-based screening was then performed to determine the linker sequences between cpGFP and Beta2 AR that maximize signal-to-noise ratio. Upon agonist binding (isoproterenol, ISO, ΙΟμΜ), we detected a 40% increase in fluorescence at the membrane of mammalian cells. The in situ affinity of this Beta2AR sensor is 1.2 nM for isoproterenol, 15 nM for epinephrine and 50 nM for norepinephrine, which is within the range of physiological relevance.

[0049] Accordingly, provided is a universal linker useful as an integrated sensor incorporated into the third intracellular loop of a G-protein-coupled receptor. In some embodiments, the universal linker has the structure of: LSSXlX2-cpGFP-X3X4DQL. In some embodiments, the universal linker has the structure of: QLQKIDLSSXlX2-cpGFP- X3X4DQL. We have demonstrated interchangeable utility of the universal GPCR integrated sensor in six structurally different and unrelated GPCRs: adrenoceptor beta 2 (ADRB2), mu ^)-type opioid receptor (OPRM), kappa (ic)-type opioid receptor (OPRK), dopamine receptor Dl (DRD1), 5-hydroxytryptamine receptor 2A (HTR2A), and melatonin receptor type IB (MTNR1B). Importantly, this group of GPCRs contains representative Gs, Gi and Gq-coupled receptors, demonstrating the universality of our approach. Upon insertion of the universal sensor, each of the six tested GPCRs was transformed into a sensor that showed positive fluorescence signal in response to the application of an agonist. Such an engineering approach is unprecedented and allows for rapid and efficient production of GPCR-sensors with applications in multiple scientific fields, from drug screening, to GPCR de-orphanization, to in vivo imaging of drug efficacy or dynamics of endogenous ligands.

[0050] The sensors described herein are capable of capturing conformational dynamics of G protein-coupled receptors, including Beta2 AR, triggered by binding of a panel of full, partial and inverse agonists. Our illustrative Beta2AR sensor has been made signaling deficient by the following mutations: F139S, S355A/S356A, in order not to interfere with endogenous cellular signaling. A similar engineering approach was successfully employed to develop sensors for monitoring the activation of the μ-opioid receptor MOR-1, the Dopamine receptor Dl and the serotonin receptor 5-HT2A. The utility of these sensors can be implemented and further characterized in vivo, e.g., in the zebrafish brain and in the mouse spinal cord. Given the structural similarity of GPCRs, our sensor design strategy represents a universal scaffold that can be readily applied generally to many different GPCRs.

[0051] Cell-based high-throughput screening assays have been the workhorse fueling G-protein coupled receptors as one of the most studied classes of investigational drug targets. However, existing high-throughput cellular screening assays are based on measuring intracellular levels of downstream signaling molecules, such as calcium and cyclic adenosine monophosphate (cAMP), which only provide a downstream binary readout (on or off) of GPCR activation. In contrast, using an integrated GPCR sensor, as described herein, allows for direct imaging of GPCR ligand binding in living cells and animals with molecular specificity and subcellular resolution, providing a platform for high-throughput cell-based screening and validation of potential therapeutics in living animal disease models. Further, the integrated GPCR sensors described herein utilize a circularly permutated fluorescent protein, and therefore employ a single wavelength of fluorescent protein, which preserves the bandwidth to engineer multi-color palette of GPCR

conformation sensors, enabling simultaneous imaging of multiple GPCRs. Moreover, when combined with optical measurement of other downstream signaling molecules such as calcium, cAMP and β-arrestin, the integrated GPCR sensors facilitate linking the conformation dynamics of GPCR with a specific downstream signaling branch, which further enhances the rigor of biased ligand detection. Additionally, the ability to detect ligand bias using the integrated sensors described herein furthers the understanding of stmcture-functional properties of drugs with allosteric and/or biased properties, which aids optimization for bias in addition to potency at the receptor, selectivity and pharmaceutical properties.

2. Fluorescent Sensors

[0052] Provided are fluorescent sensors designed to integrate into the third intracellular loop of a G protein-coupled receptor (GPCR). In some embodiments, the sensors comprise the following polypeptide structure: Ll-cpFP-L2, wherein:

(1) LI comprises a peptide linker having LSS at the N-terminus and from 5 to 13 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid;

(2) cpFP comprises a circularly permuted fluorescent protein, wherein the circularly permuted N-terminus is positioned within beta strand seven of a non-permuted fluorescent protein; and

(3) L2 comprises a peptide linker having DQL at the C-terminus and from 5 to 6 amino acid residues, wherein each amino acid residue can be any naturally occurring amino acid.

[0053] Generally, the fluorescent sensors are integrated into a GPCR, e.g. , into the third intracellular loop. The GPCR internal fluorescent sensors are polypeptides that can be produced using any method known in the art, including synthetic and recombinant methodologies. When produced recombinantly, the GPCR internal fluorescent sensor polypeptides can be expressed in eukaryotic or prokaryotic host cells.

a. Circularly Permuted Fluorescent Protein

[0054] The circularly permuted fluorescent protein (cpFP) can be from any known fluorescent protein known in the art. In some embodiments, the circularly permuted protein is from a green fluorescent protein (GFP) or a red fluorescent protein (RFP), e.g., from mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple, mKate2, mMaple, mCardinal, mNeptune, far-red single-domain cyanbacteriochrome WP 016871037 or far-red single- domain cyanbacteriochrome anacy 255 lg3. Generally, the N-terminus of the circularly permuted is an amino acid residue within the seventh beta strand of the fluorescent protein in its non-circularly permuted form. This is depicted in Figure 10. Within the seventh beta strand of the fluorescent protein, in some embodiments, the circularly permuted N-terminus of the cpFP is positioned within the motif YN(Y/F)(N/I)SHNV (SEQ ID NO:19), e.g., of a non-permuted green fluorescent protein, or within the motif

WE(A/PAO(S/L/N/T)(S/E/T)E(R/M/T/K)(M/L) (SEQ ID NO:20) of a non-permuted red fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 7 (e.g., N) of the amino acid motif YN(Y/F)(N/I)SHNV (SEQ ID NO: 19) of a non-permuted green fluorescent protein. In some embodiments, the circularly permuted N-terminus is positioned at the amino acid residue corresponding to residue 3 (e.g., (A/P/U/V/P)), 4 (e.g., (LSN)), 5 (e.g., S/T)), 6 (e.g., E) or 7 (e.g., R/M/K/T)) of the amino acid motif

WE(A/P/VXS/L/NA")(S/E/T)E(R/MyT/K)(>l/L) (SEQ ID NO:20) of a non-permuted red- fluorescent protein.

[0055] In some embodiments, the circularly permuted fluorescent protein is from a photo-convertible or photoactivable fluorescent protein. Numerous photo-convertible or photoactivable fluorescent proteins are known in the art, and their circularly permuted forms can be used in the present sensors. See, Rodriguez, et al., Trends Biochem Sci. (2016) Nov 1. pii: S0968-0004(16)30173-6; Ai, et al, NatProtoc. 2014 Apr;9(4):910-28; Kyndt, et al, Photochem PhotobiolSci. 2004 Jun;3(6):519-30; Meyer, etal., Photochem Photobiol Sci. 2012 Oct; 11(10): 1495-514. In some embodiments, the photo-convertible or photoactivable fluorescent protein is selected from the group consisting of photoactivable green fluorescent protein (paGFP; e.g., SEQ ID NO:4), mCherry (e.g., SEQ ID NOs:6-7), mEos2 (e.g., SEQ ID NO: 11), mRuby2 (e.g., SEQ ID NO:9), mRuby3, mClover3, mApple (e.g., SEQ ID NO:8), mKate2 (e.g., SEQ ID NO: 10), mMaple (SEQ ID NO: 12), far-red single-domain cyanbacteriochrome WP 016871037 and far-red single-domain cyanbacteriochrome anacy 2551g3.

[0056] In some embodiments, the circularly permuted fluorescent protein is from a fluorescent protein having at least about 90% sequence identity, e.g., at least about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a non-permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 1, wherein the tyrosine at residue position 69 of SEQ ID NO: 1 is replaced with a tryptophan (Y 69W) to generate a cyan fluorescent protein (CFP) sensor. In some embodiments, the circularly permuted fluorescent protein is from a green fluorescent protein having at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 1, wherein the threonine at residue position 206 of SEQ ID NO: 1 is replaced with a tyrosine (T206Y) to generate a yellow fluorescent protein (YFP) sensor. In some embodiments, the circularly permuted fluorescent protein has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a circularly permuted fluorescent protein selected from the group consisting of SEQ ID NOS: 15-18.

[0057] Numerous circularly permuted fluorescent proteins are described in the art, and may find use in the present fluorescent sensors. The choice of a particular circularly permuted fluorescent protein for use in a fluorescent protein sensor may depend on the desired emission spectrum for detection, and include, but is not limited to, circularly permuted fluorescent proteins with green, blue, cyan, yellow, orange, red, or far-red emissions. A number of circularly permuted fluorescent proteins are known and can be used in the present sensors. See, e.g., Pedelacq et al. (2006) Nat. Biotechnol. 24:79-88 for a description of circularly permuted superfolder GFP variant (cpsfGFP), Zhao et al. (2011) Science 333: 1888-1891 for a description of circularly permuted mApple; Shui et al. (2011) PLoS One; 6(5):e20505 for a description of circularly permuted variants of mApple and mKate; Carlson et al. (2010) Protein Science 19:1490-1499 for a description of circularly permuted red fluorescent proteins, Gautam et al. (2009) Front. Neuroeng. 2:14 for a description of circularly permuted variants of enhanced green fluorescent protein (EGFP) and mKate, Zhao et al. (2011) Science 333(6051): 1888-1891 for a description of a circularly permuted variant of mApple; Liu et al. (2011) Biochem. Biophys. Res. Commun.

412(1):155-159 for a description of circularly permuted variants of Venus and Citrine, Li et al. (2008) Photochem. Photobiol. 84(1 ): 111-119 for a description of circularly permuted variants of mCherry, and Perez- Jimenez et al. (2006) J. Biol. Chem. December 29;

281(52):40010-40014 for a description of circularly permuted variants of enhanced yellow fluorescent protein (EYFP). Further illustrative circularly permuted fluorescent proteins are described in e.g., Honda, etal, PLoSOne. 2013 May 22;8(5):e64597; Schwartzlander, et al., Biochem J. 2011 Aug 1 ;437(3):381-7; Miyawaki, et al., Adv Biochem Eng Biotechnol. 2005;95:1-15; Tantama, etal, ProgBrainRes. 2012;196:235-63; Mizuno, etal., J Am Chem Soc. 2007 Sep 19; 129(37): 11378-83; Chiang, etal., Biotechnol I Jitt. 2006

Apr;28(7):471-5; and in U.S. Patent Publication Nos. 2015/0132774; 2010/0021931; and 2008/0178309. b. N-Terminal and C-Terminal Linkers

[0058] The G protein-coupled receptor (GPCR) internal fluorescent sensors have an

N-terminal linker (LI) and a C -terminal linker (L2). In some embodiments, LI comprises a peptide linker having from 2 to 13 amino acid residues, e.g., 2 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 residues, wherein each amino acid residue can be any naturally occurring amino acid. In some embodiments, L2 comprises a peptide linker having from 2 to 5 amino acid residues, e.g., 2 to 3, 4 or 5 residues, wherein each amino acid residue can be any naturally occurring amino acid. In some embodiments, LI and L2 are peptides that independently have 2, 3, 4, 5, or 6 amino acid residues. In some embodiments, LI comprises LSSLI and L2 comprises NHDQL. In some embodiments, LI comprises LSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, LI comprises QLQKIDLSSX1X2 and L2 comprises X3X4DQL, wherein XI, X2, X3, X4 are independently any amino acid. In some embodiments, X1X2 is selected from the group consisting of leucine-isoleucine (LI), alanine-valine (AV), isoleucine-lysine (IK), serine-arginine (SR), lysine-valine (KV), leucine-alanine (LA), cysteine-proline (CP), glycine-methionine (GM), valine-arginine (VR), asparagine-valine (NV), arginine-valine (RV), arginine-glycine (RG), leucine-glutamate (LE), serine-glycine (SG), valine-aspartate (VD), alanine-phenylalanine (AF), threonine-aspartate (TD), methionine-arginine (MR), leucine-glycine (LG), arginine-glutamine (RQ), serine- tryptophan (SW), serine-glycine (SG), valine-aspartate (VD), leucine-glutamate (LE), alanine-phenylalanine (AF), serine-tryptophan (SW), arginine-glycine (RG), threonine- aspartate (TD), leucine-glycine (LG), arginine-glutamine (RQ), threonine-tyrosine (TY), leucine-leucine (LL), valine-leucine (VL), threonine-glutamine (TQ), valine-phenylalanine (VF), threonine-threonine (TT), leucine-valine (LV), valine-isoleucine (VI), valine-valine (VV), proline-valine (PV), glycine-valine (GV), serine-valine (SV), phenylalanine-valine (FV), cysteine-valine (CV), glutamate-valine (EV), glutamine-valine (QV), and lysine- valine (KV), arginine-tryptophan (RW), glycine-aspartate (GD), alanine-leucine (AL), proline-methionine (PM), glycine-arginine (GR), glycine-tyrosine (GY), isoleucine-cysteine (IC), and glycine-leucine (GL). In some embodiments, X3X4 is selected from the group consisting of asparagine-histidine (NH), threonine-arginine (TR), isoleucine-isoleucine (Π), proline-proline (PP), leucine-phenylalanine (LF), valine-threonine (VT), glutamine-glycine (QG), alanine-leucine (AL), proline-argjnine (PR), arginine-glycine (RG), threonine-leucine (TL), threonine-proline (TP), glycine-valine (GV), threonine-threonine (TT), cysteine- cysteine (CC), alanine-threonine (AT), leucine-proline (LP), tyrosine-proline (YP), tryptophan-proline (WP), serine-leucine (SL), glutamate-arginine (ER), methionine-cysteine (MC), methionine-histidine (MH), tryptophan-leucine (YL), leucine-serine (LS), arginine- proline (RP), lysine-proline (KP), tyrosine-proline (YP), tryptophan-proline (WP), serine- serine (SS), glycine-valine (GV), valine-serine (VS), glutamine-asparagine (QN), lysine- serine (KS), lysine-threonine (KT), lysine-histidine (KH), lysine-valine (KV), lysine- glutamine (KQ), lysine-arginine (KR), cysteine-proline (CP), alanine-proline (AP), serine- proline (SP), isoleucine-proline (IP), tyrosine-proline (YP), threonine-proline (TP), arginine-proline (RP), aspartate-histidine (DH), histidine-tyrosine (HY), glycine-glycine (GG), proline-histidine (PH), serine-threonine (ST), argjnine-serine (RS), arginine-histidine (RH), and tryptophan-proline (WP). In some embodiments, X1X2 comprises alanine-valine (AV) and X3X4 comprises lysine-proline (KP); threonine-arginine (TR); aspartate-histidine (DH); threonine-threonine (TT); serine-serine (SS); glycine-valine (GV); cysteine-cysteine (CC); valine-serine (VS); glutamine-asparagine (QN); lysine-serine (KS); lysine-threonine (KT); lysine-histidine (KH); lysine-valine (KV); lysine-glutamine (KQ); lysine-arginine (KR); lysine-proline (KP); cysteine-proline (CP); alanine-proline (AP); serine-proline (SP); isoleucine-proline (IP); tyrosine-proline (YP); threonine-proline (TP); or arginine-proline (RP); X1X2 comprises leucine-valine (LV) and X3X4 comprises threonine-arginine (TR), lysine-proline (KP) or valine-threonine (VT); X1X2 comprises arginine-valine (RV) and X3X4 comprises threonine-arginine (TR), lysine-proline (KP) or threonine-proline (TP); X1X2 comprises arginine-glycine (RG) and X3X4 comprises tyrosine-leucine (YL) or threonine-arginine (TR); X1X2 comprises serine-arginine (SR) and X3X4 comprises leucine-phenylalanine (LF) or proline-proline (PP); XI X2 comprises proline-methionine (PM) and X3X4 comprises proline-histidine (PH) or serine-serine (SS); X1X2 comprises valine-valine (W) and X3X4 comprises threonine-arginine (TR) or lysine-proline (KP); X1X2 comprises leucine-isoleucine (LI) and X3X4 comprises threonine-arginine (TR); X1X2 comprises threonine-tyrosine (TY) and X3X4 comprises threonine-arginine (TR); XI X2 comprises isoleucine-lysine (IK) and X3X4 comprises isoleucine-isoleucine (Π); X1X2 comprises cysteine-proline (CP) and X3X4 comprises alanine-leucine (AL); X1X2 comprises glycine-methionine (GM) and X3X4 comprises proline-arginine (PR); X1X2 comprises leucine-alanine (LA) and X3X4 comprises glutamine-glycine (QG); X1X2 comprises valine-arginine (VR) and X3X4 comprises arginine-glycine (RG); X1X2 comprises serine-glycine (SG) and X3X4 comprises tyrosine-proline (YP); X1X2 comprises valine-aspartate (VD) and X3X4 comprises tryptophan-proline (WP); XI X2 comprises leucine-glutamate (LE) and X3X4 comprises leucine-proline (LP); X1X2 comprises alanine-phenylalanine (AF) and X3X4 comprises serine-leucine (SL); X1X2 comprises serine-tryptophan (SW) and X3X4 comprises argjnine-proline (RP); XI X2 comprises threonine-aspartate (TD) and X3X4 comprises glutamate-arginine (ER); X1X2 comprises leucine-glycine (LG) and X3X4 comprises methionine-histidine (MH); X1X2 comprises arginine-glutamine (RQ) and X3X4 comprises leucine-serine (LS); X1X2 comprises methi oni ne-arginine (MR) and X3X4 comprises methionine-cysteine (MC); X1X2 comprises leucine-leucine (LL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-leucine (VL) and X3X4 comprises threonine-arginine (TR); X1X2 comprises threonine-glutamine (TQ) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-phenylalanine (VF) and X3X4 comprises threonine-arginine (TR); XI X2 comprises threonine-threonine (TT) and X3X4 comprises threonine-arginine (TR); X1X2 comprises valine-isoleucine (VI) and X3X4 comprises threonine-arginine (TR); X1X2 comprises proline-valine (PV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glycine-valine (GV) and X3X4 comprises lysine-proline (KP); X1X2 comprises serine- valine (SV) and X3X4 comprises lysine-proline (KP); X1X2 comprises asparagjne-valine (NV) and X3X4 comprises lysine-proline (KP); X1X2 comprises phenylalanine-valine (FV) and X3X4 comprises lysine-proline (KP); X1X2 comprises cysteine-valine (CV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glutamate-valine (EV) and X3X4 comprises lysine-proline (KP); X1X2 comprises glutamine-valine (QV) and X3X4 comprises lysine-proline (KP); X1X2 comprises lysine-valine (KV) and X3X4 comprises lysine-proline (KP); X1X2 comprises arginine-tryptophan (RW) and X3X4 comprises histidine-tyrosine (HY); X1X2 comprises glycine-aspartate (GD) and X3X4 comprises glycine-glycine (GG); X1X2 comprises alanine-leucine (AL) and X3X4 comprises asparagine-histidine (NH); X1X2 comprises glycine-arginine (GR) and X3X4 comprises serine-threonine (ST); X1X2 comprises glycine-tyrosine (GY) and X3X4 comprises arginine-serine (RS); X1X2 comprises isoleucine-cysteine (IC) and X3X4 comprises argjnine-histidine (RH); or X1X2 comprises glycine-leucine (GL) and X3X4 comprises tryptophan-proline (WP). In some embodiments, LI comprises LSSLIX1 and L2 comprises X2NHDQL, wherein XI, X2 are independently any amino acid. In some embodiments, XI is selected from the group consisting of L W, V, L, F, P, N, Y and D; and X2 is selected from the group consisting of G, N, M, R T, S, K, L, Y, H, F, E, I and W. In some embodiments, XI is I and X2 is N or S; XI is W and X2 is M, T, F, E or I; XI is V and X2 is R, H or T; Xl is L and X2 is T; Xl is F and X2 is S; XI is P and X2 is K or S; XI is Y and X2 is S, L; or XI is D and X2 is W. 3. G Coupled Protein Receptors With Integrated Sensors

[0059] In some embodiments, the fluorescent sensors are incorporated or integrated into the third intracellular loop of a G protein-coupled receptor (GPCR). This can be readily accomplished employing recombinant techniques known in the art. Generally, any amino acid within the third loop region of a GPCR may serve as an insertion site for a cpFP (e.g., before or after, or as a replacement). In some embodiments, the cpFP sensor is inserted between two amino acid residues within the middle third of the third intracellular loop of a G protein-coupled receptor (GPCR). As necessary or appropriate, one, two, three, four, or more, amino acid residues within the third intracellular loop of the wild-type G protein-coupled receptor may be removed in order that the loop can accommodate the sensor. In some embodiments for inserting a cpFP into the third intracellular loop, the third intracellular loop and part of the sixth transmembrane sequence (TM6) (e.g., for a beta2 adrenergic receptor RQLQ— cpFP CWLP) can be used as a module system to transfer to other GPCRs.

[0060] As is standard or customary in the art, the "third intracellular loop" or "third cytoplasmic loop" is with reference to N-terminus of the GPCR that is integrated into the extracellular membrane of a cell and refers to the third segment of a GPCR polypeptide that is located in the cytoplasmic or intracellular side of the extracellular membrane. It is phrase commonly used by those of skill in the art. See, e.g., Kubale, et al., IntJMol Sci. (2016) Jul 19; 17(7); Clayton, et al., J Biol Chem. (2014) Nov 28;289(48):33663-75; Gomez-Mouton, etal, Blood. (2015) Feb 12; 125(7): 1116-25; Terawaki, etal, Biochem BiophysRes Commun. 2015 Jul 17-24;463(l-2):64-9; Gabl, et al., PLoS One. 2014 Oct

10;9(10):el09516; Fukunaga, et al, Mol Neurobiol 2012 Feb;45(l): 144-52; Nakatsuma, et al, BiophysJ. 2011 Apr 20; 100(8): 1874-82; Shioda, etal., J Pharmacol Sci.

2010;114(1):25-31; Shpakov, et al., Dokl Biochem Biophys. 2010 Mar-Apr;431:94-7;

Takeuchi, et al., JNeurochem. 2004 Jun;89(6): 1498-507. The third intracellular loop of various G protein-coupled receptors (GPCRs) is identified in Figures 11 A-E.

[0061] Accordingly, provided are G protein-coupled receptors comprising a cpFP sensor, as described above and herein, wherein the sensor is integrated into the third intracellular loop of the G protein-coupled receptor.

[0062] In some embodiments, the G protein-coupled receptor is a class A type or alpha G protein-coupled receptor. In some embodiments, the G protein-coupled receptor is selected from the group consisting of an adrenoceptor or adrenergic receptor, an opioid receptor, a 5-Hydroxytryptamine (5-HT) receptor, a dopamine receptor, a muscarinic acetylcholine receptor, an adenosine receptor, a glutamate metabotropic receptor, a gamma- aminobutyric acid (GABA) type B receptor, corticotropin-releasing factor (CRF) receptor, a tachykinin or neurokinin (NK) receptor, an angiotensin receptor, an apelin receptor, a bile acid receptor, a bombesin receptor, a bradykinin receptor, a cannabinoid receptor, a chemokine receptor, a cholecystokinin receptor, a complement peptide receptor, an endothelin receptor, a formylpeptide receptor, a free fatty acid receptor, a galanin receptor, a ghrelin receptor, a glycoprotein hormone, a gonadotrophin-releasing hormone receptor, a G protein-coupled estrogen receptor, an histamine receptor, a leukotriene receptor, a lysophospholipid (LP A) receptor, a lysophospholipid (SIP) receptor, a melanocortin receptor, a melatonin receptor, a neuropeptide receptor, a neurotensin receptor, an orexin receptor, a P2Y receptor, a prostanoid or prostaglandin receptor, somatostatin receptor, a tachykinin receptor, a thyrotropin-releasing hormone receptor, a urotensin receptor, and a vasopressin/oxytocin receptor. In some embodiments, the G protein-coupled receptor is selected from the group consisting of an adrenoceptor beta 1 (ADRBl), adrenoceptor beta 2 (ADRB2), adrenoceptor alpha 2A (ADRA2A), a mu (M)-type opioid receptor (OPRM), a kappa (ic)-type opioid receptor (OPRK), a delta (6)-type opioid receptor (OPRD), a dopamine receptor Dl (DRD1), a 5-hydroxy-tryptamine receptor 2A (5-HT ₂ A), a melatonin receptor type IB (MTNR1B), an adenosine Al receptor (ADORAl), a cannabinoid receptor (type-1) (CNR1), a histamine receptor HI (HRH1), a neuropeptide Y receptor Yl (NPY1R), a cholinergic receptor muscarinic 2 (CHRM2), a hypocretin (orexin) receptor 1 (HCRTR1), a tachykinin receptor 1 (TACR1) {a.k.a. neurokinin 1 receptor (NK1R)), a corticotropin releasing hormone receptor 1 (CRHRl), a glutamate metabotropic receptor 1 (GRMl), and a gamma-aminobutyric acid (GABA) type B receptor subunit 1 (GABBRl). In some embodiments, the G protein-coupled receptor is selected from the group consisting of:

Metabotropic Glutamate Receptor type-3 (MGLUR3); Metabotropic Glutamate Receptor type-5 (MGLUR5); Gamma-aminobutyric acid Receptor type-2 (GABABl); Gamma- aminobutyric acid Receptor type-2 (GABAB2); Cannabinoid Receptor type-1 (CB1);

Gonadotropin-Rel easing Hormone Receptor (GNRHR); Vasopressin Receptor type- 1

(VIA); Oxytocin Receptor (OTR); Adenosine Receptor type-2 (A2A); Beta-2 Adrenergic Receptor (B2AR); Dopamine Receptor type-1 (DRDl); Dopamine Receptor type-2

(DRD2); Acetylcholine Muscarinic Receptor type-2 (M2R); Histamine Receptor type-1 (H1R); Serotonin Receptor type-2A (5HT2A); Serotonin Receptor type-2B (5HT2B);

Tachykinin Receptor type-1 (NK1); Tachykinin Receptor type-2 (NK2); Tachykinin

Receptor type-3 (NK3); Melatonin Receptor type-IB (MTNR1B); P2 purinoceptor type Yl (P2Y1); Angiotensin-n Receptor type-1 (ATI); Kappa Opioid Receptor type-1 (KOR1); Mu Opioid Receptor type-1 (MORI); and Delta Opioid Receptor type-1 (DOR1).

[0063] In some embodiments, the receptor is mutated to be signaling incompetent or incapable. To prevent internalization and arrestin-dependent signaling for any GPCR, GRK6 phosphorylation sites can be replaced with alanine residues. The residue numbers and location of the G protein-coupled receptor kinase 6 (GRK6) residues vary between different GPCRs. On the Beta2AR, the GRK6 residues are SS355, 356 (residues 624-625 of SEQ ID NO: 22). Alternatively or additionally, G-protein dependent signaling can be prevented or inhibited by mutating a specific residue that is mostly conserved among many GPCRs. This residue corresponds to Phenylalanine (F) 139 (residue F163 of SEQ ID NO: 22) on the Beta2AR. This conserved residue that facilitates G protein dependent signaling varies from GPCR to GPCR, but the sequence alignment in Figure 11 shows its

correspondent residue on other GPCRs.

[0064] In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a beta2 adrenergic receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 22 or SEQ ID NO:32. In some embodiments, the sensor replaces one or more or all of amino acid residues QLQKIDKSEGRFHVQNLS (residues 253-270 of SEQ ID NO:22) and the carboxy-terminus of L2 abuts KEHK (residues 536-539 of SEQ ID NO: 22). In some embodiments, the sensor replaces one or more or all of amino acid residues QLQKIDKSEGRFHVQNLS (residues 253-270 of SEQ ID NO:22) and the carboxy-terminus of L2 abuts FCLK (residues 533-536 of SEQ ID NO:22). In some embodiments, one or more of amino acid residues F139, S355 and S356 (residues 163 and 624-625 in SEQ ID NO: 22) of the beta2 adrenergic receptor are replaced with alanine residues to render the beta2 adrenergic receptor signaling incompetent. In some

embodiments, X at amino acid residue 163 in SEQ ID NO: 22 or at residue 139 of SEQ ID NO:32 is any amino acid or an amino acid selected from the group consisting of A, F, G, L L, M, S, T and V, particularly A. In some embodiments when the G protein-coupled receptor is a beta2 adrenergic receptor, the cpFP sensor is inserted into the third intracellular loop between residues AKRQ and LQKL e.g., between residues 253 and 254 of SEQ ID NO:22. In some embodiments, the insertion sites of the cpGFP into a beta2 adrenergic receptor can be any amino acids in the region of KSEGRFHVQLSQVEQDGRTGHGL of the third loop. In some embodiments when the G protein-coupled receptor is a beta2 adrenergic receptor, the cpFP sensor is inserted into the third intracellular loop between residues QNLS and AEVK, e.g., between residues 270 and 271 of SEQ ID NO:22. In some embodiments when the G protein-coupled receptor is a beta2 adrenergic receptor, the cpFP sensor is inserted into the third intracellular loop between residues EAKR and QLQK, e.g., between residues 252 and 253 of SEQ ID NO:22. In some embodiments when the G protein-coupled receptor is a beta2 adrenergic receptor, the cpFP sensor is inserted into the third intracellular loop between residues KRQL and QKID, e.g., between residues 254 and 255 of SEQ ID NO:22. In some embodiments when the G protein-coupled receptor is a beta2 adrenergic receptor, LI of the cpFP sensor is alanine-valine (AV) and L2 of the cpFP sensor is threonine-arginine (TR) or lysine-proline (KP).

[0065] In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a mu ^)-type opioid receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:24 or SEQ ID NO:37. In some embodiments, amino acid residue V199 (residue 199 in SEQ ID NO: 24) of the mu (μ)^ρβ opioid receptor is replaced with an alanine residue to render the mu (μ)-ί)φβ opioid receptor signaling incompetent. In some embodiments, X at amino acid residue 199 in SEQ ID NO: 24 or at residue 175 of SEQ ID NO:37 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments when the G protein-coupled receptor is a mu (μ)-ί)φβ opioid receptor, the cpFP sensor is inserted into the third intracellular loop between residues RMLS and GS, e.g., between residues 292 and 293 of SEQ ID NO:24. In some embodiments when the G protein-coupled receptor is a mu ^)-type opioid receptor, LI of the cpFP sensor is isoleucine-lysine (IK) and L2 of the cpFP sensor is isoleucine-isoleucine (Π).

[0066] In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a dopamine receptor Dl (DRD1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 26 or SEQ ID NO:30. In some embodiments, the N-terminus of LI abuts IAQK (residues 244-247 of SEQ ID NO:26), the C-terminus of L2 abuts KRET (residues 534-537 of SEQ ID NO:26), the sensor replacing residues 248 to 533 of SEQ ID NO:26. In some embodiments, amino acid residue F129 (residue 153 in SEQ ID NO: 26 or residue 129 of SEQ ID NO:30) of the dopamine receptor Dl (DRD1) is replaced with an alanine residue to render the dopamine receptor Dl (DRD1) signaling incompetent. In some embodiments, X at amino acid residue 153 in SEQ ID NO: 26 or at residue 129 of SEQ ID NO:30 is any amino acid or an amino acid selected from the group consisting of A, F, G, L _. L, M, S, T and V, particularly A. In some embodiments when the G protein- coupled receptor is a dopamine receptor Dl (DRDl), the cpFP sensor is inserted into the third intracellular loop between residues AKNC and QTTT, e.g., between residues 265 and 266 of SEQ ID NO:21. In some embodiments when the G protein-coupled receptor is a dopamine receptor Dl (DRDl), LI of the cpFP sensor is serine-arginine (SR) and L2 of the cpFP sensor is proline-proline (PP).

[0067] In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a 5 hydroxy-tryptamine 2A (5-HT2A) receptor having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 28 or SEQ ID NO:33. In some embodiments, the N-terminus of LI abuts SLQK (residues 284-287 of SEQ ID NO:28), the C-terminus of L2 abuts NEQK (residues 586-589 of SEQ ID NO:28), the sensor replacing residues 288 to 585 of SEQ ID NO:28. In some embodiments, amino acid residue 1181 (residue 205 in SEQ ID NO: 28) of the 5-hydroxy-tryptamine 2A (5-HT2A) receptor is replaced with an alanine residue to render the 5-hydroxy-tryptamine 2A (5-HT2A) receptor signaling incompetent. In some embodiments, X at amino acid residue 205 in SEQ ID NO: 28 or at residue 181 of SEQ ID NO:33 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments when the G protein-coupled receptor is a 5-hydroxy-tryptamine 2A (5-HT2A) receptor, the cpFP sensor is inserted into the third intracellular loop between residues TRAK and LASF, e.g., between residues 301 and 302 of SEQ ID NO:23. In some embodiments when the G protein-coupled receptor is a 5-hydroxy-tryptamine 2A (5-HT2A) receptor, LI of the cpFP sensor is serine-argjnine (SR) and L2 of the cpFP sensor is leucine-phenylalanine (LF).

[0068] In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises an adrenoceptor beta 1 (ADRBl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:31. In some embodiments, X at amino acid residue 164 in SEQ ID NO: 31 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises an adenosine A2a receptor (ADORA2A) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 34. In some embodiments, X at amino acid residue 110 in SEQ ID NO: 34 is any amino acid or an amino acid selected from the group consisting of A, F, G, L _. L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises an adrenoceptor alpha 2A (ADRA2A) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 35. In some embodiments, X at amino acid residue 139 in SEQ ID NO: 35 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein coupled- receptor comprising an integrated cpFP sensor comprises a kappa receptor delta 1 (OPRKl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 36. In some embodiments, X at amino acid residue 164 in SEQ ID NO: 36 is any amino acid or an amino acid selected from the group consisting of A, F, G, L L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises an opioid receptor delta 1 (OPRDl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 38. In some embodiments, X at amino acid residue 154 in SEQ ID NO: 38 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein couple receptor comprising an integrated cpFP sensor comprises a melatonin receptor IB (MTNR1B) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 39. In some embodiments, X at amino acid residue 146 in SEQ ID NO: 39 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a cannabinoid receptor type 1 (CNRl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 40. In some embodiments, X at amino acid residue 222 in SEQ ID NO: 40 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a histamine receptor HI (HRHl) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 41. In some embodiments, X at amino acid residue 133 in SEQ ID NO: 41 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a neuropeptide Y receptor Yl (NPYIR) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 42. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a muscarinic cholinergic receptor type 2 (CHRM2) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 43. In some embodiments, X at amino acid residue 129 in SEQ ID NO: 43 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. In some embodiments, the G protein-coupled receptor comprising an integrated cpFP sensor comprises a hypocretin (orexin) receptor 1 (HCRTR1) having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 44. In some embodiments, X at amino acid residue 152 in SEQ ID NO: 44 is any amino acid or an amino acid selected from the group consisting of A, F, G, I, L, M, S, T and V, particularly A. 4. Production of Circularly Permuted Fluorescent Protein Sensors and GPCRs with an Integrated cpFP Sensor

[0069] Fluorescent protein sensors can be produced in any number of ways, all of which are well known in the art. In one embodiment, the fluorescent protein sensors are generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode the desired polypeptides using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), 2012, Cold Spring Harbor Laboratory Press and Ausubel, et al., eds. Current Protocols in Molecular Biology, 1987-2016, John Wiley & Sons

(http://onlinelibrary.wiley.coni/book/10.1002/0471142727), for a description of techniques used to obtain, isolate and manipulate nucleic acids. In some embodiments, Circular Polymerase Extension Cloning (CPEC) can be used to insert a polynucleotide encoding a cpFP sensor into a polynucleotide encoding a GPCR. See, e.g., Quan, et al., NatProtoc, 2011. 6(2): p. 242-51.

[0070] The sequences encoding polypeptides can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311;

Stemmer et al. (1995) Gene 164:49-53.

[0071] Recombinant techniques are readily used to clone sequences encoding polypeptides useful in the present fluorescent protein sensors that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409.

[0072] Once coding sequences have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs which operably link, in various combinations,

polynucleotides encoding polypeptides having deletions or mutations therein.

[0073] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage .lamda. (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, Green and Sambrook, supra; and Ausubel, supra.

[0074] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit).

[0075] Plant expression systems can also be used to produce the fluorescent protein sensors described herein. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139:1-22. [0076] Viral systems, such as a vaccinia based infection/transfection system, as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74: 1103-1113, will also find use. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s). Other viral systems that find use include adenovirus, adeno-associated virus, lentivirus and retrovirus.

[0077] The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. Both the naturally occurring signal peptides and heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honey bee mellitin signal sequence.

[0078] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0079] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0080] In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, generally, Green and Sambrook, supra; and Ausubel, supra.

[0081] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, HEK 293T cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorphs, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti,

Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugjperda, and Trichoplusia ni.

[0082] Depending on the expression system and host selected, the fluorescent protein sensors are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.

S. Polynucleotides, Expression Cassettes, Vectors

[0083] Accordingly, provided are polynucleotides that encode the cpFP sensors, as described above and herein. In some embodiments, the polynucleotide encodes a cpFP sensor (Ll-cpFP-L2), wherein the circularly permuted fluorescent protein has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a circularly permuted fluorescent protein selected from the group consisting of SEQ ID NOS: 15-18 (e.g., cpGFP, cpmRuby2, cpmApple and cpmEos2). In some embodiments, the polynucleotide encodes a cpFP sensor (Ll-cpFP-L2), wherein the polynucleotide encoding the circularly permuted fluorescent protein has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 29 (a cpGFP). [0084] Further provided are polynucleotides encoding a GPCR comprising a cpFP sensor integrated into its third intracellular loop. In some embodiments, the polynucleotide encodes a beta2 adrenergic receptor comprising an integrated cpFP sensor, the protein having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 22. In some embodiments, the polynucleotide encodes a beta2 adrenergic receptor comprising an integrated cpFP sensor, the polynucleotide having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 16. In some embodiments, the polynucleotide encodes a mu M-type opioid receptor comprising an integrated cpFP sensor, the protein having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 24. In some embodiments, the polynucleotide encodes a mu (μ)^ρε opioid receptor comprising an integrated cpFP sensor, the polynucleotide having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a dopamine receptor Dl (DRD1) comprising an integrated cpFP sensor, the protein having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 26. In some embodiments, the polynucleotide encodes a dopamine receptor Dl (DRD1) comprising an integrated cpFP sensor, the protein having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a 5 hydroxy-tryptamine 2 A (5-HT2A) receptor comprising an integrated cpFP sensor, the protein having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 28. In some embodiments, the polynucleotide encodes a 5 hydroxy-tryptamine 2A (5-HT2A) receptor comprising an integrated cpFP sensor, the polynucleotide having at least 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 22.

[0085] Further provided are expression cassettes comprising the polynucleotides encoding the cpFP sensors or GPCRs comprising a cpFP sensor integrated into its third intracellular loop, as described above and herein. The expression cassettes comprise a promoter operably linked to and capable of driving the expression of the cpFP sensor or GPCR comprising a cpFP sensor integrated into its third intracellular loop. In some embodiments, the promoters can promote expression in a prokaryotic or a eukaryotic cell, e.g., a mammalian cell, a fish cell. In some embodiments, the promoter is constitutive or inducible. In some embodiments, the promoter is organ or tissue specific. In some embodiments, the expression cassettes comprise a synapsin, CAG (composed of: (C) the cytomegalovirus (CMV) early enhancer element; (A) the promoter, the first exon and the first intron of chicken beta-actin gene; (G) the splice acceptor of the rabbit beta-globin gene), cytomegalovirus (CMV), glial fibrillary acidic protein (GFAP), Calcium/calmodulin- dependent protein kinase II (CaMKH) or Cre-dependent promoter (e.g., such as FLEX-rev) operably linked to and driving the expression a polynucleotide encoding a GPCR comprising a cpFP sensor integrated into its third intracellular loop, to direct expression in neurons. Subcellular targeting of GPCR comprising cpGFP is also possible using genetic strategy or intrabodies.

[0086] Further provided are plasmid and viral vectors comprising the

polynucleotides encoding the cpFP sensors or GPCRs comprising a cpFP sensor integrated into its third intracellular loop, as described above and herein. Viral vectors of use include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors and vaccinia viral vectors.

6. Cells

[0087] Further provided are cells comprising the polynucleotides encoding the cpFP sensors or GPCRs comprising a cpFP sensor integrated into its third intracellular loop, as described above and herein. In some embodiments, the polynucleotides encoding the cpFP sensors or GPCRs comprising a cpFP sensor integrated into its third intracellular loop may be episomal or integrated into the genome of the cell. In some embodiments, the host cells are prokaryotic or eukaryotic. Illustrative eukaryotic cells include without limitation mammalian cells (e.g., Chinese hamster ovary (CHO) cells, HeLa cells, HEK 293T cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells) fish cells (e.g., zebra fish cells) and insect cells.

[0088] In addition, GPCRs having an integrated cpFP sensor can be expressed in iPSC-derived cells or primary cells, such as dissociated neuronal culture. For example, in some embodiments with patient iPSC-derived brain cells (e.g., neurons and astrocytes), can be transformed with a polynucleotide encoding a GPCR having an integrated cpFP sensor. GPCRs comprising a cpFP sensor can be used for evaluating mechanistic action of neuropharmacological drugs.

[0089] In certain embodiments, the GPCRs having an integrated cpFP sensor can be wholly or partially purified and/or solubilized from the host cell, using methodologies for wholly or partially purifying and/or solubilizing the wild-type GPCRs known in the art. In some embodiments, the GPCRs having an integrated cpFP sensor are produced as partially or substantially purified nanodiscs solubilized-proteins. The method of solubilizing membrane proteins on nanodiscs has been well-established, especially in large scale production of GPCRs for crystallization purposes. Production of nanodisc-solubilized GPCR is described, e.g., in Manglik, et al. "Crystal structure of the μ -opioid receptor bound to a morphinan antagonist" Nature. (2012) 485(7398):321-6. Nanodiscs attached to membrane proteins are further described, e.g., in Parker, etal., Biochemistry (2014) 53(9):1511-20; Bertram, etal., Langmuir (2015) 31(30):8386-91; and Ma, etal., Anal Chem. (2016) 88(4):2375-9. Nanodiscs are reviewed in, e.g., Viegas, et al., Biol Chem. (2016) 397(12): 1335-1354; Malhotra, et al., Biotechnol Genet Eng Rev. (2014) 30(l-2):79- 93; Schuler, et al., Methods Mol Biol. (2013) 974:415-33; and Inagaki, et al , Methods. (2013) 59(3):287-300. Among many advantages, substantially purified (and solubilized) sensor proteins can have a long shelf life, e.g., of about 1-2 months (when refrigerated and stored properly) and can be immobilized onto a chip (e.g., a nanodisc) for the production of diagnostics or similar devices to be used, e.g., in clinical medicine, sport medicine or for a variety of other applications. In some embodiments, nanodisc-immobilized and solubilized GPCRs having an integrated cpFP sensor can be substantially purified and delivered to live cells and tissues to monitor the local drug effect.

7. Transgenic Animals

[0090] Further provided are transgenic animals comprising one or more G Coupled-

Protein Receptors (GPCRs) comprising a cpFP sensor integrated into its third intracellular loop, as described above and herein. In some embodiments, the transgenic animal is a mouse, a rat, a worm, a fly or a zebrafish. Depending on the promoter or promoters driving the expression of the G Coupled-Protein Receptors (GPCRs) comprising a cpFP sensor integrated into its third intracellular loop, the GPCR may be expressed only in select tissues or organs, or may be inducible. In some embodiments, a polynucleotide encoding a GPCRs having an integrated a cpFP sensor can be integrated into any locus in the genome of a non- human animal, e.g., via CRISPR/Cas9 or equivalent techniques. As described above and herein, the GPCR transgenes may be altered such that the expressed GPCR is signaling incompetent. In certain embodiments, a polynucleotide encoding a G Coupled-Protein Receptors (GPCRs) comprising a cpFP sensor integrated into its third intracellular loop is administered to a non-human animal in a viral vector.

[0091] Methods for making transgenic mice are known in the art, and described, e.g., in Behringer and Gertsenstein, "Manipulating the Mouse Embryo: A Laboratory Manual," Fourth edition, 2013, Cold Spring Harbor Laboratory Press; Pinkert, "Transgenic Animal Technology, Third Edition: A Laboratory Handbook," 2014, Elsevier; Hofker and Van Deursen, "Transgenic Mouse Methods and Protocols (Methods in Molecular

Biology)," 2011 , Humana Press.

[0092] Methods for making transgenic rats are known in the art, and described, e.g., in Li, et al, "Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon," SciRep. 2016 Sep 14;6:33225. doi: 10.1038/srep33225 (PMID: 27624004); Kawamata, et al, "Gene-manipulated embryonic stem cells for rat transgenesis," Cell Mol Life Sci. 2011 Jun;68(l 1): 1911-5; Pradhan, et al, "An Efficient Method for Generation of Transgenic Rats Avoiding Embryo Manipulation," Mol Ther Nucleic Acids. 2016 Mar 8;5:e293; Kanatsu-Shinohara, et al., "Production of transgenic rats via lentiviral

transduction and xenogeneic transplantation of spermatogonial stem cells," Biol Reprod. 2008 Dec;79(6): 1121-8.

[0093] Methods for making transgenic zebrafish are known in the art, and described, e.g., in Kimura, et al., "Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering," Nature Scientific Reports 4, Article number: 6545 (2014); Allende, "Transgenic Zebrafish Production," 2001, John Wiley & Sons, Ltd.; Bernardos and Raymond, "GFAP transgenic zebrafish," Gene Expression Patterns, (2006) 6(8): 1007-1013; Clark, et al, "Transgenic zebrafish using transposable elements," Methods Cell Biol. 2011;104:137-49; Lin, "Transgenic zebrafish," in Developmental Biology Protocols: Volume Π, Volume 136 of the series Methods in Molecular Biology™ pp 375-383, 2000, Humana Press.

[0094] Methods for making transgenic worms (e.g., Caenorhabditis elegans) are known in the art, and described, e.g., in Praitis, et al., Methods in Cell Biology (2011) 106:159-185; Hochbaum, etal., "Generation of Transgenic C. elegans by Biolistic

Transformation," J Vis Exp. 2010; (42): 2090, video atjove.com/video/2090/generation-of- transgenic-c-elegans-by-biolistic-transformation; Berkowitz, etal., "Generation of Stable Transgenic C. elegans Using Microinjection," J Vis Exp. 2008 Aug 15;(18). pii: 833, video atjove.com/video/833/generation^f-stable4ransgenic^^legans-u sing-microinjection; and Wu, etal., Curr Alzheimer Res. (2005) 2(l):37-45. Additionally, Knudra Transgenics provides services for creating transgenic C. elegans (www. knudra.com).

[0095] Methods for making transgenic flies (e.g., Drosophila) are known in the art, and described, e.g., in Fujioka, et al., "Production of Transgenic Drosophila," in

Developmental Biology Protocols: Volume II, Volume 136 of the series Methods in Molecular Biology™ pp 353-363; Fish, et al, NatProtoc. (2007) 2(10):2325-31; Jenett, et al, Cell Rep. (2012) 2(4): 991-1001; and at the Transgenic Fly Virtual Lab

(hlmii.org/biointeractive/transgenic-fly-virtual-lab). Further, Genetics Services, Inc.

provides services for creating transgenic Drosophila

(geneticservices.com/injection/drosophila-injections/).

[0096] In addition, virus encoding the GPCR having an integrated cpFP can also be injected into a transgenic mammal (e.g., rodents such as mice and rats) for transient, local expression followed by live imaging. UAS-GAL4 systems can also be used for specific expression of GPCR having an integrated cpFP into other animals, including worms, flies and zebrafish. See, e.g., DeVorkin, etal., Cold Spring HarbProtoc. (2014) 2014(9):967-72 (Drosophila); Jeibmann, et al., Int J Mol Sci (2009) (Drosophila); Halpera, et al., Zebrafish. 2008 Summer;5(2):97-110 (zebrafish); Scheer, et al. , Mechanisms of Development (1999) 80(2): 153- 158 (zebrafish); Auer, etal., Nature Protocols (2014) 9:2823-2840 (zebrafish).

8. Methods of Determining G Coupled-Protein Receptor (GPCR) Activation

[0097] The G Coupled-Protein Receptors (GPCRs) having a fluorescent sensor integrated into their third intracellular loop are useful to detect binding (and activation or inactivation) of ligands (e.g. agonists, inverse agonist and antagonists), in both in vitro and in vivo model systems. Accordingly, provided are methods of detecting binding of a ligand to a G protein-coupled receptor. In some embodiments, the methods comprise:

a) contacting the ligand with a GPCR, as described above and herein, under conditions sufficient for the ligand to bind to the GPCR; and

b) determining a change, e.g., increase or decrease, in an optics signal from the sensor integrated into the third intracellular loop of the GPCR, wherein a detectable change in fluorescence signal indicates binding of the ligand to the GPCR. In some embodiments, the optics signal is a linear optics signal. In some embodiments, the linear optics signal comprises fluorescence. In some embodiments, the change, e.g., increase or decrease, in fluorescence signal comprises a change in intensity of the fluorescence signal. In some embodiments, the change, e.g., increase or decrease, in fluorescence intensity is at least about 10% over, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, or more, over baseline, in the absence of ligand binding. In some embodiments, the change in

fluorescence signal comprises a change in color (spectrum or wavelength) of the fluorescence signal. In some embodiments, the optics signal is a non-linear optics signal. In some embodiments, the non-linear optics signal is selected from the group consisting of fiber optics, miniature fiber optics, fiber photometry, one photon imaging, two photon imaging, and three photon imaging. The use of non-linear optics in biological imaging is known in the art and can find use in the present methods. Fiber optic fluorescence imaging is described in Flusberg, etal, Nat Methods. 2005 Dec;2(12):941-50. Further illustrative publications relating to the use of non-linear optics in biological imaging include without limitation Javadi, etal, Nat Common. (2015) 6:8655; Qian, etal, Adv Mater. (2015) 27(14):2332-9; Kirmani, etal, Science. (2014) 343(6166):58-61; Kierdaszuk, JFluoresc. (2013) 23(2):339-47; Ware, Biotechniques. (2014) 57(5):237-9; Mandal, et al, ACSNano. (2015) 9(5):4796-805; Guo, etal, Biomed Opt Express. (2015) 6(10):3919-31; Miyamoto, et al, Neurosci Res. (2016) 103:1-9.

[0098] In some embodiments, a binding ligand further indicates activation of intracellular signaling from the GPCR. In some embodiments, the ligand is a suspected agonist of the GPCR. In some embodiments, the ligand is a suspected inverse agonist of the GPCR. In some embodiments, the ligand is a suspected antagonist of the GPCR. In some embodiments, the GPCR is in vitro, e.g., integrated into the extracellular membrane of a cell. In some embodiments, the GPCR is in vivo, e.g., expressed in a transgenic animal. In some embodiments, the GPCRs are altered to be signaling incompetent, as described above and herein.

[0099] In certain embodiments, the in vitro binding detection methods can be performed by measuring the fluorescence intensity of host cells expressing the G Coupled- Protein Receptors (GPCRs) having a fluorescent sensor integrated into their third intracellular loop employing microscopy, e.g., using a perfusion chamber to efficiently wash cultured cells in an isotonic buffer. For performing the methods in vivo, the one or more ligands suspected or known to be an agonist, inverse agonist or antagonist of the GPCR.

9. Methods of Library Screening [0100] The G Coupled-Protein Receptors (GPCRs) having a fluorescent sensor integrated into their third intracellular loop are further useful for screening of a plurality of ligands that are suspected agonists, inverse agonist and antagonists of the GPCR, particularly in in vitro model systems. Accordingly, provided are methods of screening for binding of a ligand to a G protein-coupled receptor and/or activation of a GPCR by a ligand. The methods can be performed for high throughput screening. In some embodiments, the methods comprise: a) contacting a plurality of members from a library of ligands with a plurality of GPCRs, as described above and herein, under conditions sufficient for the ligand members to bind to the GPCRs, wherein the plurality of GPCRs are arranged in an array of predetermined addressable locations; and b) determining a change, e.g., increase or decrease, in one or more optics signals from the sensor integrated into the third intracellular loop of the plurality GPCRs, wherein a detectable change in the one or more fluorescence signals indicates binding of one or more members of the library of ligands to at least one of the plurality GPCR.

[0101] In some embodiments, the one or more optics signals comprise a linear optics signal. In some embodiments, the linear optics signal comprises fluorescence. In some embodiments, the one or more fluorescence signals fluoresce at the same wavelength. In some embodiments, the one or more fluorescence signals fluoresce at different wavelengths. In some embodiments, the change in fluorescence signal comprises a change, e.g., increase or decrease, in intensity of the fluorescence signal. In some embodiments, the change, e.g., increase or decrease, in fluorescence intensity is at least about 10% over, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, or more, over baseline, in the absence of ligand binding. In some embodiments, the change in fluorescence signal comprises a change in color (spectrum or wavelength) of the fluorescence signal. In some embodiments, the optics signal is a non-linear optics signal. In some embodiments, the non-linear optics signal is selected from the group consisting of fiber optics, miniature fiber optics, fiber photometry, one photon imaging, two photon imaging, and three photon imaging. In some embodiments, a binding ligand further indicates activation of intracellular signaling from the GPCR. In some embodiments, two or more members of the plurality of GPCRs are different. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different type of GPCR. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different subtype of a GPCR. In some embodiments, two or more members of the plurality of GPCRs comprise a sensor that fluoresces at a different wavelength.

[0102] In some embodiments, the one or more fluorescence signals fluoresce at the same wavelength. In some embodiments, the one or more fluorescence signals fluoresce at different wavelengths. In some embodiments, the change in fluorescence signal comprises a change in intensity of the fluorescence signal. In some embodiments, the change in fluorescence intensity is at least about 10% over baseline, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, or more, in the absence of ligand binding. In some embodiments, the change in fluorescence signal comprises a change in color (spectrum or wavelength) of the fluorescence signal. In some embodiments, the change in fluorescence signal comprises both a change in intensity and a change in color (spectrum or wavelength) of the fluorescence signal. In some embodiments, a binding ligand further indicates activation of intracellular signaling from the G protein-coupled receptor. In some embodiments, two or more members of the plurality of G protein-coupled receptors are different. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different type of G protein-coupled receptor. In some embodiments, two or more members of the plurality of G-protein coupled receptors are a different subtype of a G protein- coupled receptor. In some embodiments, two or more members of the plurality of G-protein coupled receptors comprise a sensor that fluoresces at a different wavelength. In some embodiments, the plurality of ligands comprises suspected or known agonists of the G protein-coupled receptor. In some embodiments, the plurality of ligands comprises suspected or known inverse agonists of the G protein-coupled receptor. In some embodiments, the plurality of ligands comprises suspected or known antagonists of the G protein-coupled receptor. In some embodiments, the GPCRs are altered to be signaling incompetent, as described above and herein. In some embodiments, the screening methods are performed in a multiwell plate.

10. Kits

[0103] Provided are kits comprising one or more circularly permuted fluorescent protein sensors as described above and herein, e.g., in polynucleotide form, e.g., in a vector such as a plasmid or viral vector, suitable to integrate into a G Coupled-Protein Receptor (GPCR). Further provided are kits comprising one or more G Coupled-Protein Receptors (GPCRs) having a circularly permuted fluorescent protein sensor integrated into its third intracellular loop as described above and herein, e.g., in polypeptide and/or polynucleotide form. When provided in polynucleotide form, the polynucleotides may be lyophilized. In some embodiments, the kits comprise expression cassettes, plasmid vectors, viral vectors or cells comprising a polynucleotide encoding a G Coupled-Protein Receptor (GPCR) having a circularly permuted fluorescent protein sensor integrated into its third intracellular loop, as described above and herein. In kits comprising cells, the cells may be suspended in a glycerol solution and frozen. The kits may further comprise buffers, reagents, and instructions for use. In some embodiments, the kits comprise one or more transgenic animals having a transgene for expressing a G Coupled-Protein Receptor (GPCR) with a cpFP sensor integrated into its third intracellular loop, as described above and herein. EXAMPLES

[0104] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

An Optogenetic Platform For Monitoring G Coupled-Protein Receptor (GPCR)

Activation

MATERIALS AND METHODS:

[0105] Computational analysis. PyRosetta3 was used to extract every Phi dihedral angles for each residue from the active and inactive structure of Beta-2 Adrenergic receptor (PDB: 3P0G and 2RH1 respectively). The difference of phi angles was plotted along the residue sequence to identify the most dramatic change of phi angles between active and inactive state.

[0106] Molecular cloning. Codon optimized geneblocks were ordered from

Integrated DNA Technologies (IDT) for each of the following GPCRs: Beta-2 Adrenergic Receptor (Beta2AR), Mu-type Opioid Receptor- 1 (MOR-1), Dopamine Receptor Dl (DRDl) and Serotonin Receptor-2A (5-HT2A). Briefly, the geneblocks contained the following in order from the beginning: Hind-ΙΠ site, hemagglutinin secretion motif, flag tag, the full length human GPCR coding sequence (with the exception of DRDl for which the sequence corresponding to amino acids 1-377 was used), Not-I cut site. In the case of the DRDl sensor, an ER export motif (FCENEV) was added at the C-terminus to aid surface expression. The geneblocks were cloned into pEGFP Nl , a plasmid carrying a CMV promoter, using the Hind-ΙΠ and Not-I sites. To linearize the vector for CPEC we used the following primers: [0107] Beta2AR vector:

FWD: 5 '-GTC AGTTTTT ACGTTCCTCTGGTT ATT ATG G-3',

REV: 5'-CATGATAATTCCAAGCGTCTTCAGCG-3';

[0108] mu-type opioid receptor, MOR-1 vector:

FWD: 5'- GGC AGC AAGGAGAAGGACCGC-3

REV: 5 '- ACTGAGC ATTCGAACTGATTTGAGCC-3 ';

[0109] DRDl vector:

FWD: 5'-CAGACCACCACAGGTAATGGAAAGCCTG-3\

REV: 5'-GCAATTCTTGGCGTGGACTGCTGC-3 ';

[0110] 5-HT2A vector:

FWD: 5'-CTTGCCAGCTTCTCATTCCTTCCCC-3',

REV: 5'- TTTGGCCCGAGTGCCGAGGTC-3 '.

[0111] To create a library of insert variants with randomized linkers cpGFP was amplified from a GCaMP6 template using the following primers:

[0112] Beta2AR cpGFP insert library:

FWD: 5'-GGACGCTTTCATGTGCAGAATCTTTCANNKNNKAACGTCTATATCAAG GCCGACAAGCA -3',

REV: 5'-CTGTGCGTCCGTCCTGTTCAACTTGMNNMNNGTTGTACTCCAGCTTGTG CCCCAG- 3';

[0113] MOR-1 cpGFP insert library:

FWD 5 '-1TGGAAGCGGAAACTGCTCCTCTGCC ANNKNNKaacGTCTAT ATC AAGG CCGAC-3',

REV: 5'-GCGGCCGCTGTACATCAGGTTGTCAMNNMNNGTTGTACTCCAGCTTGT G-3';

[0114] DRDl cpGFP insert library:

FWD: 5 '-GC AGC AGTC C AC GC C A AGAATTGCNNKNNK A AC GTC TAT ATC AAGGCC GACAAGC -3', REV 5'-CAGGCTTTCCATTACCTGTGGTGGTCTGMNNMNNGTTGTACTCCAGCTT GTGCCCCAG-3';

[0115] 5-HT2A cpGFP insert library:

FWD: 5'-GACCTCGGCACTCGGGCCAAANNKNNKAACGTCTATATCAAGGCCGA CAAGCAG-3',

REV: 5'-GGGGAAGGAATGAGAAGCTGGCAAGMNNMNNGTTGTACTCCAGCTTG TGCCCC AGGATG-3 '.

[0116] With reference to the above-described primers, N means any nucleic acid base; K means either C or T; M means either C or A.

[0117] Cell culture, confocal imaging and quantification. For sensor screening,

HEK293T cells (ATCC #1573) were cultured at 37°C either on glass-bottomed 3.5 cm dishes (Mattek) or on glass-bottomed 12-well plates (Fisher Scientific) in the presence of DMEM supplemented with 10% Fetal Bovine Serum and 100 U/ml Penicillin/Streptomycin (all from Life Technologies). Cells were transfected at 60% confluency using Effectene reagent (Qiagen).

[0118] For sensor characterization, primary hippocampal neurons were freshly isolated according to a previously published protocol and co-cultured with astrocytes in Neurobasal medium with 2% 50x B27, 1% lOOx glutamax, 5% FBS and 0.01% gentamicin (lOmg/mL) (all reagents from Life Technologies). After 5-7 days in vitro

fluorodexoyuridine (FUdR) is added to cultures to inhibit mitotic growth of glia. Neuronal cultures were prepared on glass-bottomed dishes (Matteks) coated with Poly- Omithine/Laminine (20 μ¾½1 and 5μ¾¾η1 respectively). Neurons were infected at DIV7 and imaged at DIV14-20 using a 40X oil-based objective on an inverted Zeiss Observer LSN710 confocal microscope with 488/513 ex/em wavelengths. Cells were washed immediately prior to imaging using HBSS (Life Technologies) buffer supplemented with 1 mM CaCl ₂ and MgC-2. The sensor performance was analyzed as fluorescence signal change (AFF) after the addition of the Beta2AR agonist isoproterenol diluted in HBSS (10 uM). During drug titrations, a dual buffer gravity-driven perfusion system was used to exchange buffers between different drug concentrations. To determine AFF, ROIs were selected at the cell membrane signal was extracted using Fiji. For drug/response curves, data were plotted and fit using a One Phase Association curve on GraphPad Prism. RESULTS:

Sensor design and screening

[0119] Computationally-guided design of optimal insertion site of cpGFP into

Beta2AR. In general, a protein-based biosensor consists of at least a recognition element and a reporter element. Here we choose circular permuted GFP as a reporter element. When properly inserted to the recognition element (i.e. GPCRs), ligand binding induces conformational adjustments of the receptor, which will result in changes in the chromophore environment, thus transforming the ligand-binding event into a fluorescence change. As a prototype to test our sensor design strategy, we chose the Beta2AR, a well-studied GPCR for which a wealth of structural information is available [3]. Using PyRosetta we compared the active and inactive structures of Beta2AR and extracted the Phi torsion angle values for each residue of the structure (Fig. 1). This approach highlighted Loop2 and Loop3 of Beta2AR as the regions that undergo the most dramatic conformational changes during receptor activation. This is in line with the structural information of the two transmembrane domains (TM5, TM6) adjacent to the third intracellular loop (Loop3) undergo an outward movement during the activation process [2, 14]. We therefore chose to insert circularly permuted GFP (cpGFP) within the third intracellular loop of Beta2AR (see Fig. 2).

[0120] To identify the best position for cpGFP insertion within the Loop3 multiple insertion modalities were tested, comprising both different insertion sites and deletions of part of the Loop3 sequence (Table 1). Circular Polymerase Extension Cloning (CPEC) was used to insert cpGFP into the GPCR (for details about this technique see [15]). Briefly primers were designed to PCR a cpGFP insert from GCaMP6 (including original linker sequences: LE-LP) containing overhangs that overlap with the chosen Beta2AR insertion site sequence. Primers were also designed to open the Beta2AR-containing vector DNA. Finally the two products were Dpnl digested and mixed together for CPEC. The different sensor variants were separately transfected using Effectene transfection reagent (Qiagen) onto HEK293T cells cultured in 12-well glass bottomed plates. After 24 hours of expression, cells were imaged using a confocal microscope at 488 nm excitation and 513 nm emission wavelengths. During time lapse, images were continuously taken approximately every 2 seconds. Fluorescence at the cell membrane was monitored upon addition of saturating concentration of the Beta2AR full agonist isoproterenol (ISO, 10uM). ROIs were manually selected using Fiji. The dynamic range of the sensor variants (AFF) was calculated as the fractional difference of the fluorescence change over baseline fluorescence. The largest change in fluorescence was achieved when the complete sequence of Loop3 was maintained while cpGFP was inserted between amino acids S246 and Q247 (AF/F= -35%).

TABLE 1

[0121] Table 1 shows a list of the tested modalities of cpGFP insertion into the intracellular Loop3 of the Beta2AR and the corresponding AFFs. The residues indicated in the insertion site represent the 4 amino acid residues before and after cpGFP insertion (which occurs where the - symbol is). In two variants, a portion of the Beta2AR Loop3 (originally contained between the shown amino acids) was deleted.

[0122] In situ high-throughput screening for optimizing the dynamic range of

Beta2AR with a cpGFP integrated into the third intracellular loop. Through engineering GCaMP and iGluSnFR, we learned that both linker regions between Beta2AR and cpGFP are critical for a sensor's dynamics and kinetics. Therefore, we created 6 different types of linker libraries by randomizing the first and last 2-amino acids of the cpGFP (using NNK and MNN codons in our cpGFP forward and reverse primers respectively, XX-NV; -XX). In addition, rationale design of linker sequences was also employed. We set up a cell-based high-throughput screening method measuring changes in fluorescence in the absence and presence of agonists. The linkers of lead variants showing best AF/F from a library of -200 variants were sequenced and are shown in Table 2. In particular, we identified a variant (linker 1 sequence: AV; linker 2 sequence: TR) with a strong increase in fluorescence intensity upon activation and high photostability (defined as a fluorescence decay of less than 10% while illuminated in its active state at 1% laser power for ten minutes), turning our construct from negative to positive sensor (AFF= +40%). The dynamic range of Beta2AR with a cpGFP integrated into the third intracellular loop can be increased (or decreased) by employing rational design and direct evolution. TABLES 2A-G LI and L2 Linkers

[0123] Tables 2A-B list different linker variants flanking the N- and C-termini of the integrated circularly permuted fluorescent protein, separated in two groups: positive variants (which show positive fluorescent signal change, AFF) and the negative variants (which show a negative AFF). Variant (linkers AV-TR) were further characterized, as shown in Tables 2C-D. For each variant the AFF value, the amino acid sequence for both Linker 1 and Linker 2 and the photobleaching properties are shown. No photobleaching is defined as a fluorescence decay of less than 10% while the sensor expressed on cells is illuminated in its active state at 1% laser power for ten minutes.

[0124] Calibrate affinity, specificity mid dynamic range of Beta2AR with a cpGFP integrated into the third intracellular loop in mammalian 293 cells. We next set out to determine the specificity and sensitivity of Beta2AR with a cpGFP integrated into the third intracellular loop (driven by CMV promoter) by measuring the fluorescence of HEK293 cells with confocal microscopy, using a perfusion chamber to efficiently wash cultured cells in Hank's balanced salt solution (HBSS)-agonists solutions. A series of agonist solutions ranging from lnM to 10μΜ were made, covering three full agonists (isoproterenol (ISO), epinephrine (EPI) and norepinephrine (NE)) (Fig 3). The in situ affinity and response linearity of the sensors were determined to see whether it fits the range expected to be physiologically relevant for measuring neuromodulator release. To determine the specificity, a series of other neurotransmitters including dopamine and serotonin, were used for titration. To determine the kinetics, we either washed out the agonists using HBSS or used a published concentration (50 μΜ) of a Beta2AR inverse agonist (CGP-12177), known to counteract the effects of saturating full agonist in cell cultures [16]. The in situ Kd of the sensor for isoproterenol, epinephrine and norepinephrine are 1.2 nM, 15 nM and 50nM respectively, whose ratios are in line with the known affinities of these drugs for the Beta2AR [17] (Fig 4A). We further characterized the kinetics of drug-sensor interaction by determining the time constants (τ1/2) of association and dissociation for the three different full agonists tested (Table 3). In addition, we did not detect any specific response of the sensor when incubated with 50 μΜ Serotonin and Dopamine (two drugs that should not activate Beta2AR) (Fig 4B). The apparent decrease in fluorescence during Serotonin application is non-specific and due to the fluorescence quenching properties of the drug itself (i.e. it can be seen also when applying Serotonin onto control GFP expressing cells). These results suggest that Beta2AR with a cpGFP integrated into the third intracellular loop is capable to detect physiologically relevant changes of neuromodulator with high signal-to- noise ratio and specificity.

TABLE 3

[0125] Table 3 shows the time constants of association (τ1/2 ON) and dissociation

(τ1/2 OFF) as well as the affinity values (Kd) of the three different full agonists tested on Beta2 AR with a cpGFP integrated into the third intracellular loop. The time constants were calculated by interpolating the titration curve values under saturating conditions (luM drug) to determine the y value corresponding to the half-time of the association or dissociation phase respectively: y = (Xj-Xo)/2. The affinity values were calculated by fitting the respective drug/response curves with a one-site total binding fit curve using GraphPad Prism 6.

[0126] Determining the conformational dynamics of Beta2AR in response to drugs using Beta2AR with a cpGFP integrated into the third intracellular loop. A G protein- coupled receptor with a cpFP integrated into the third intracellular loop represents a new method to visualize the conformational dynamics of GPCR in the presence and absence of drugs. It remains less understood at the molecular level how drugs stimulate the signaling activity of a GPCR at different potency. Visualizing the structural rearrangement of GPCR triggered by binding of ligands in real-time will aid in screening and design of new GPCR- targeted drugs with tailored pharmacological efficacy. We thus tested the utility of Beta2AR with a cpGFP integrated into the third intracellular loop in reporting conformation dynamics of Beta2AR triggered by different classes of agonists. We compared the structural rearrangement of Beta2AR in response to a panel of drugs, including 3 full agonists 4 partial agonists and an antagonist (Fig 5). Interestingly, we observed a drug-specific fluorescent response using Beta2AR with a cpGFP integrated into the third intracellular loop, with the full-agonists being able to activate the sensor's fluorescence change to fully, while the partial agonists performed less than the full agonists and to various degrees. Of particular interest we observed a decrease in fluorescence using the antagonist Sotalol, which indicated that the true dynamic range of Beta2AR with a cpGFP integrated into the third intracellular loop is even larger than what was characterized using full agonists (Fig S). The different responses of the sensor to unrelated partial agonists indicate that our sensor is capable of distinguishing among different conformational states or structural rearrangement of the receptor triggered by different drugs and highlight the versatility of its use in drug screenings. We thus conclude that our sensor can be used as a tool for testing affinity, specificity, to predict the pharmacological action of different drugs targeting GPCR, especially orphan GPCRs, to reveal more subtle molecular mechanisms underlying GPCR activation, and to unveil new opportunities for the development of more selective clinical therapies, such as biased ligands.

[0127] Characterization o/Beta2AR sensors in dissociated neuronal culture and in vivo in zebrqfish and mouse brain. Neuroscience faces two great interrelated challenges: to develop better therapeutic neural drugs, and to alleviate the damage done by addictive drugs. To address these, it is desirable to better understand the mechanisms of action of existing drugs, at the level of molecular and cell biology, so that the field can exploit this knowledge to design even better therapeutic reagents. GPCRs are target of a series of drugs including antidepressants, antipsychotics, opiates and neuroprotective drugs. A G protein- coupled receptor with a cpFP integrated into the third intracellular loop represents a novel toolbox to do so and we therefore characterize the sensor's performance in living neurons.

[0128] To characterize the expression of the sensor in neurons, Beta2AR with a cpGFP integrated into the third intracellular loop was sub-cloned into an HIV-based lentiviral vector under the control of the synapsin promoter. Primary hippocampal neurons were cultured in the presence of astrocytes on glass-bottomed dishes (Matteks) coated with Poly-Ornithine/Laminine (20 μg/ml and 5 μg/ml respectively). Neurons were infected at DIV7 and imaged at DIV14-20. As an advantage of using the synapsin promoter, the expression of the sensor is restricted to neurons. Although quite dim, seven days postinfection neurons clearly showed visible fluorescence at the plasma membrane, indicating that the sensor expresses, folds and inserts into the membrane properly. Upon application of a saturating dose of the agonist ISO (10 μΜ) we observed a 25% increase in fluorescence intensity (Fig. 6). The DFF in neurons appears lower than what was observed in HEK293T cells due to the lower expression levels of the sensor itself.

[0129] The superior signal-to-noise ratio of a G protein-coupled receptor with a cpFP integrated into the third intracellular loop permits mapping spatiotemporal dynamics of neuromodulators and neural drugs in living brain. In order to prove that our sensor can achieve such goal, we chose to test it in the nervous system of two different vertebrate model organisms: the zebrafish and the mouse. Our current work is focused on testing the utility of our sensors in detecting the spatial action and effective concentrations of pharmacological drugs in brain with single cell and single synapse in vivo.

[0130] Design of a signaling-incompetent G protein-coupled receptor with a cpFP integrated into the third intracellular loop sensor. To be suitable for in vivo expression an ideal sensor should not alter or interfere with endogenous cellular signaling. GPCRs are known to activate cellular signaling through both G-protein and β-Arrestin-dependent pathways [18]. Structural studies have highlighted one particular well conserved residue on GPCRs (F139 for the Beta2AR) that plays a critical role in mediating the interaction with G proteins. In addition, phosphorylation of two G-protein coupled receptor kinase-6 (GRK6) sites on the Beta2AR C-terminus (S355, S356) is known to be a critical determinant for β- Arrestin recruitment and signaling. To eliminate the possibility of our sensor interfering with endogenous cellular signaling, we mutated both F139 and S355, S366 residues to Alanine in our sensor. Importantly the mutations we introduced in our sensor have no effect on either sensor brightness or response to agonist. We compared mutated Beta2AR with a cpGFP integrated into the third intracellular loop with wild-type Beta2AR for the ability to recruit G-proteins upon activation using an established assay that measures membrane recruitment of M>80 in HEK293T cells [16]. The results confirmed that our mutated sensor cannot recruit Nb80 upon activation (Fig.7 A-D). We further confirmed lack of G-protein dependent signaling in our mutated sensor (using a TYG/TSG mutation to abolish chromophore of cpGFP) [19] by measuring intracellular production of the second messenger cAMP upon receptor activation using a luciferase based assay. For those applications in which the sensor's signaling must be completely abolished, we propose to use a different set of mutations (Beta2 ART68F, Y 132G, Y219 A, or Beta2ARTYY), which are known to completely abolish cAMP production by the Beta2AR [20]; however those mutations may affect surface expression of the GPCR. To test that our mutated Beta2AR with a cpGFP integrated into the third intracellular loop does not interfere with β-Arrestin signaling, we tested it for internalization, a well-known β-Arrestin-dependent phenomenon that occurs rapidly after GPCR activation and that shifts its signaling to endosomes [16, 21, 22]. To test for internalization, we compared our mutated Beta2AR with a cpGFP integrated into the third intracellular loop to GFP-labeled wildtype Beta2AR using total-internal reflection (TIRF) microscopy and confirmed that the sensor does not undergo

internalization. Taken together, these data prove that our mutated sensor does not interfere with cellular signaling and is thus suitable for in vivo applications.

[0131] Multiplex imaging. The preserved spectrum bandwidth of single-FP indicators can allow for multiplex imaging with other optogenetic sensors including calcium, and cAMP or use alongside optogenetic effectors such as channel rhodopsin. We have also expanded the color-spectrum of GPCR based sensor, which allow multiplex imaging of activation of different types of GPCR simultaneously.

[0132] Towards engineering circularly permuted fluorescent protein sensors for other types of GPCRs. A similar sensor design strategy can be extend to other GPCRs, for example, μ-opioid receptor 1 (MOR-1), dopamine receptor Dl (DRDl), 5- Hydroxytryptamine (5-HT) receptor. We have already obtained a MOR-1 sensor variant that displays 25% positive AF/F in response to 10μΜ DAMGO (Fig. 8).

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Example 2

Discovering A Universal Module For GPCR Sensor Engineering

[0133] This example describes the additional sequences for linkers LI and L2 in the circularly permuted fluorescent protein sensors, including linker sequences that allow for the construction of a universal GPCR sensor, that can be integrated into the third intracellular loop of any GPCR. Importantly, the prototype GPCRs we tested belong to each of the three different GPCR types available: Gs-coupled (B2AR, DRD1), Gq-coupled (MT2R, 5HT2A) and Gi-coupled (A2AR, KOR). In addition, our prototype sensors show the applicability of our universal module to GPCRs that bind ligands of different nature: monoamines for B2 AR, A2AR, DRD 1 , MT2R, 5HT2A and neuropeptides for KOR. The data are consistent with the conclusion that the identified universal cpFP sensor modules described herein can be integrated into and successfully used to evaluate signaling of all GPCR types.

[0134] We have identified minimal sequences for LI and L2 for the construction of a universal linker that could be inserted into the third loop of GPCRs generally to readily produce a positive sensor.

[0135] Universal cpFP sensor module 1 : LI contains the 11 amino acids

QLQKIDLSSX1X2 and L2 contains the 5 amino acids X3X4DQL. In some embodiments, X1X2 can be amino acid LI (Leucine-Isoleucine) and X3X4 can be NH (Asparagine- Histidine). In a particular embodiment, universal module 1 is QLQKIDLSSLI-cpGFP- NHDQL.

[0136] Universal cpFP sensor module 2: LI contains the 5 amino acids LSSX1 X2 and L2 contains the 5 amino acids X3X4DQL. In some embodiments, X1X2 can be amino acid LI (Leucine-Isoleucine) and X3X4 can be NH (Asparagine-Histidine). In a particular embodiment, universal module 2 is LSSLI-cpGFP-NHDQL.

[0137] In some embodiments, universal cpFP sensor modules can be inserted into or can replace the third loop of any GPCR. As proof of principle, we demonstrated that this universal module can inserted into or replace the third loop of MT2R: melatonin receptor type IB (NCB I Reference Sequence: NP_005950.1); KOR1 : Kappa Opioid Receptor type- 1 (GenBank: AAC50158.1); 5HT2A: Serotonin Receptor type-2A (NCBI Reference

Sequence: NP_000612.1); A2AR: Alpha-2C Adrenergic Receptor (NCBI Reference Sequence: NP 000674.2); B2AR: Beta-2 Adrenergic Receptor (GenBank: AAB82151.1); and DRDl : Dopamine Receptor type-1 (GenBank: AAH96837.1) to transform these GPCRs into sensors that give a positive fluorescent signal in response to ligand binding. See, Fig 14).

[0138] Figure 14 demonstrates that the universal module 1 can be inserted into

EAKR-deleted third intracellular loop residues-KEHK of B2AR to obtain a sensor with 150% AF/F in response to 10 μΜ norepinephrine (NE). See, e.g., Figure 12. We fully characterized this B2AR variant with a panel of different B2AR drugs, including full, partial and inverse agonists as well as antagonists. See, Fig 13.

[0139] Universal module 1 can be used to replace the whole third intracellular loop of GPCRs to produce positive sensors to various degrees of AF/F out of all the GPCR tested, including 5HT2A, DRD1, MT2R, KOR and A2AR, confirming the development of a universal cpFP sensor that can be integrated into or replace the third cellular loop of any GPCR (Fig 14).

[0140] Universal module 2 can be inserted into EAKR-deleted third intracellular loop residues-KEHK of B2AR (100% AF/F) or replace the third intracellular loop of DRD 1 (IAQK-deleted third intracellular loop residues-KRET) to make a sensor that responds with -230% AF/F to dopamine; into SLQK-deleted third intracellular loop residues-NEQK of 5HT2A receptor to make a sensor that responds with -30% to serotonin; into RLKS-deleted third intracellular loop residues-REKD of KOR to make a sensor that responds with -40% to the kappa-opioid agonist U-50488 (Fig 15).

[0141] More precisely, the sequences flanking the deletion site are summarized as follows and depicted in Figure 12:

A2AR: IAKR - deleted third intracellular loop residues - REKR

MT2R: QARR- deleted third intracellular loop residues - KPSD DRD 1 : IAQK - deleted third intracellular loop residues - KRET

5HT2A: SLQK - deleted third intracellular loop residues - NEQK

KOR: RLKS - deleted third intracellular loop residues - REKD

[0142] For B2AR, in some embodiments, the universal cpFP sensor modules can be inserted into QLQKIDKSEGRFHVQNLS- deleted third intracellular loop residues -KEHK where LI -cpGFP-L2 can replace any part of QLQKIDKSEGRFHVQNLS. In some embodiments, the universal cpFP sensor modules can be inserted into

QLQKIDKSEGRFHVQNLS -deleted-FCLK where Ll-cpGFP-L2 can replace any part of QLQKIDKSEGRFHVQNLS.

[0143] Generally, in determining the location where to delete residues of the GPCR third intracellular loop, we consider the degree of sequence homology to the original B2AR deletion site (EAKR - deleted third intracellular loop residues - KEHK). Depending on the GPCR sequence, we select as starting point for the Loop deletion a position between 0 and +2 residues from the last positively charged amino acid residue (reading the sequence N- terminal to C-terminal) in the sequence that aligns with B2AR sequence EAKR. To precisely determine the end of the deletion, we select a position between 0 and -3 amino acids (reading the sequence N-terminal to C-terminal) away from the first charged amino acid (reading the sequence left to right) in the region that aligns with the B2AR sequence KEHK.

Example 3

Determining the conformational dynamics of β2ΑΚ in the brain of behaving mice.

[0144] We sought to test the utility of β2ΑΚ. conformational sensor in response to endogenous cortical norepinephrine (NE) release triggered by running in mice. After introducing the genetically-encoded sensors in the mouse motor cortex with the use of an adeno-associated virus (AAV) we could observe for the first time spontaneous

norepinephrine release that correlated well with the running activity of the mice {See Fig 16 in collaboration with Axel Nimmerjahn, Salk Institute). This affirmatively demonstrates that our sensors are capable not only to enabling breakthrough discoveries, but also to visualize the presence and dynamics of a GPCR ligand in a previously inaccessible system (living brain tissue).

[0145] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.