BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention generally relates to amino acid sequences that can interact with specified glutamate receptors. In one aspect, the invention features purified polypeptides that can interact with the glutamate receptors. In another aspect, the invention relates to isolated polynucleotides that encode the polypeptides. The present invention has a variety of applications, including use in detecting disorders in the nervous or reproductive system of a subject.

2. Background The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins.

Synapses are specialized areas of cell-cell contact that are optimized for the efficient transduction of signals between neurons in the brain. Both the pre-and postsynaptic membrane have an elaborate cytoarchitecture that is essential for the functional organization of the synapse. Neurons have many different types of synapses and the synaptic cytoskeleton is likely to play an important role in the localization of different receptors and ion channels to their appropriate synapses. A variety of studies on the neuromuscular junction and inhibitory synapses in spinal cord have shown that the cytoskeleton is intimately involved in the clustering of synaptic components at the postsynaptic membrane [Froehner, Annul Rev. Neurosci.,

16: 347-368 (1993), Hall et al., Cell/Neuron, 72: 99-121 (1993), Gautam et al., Nature, 377: 232-236 (1995), Kirsch et al., Nature, 266: 745-748 (1993), Meyer et al., Neuron, 15: 563-572 (1993), Kirsch et al., J. Neuroscience, 15: 41484156 (1995)] For example, the synaptic peripheral membrane proteins rapsyn and gephryin have been shown to play a critical role in the synaptic targeting of nicotinic acetylcholine receptors and glycine receptors, respectively [Froehner, Annul Rev. Neurosci., 16: 347- 368 (1993), Hall et al., Cell/Neuron, 72: 99-121 (1993), Gautam et al., Nature, 377: 232-236 (1995), Kirsch et al., Nature, 266: 745-748 (1993), Meyer et al., Neuron, 15: 563-572 (1995), Kirsch et al., J. Neuroscience, 15: 41484156 (1995)]. The cytoskeletal proteins involved in the formation of excitatory synapses in the central nervous system, however, have only recently begun to be identified [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996), Ehlers et al., Curr. Opin. in Cell Biol., 8: 490495 (1996)].

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system and play important roles in the synaptic plasticity underlying learning and memory and neural development, and in the excitotoxicity associated with stroke and other neurological disorders [Hollmann et al., Annul Rev. Neurosci., 17: 31-108 (1994), Seeburg, The TINS/TIPSLecture.

Trends Neurosci., 16: 359-365 (1993), Bliss et al., Nature, 361: 31-39 (1993), Linden, , Veuron, 12: 457472 (1994), Choi, Trends Neurosci., 18: 58-60 (1995)]. These receptors can be divided into three different subclasses, AMPA (a-amino-3-hydroxy- 5-methyl-isoxazole4-propionic acid), kainate and NMDA (N-methyl-D-aspartate) receptors, based on their physiological and pharmacological properties [Hollmann et al., Annul Rev. Neurosci., 17: 31-108 (1994), Seeburg, The TINS/TIPSLecture.

Trends Neurosci., 16: 359-365 (1993)]. AMPA and kainate receptors mediate rapid synaptic transmission while NMDA receptors are important in activity-dependent plasticity in the nervous system and in excitotoxicity. These receptors are ligand-gated ion channels consisting of oligomeric complexes of homologous subunits [Hollmann et al., Annul Rev. Neurosci., 17: 31-108 (1994), Seeburg., The TINSlTIPSLecture.

Trends Neurosci., 16: 359-365 (1993)]. Molecular cloning studies have demonstrated that each receptor subclass is composed of several distinct subunits. The GluR14 subunits belong to the AMPA subclass, the GluR5-7 and KA1-2 subunits belong to

the kainate subclass, while the NR1 and NR2A-D subunits belong to the NMDA subclass of ionotropic glutamate receptors [Hollmann et al., Annul Rev. Neurosci., 17: 31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16: 359-365 (1993)]. These subunits have been proposed to have a large extracellular N-terminal domain, three transmembrane domains, and an intracellular C-terminal domain [Molnar et al., Neuroscience, 53: 307-326 (1993), Tingley et al., Nature, 364: 70-73 (1993), Wo et al., Proc. Natl. Acad. Sci. USA, 91: 7154-7158 (1994), Hollmann et al., Neuron, 13: 1331-1343 (1994), Stern-Bach et al., Neuron, 13: 1345-1357 (1994), Bennett et al., Neuron, 14: 373-384 (1995), Wood et al., Proc. Natl. Acad. Sci., USA, 92: 4882-4886 (1995), Roche et al., Neuron, 16: 1179-1188 (1996)] Glutamate receptor diversity is generated through the differential combination of subunits as well as alternative splicing and editing of glutamate receptor mRNAs [Hollmann et al., Annul Rev. Neurosci., 17: 31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16: 359-365 (1993)].

Iontophoretic mapping, immunocytochemistry and immunoelectron microscopy studies have demonstrated that ionotropic glutamate receptors are specifically localized to the postsynaptic membranes of CNS neurons [Jones et al., Neuron, 7: 593-603 (1991), Petralia et al., Neurol. 318: 329-354 (1992), Craig et al., Neuron, 10: 1055-1068 (1993)., Huntley et al., J. Neurosci., 14: 3603-3619 (1994), Martin et al., Neuroscience, 53: 327-358 (1993), Petralia et al., J. Comp. Neurol., 349: 85-110 (1994a), Petralia et al., J. Neurosci., 14: 667-696 (1994b), Petralia et al., J.

Neurosci., 14: 6102-6120 (1994c), Tachibana et al., J. Comp. Neurol., 344: 431-454 (1994), Lau et al., J. Biol. Chem., 270: 20036-20041 (1995), Roche et al., Neuroscience, 69: 383-393 (1995), Craig et al., Proc. Natl. Acad. Sci. USA, 91: 2373- 12377 (1994), Nusser et al., Neuroscience, 61: 421 427 (1994)]. Although both AMPA and NMDA glutamate receptors are clustered at excitatory synapses, neither is present at inhibitory synapses enriched with GABAA receptors [Petralia et al., J.

Neurosci., 14: 667-696 (1994b), Lau et al., J. Biol. Chem., 270: 20036-20041 (1995), Craig et al., Proc. Natl. Acad. Sci. USA, 91: 2373-12377 (1994)]. This specific concentration and segregation of excitatory and inhibitory neurotransmitter receptor subunits within a neuron requires innervation of the postsynaptic cell but does not require synaptic activity [Craig et al., Proc. Natl. Acad. Sci. USA, 91: 2373-12377

(1994)]. Several recent studies have suggested that NMDA receptors directly or indirectly interact with the neuronal cytoskeleton. For example, actin filament stabilization prevents CA2+-dependent inactivation of NMDA channels [Rosenmund et al., Neuron, 10: 805-816 (1993)], and NMDA receptor responses are sensitive to cytoskeletal strain [Paoletti et al., Neuron, 13: 645-655 (1994)]. In addition, NMDA receptor subunits are enriched in the PSD and are resistant to solubilization in nonionic detergents [Lau et al., J. Biol. Chem., 270: 20036-20041 (1995), Brose et al., J. Biol. Chem., 268: 22663-22671 (1993)]. Recent experiments have shown that the C-termini of NMDA receptors are involved in the subcellular targeting of the receptors and directly interact with synaptic cytoskeletal proteins [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996), Ehlers et al., Science, 269: 1734-1737 (1995)]. Studies on NMDA receptor expression in fibroblasts have demonstrated that the NR1 subunit is clustered into highly concentrated receptor rich domains near the plasma membrane [Ehlers et al., Science, 269: 1734-1737 (1995)]. An alternatively spliced region within the C- terminus of the NR1 subunit, the Cl cassette, is both necessary and sufficient for the localization of NR1 to these receptor-enriched domains, suggesting that the Cl cassette directly interacts with cytoskeletal proteins [Ehlers et al., Science, 269: 1734- 1737 (1995)]. Interestingly the Cl cassette contains several phosphorylation sites [Tingley et al., Nature, 364: 70-73 (1993)] and PKC phosphorylation of this region rapidly disperses the NR1 clusters, perhaps by disrupting the association of NR1 with the cytoskeleton [Ehlers et al., Science, 269: 1734-1737 (1995). Kornau et al., Science, 269: 1737-1740 (1995)] have recently demonstrated that specific subunits of the NMDA receptor directly interact with the synaptic cytoskeletal associated protein PSD-95 or SAP90 [Cho et al., Neuron, 9: 929-942 (1992), Kistner et al., J. Biol.

Chem., 268: 45804583 (1993)]. PSD95/SAP90 specifically binds to the C-termini of the NR2A, NR2B and NR2D subunits and certain splice variants of the NR1 subunit (NRld, NRle) [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J.

Neurosci., 16: 2157-2163 (1996)]. PSD-95/SAP90 is colocalized with the NR2B subunit in excitatory synapses in neurons and is highly enriched in the postsynaptic density [Kornau et al., Science, 269: 1737-1740 (1995), Cho et al., Neuron, 9: 929-942 (1992)], where it is well-poised to anchor or target NMDA receptors. PSD-95/SAP90

has an intriguing structure which includes three repeats in the N-terminal region of a newly identified protein motif called a PDZ domain [Cho et al., Neuron, 9: 929-942 (1992)], named after three proteins containing this motif, PSD-95, Dlg-A and ZO-1 [Cho et al., Neuron, 9: 929-942 (1992), Kennedy, Trends in Biochem. Sci., 20: 350 (1995), Gomperts, Cell, 84: 659-662 (1996)]. These domains have also been called discs-large-homology regions (DHR) or GLGF repeats (referring to a conserved amino acid sequence in the repeat) [Cho et al., Neuron, 9: 929-942 (1992), Kennedy, Trends in Biochem. Sci., 20: 350 (1995), Gomperts, Cell, 84: 659-662 (1996)]. In addition, PSD95/SAP90 has a arc-homology 3 (SH3) domain and a domain homologous to a yeast guanylate kinase in the C-terminal region [Cho et al., Neuron, 9: 929-942 (1992), Kistner et al., J. Biol. Chem., 268: 45804583 (1993), Kennedy, Trends in Biochem. Sci., 20: 350 (1995), Ponting et al., Trends in Biol. Sci., 20: 102- 103 (1995), Kim, Cell Biol., 7: 641-649 (1995), Gomperts, Cell, 84: 659-662 (1996)].

PDZ domains are motifs of approximately 90 amino acids [Cho et al., Neuron, 9: 929-942 (1992)] present in a number of homologous proteins including the Drosophila septate junction discs-large protein (Dlg-A) [Woods et al., Cell, 66: 451464 (1991)], the mammalian tight junction protein ZO-1 [Itoh et al., J. Cell Biol., 121: 491-502 (1993)], the C. elegans epithelial cell junction proteins LIN-2A [Hoskins et al., The C. elegans vulval induction gene lin-2 encodes a member of the MAGUKfamily of cell junction proteins Development, 122: 97-111 (1996)] andin-7 [Simske et al., Cell, 85: 195-204 (1996)] as well as a variety of other proteins including protein tyrosine phosphatases, nitric oxide synthase, and the mammalian neuromuscular junction protein syntrophin [Ponting et al., Trends in Biol. Sci., 20: 102-103 (1995)]. PDZ domains are now thought to mediate a variety of protein- protein interactions [Kornau et al., Science, 269: 1737-1740 (1995), Gomperts, Cell, 84: 659-662 (1996)].

The interaction of NMDA receptor subunits with PSD-95/SAP90 occurs between the final 7 C-terminal amino acids of the NMDA receptor subunits and the first and second domains of PSD-95 [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996)]. The common motif present in the C-termini of NMDA receptor subunits which interact with the PDZ domains consists of a threonine or serine followed one amino acid later by a valine (referred to

as a T/SXV motif) [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J Neurosci., 16: 2157-2163 (1996)]. Interestingly, such motifs are found in a wide variety of cell surface receptors and ion channels [Kornau et al., Science, 269: 1737- 1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996)]. Indeed, Kim et al. [Kim et al., Nature, 378: 85-88 (1995)] have shown that such a motif in the Shaker- type K+-channel subunit Kvl. 4 interacts with the PDZ1 and PDZ2 domains of PSD- 95/SAP90, with mutations of the conserved threonine completely abolishing the interaction.

It has become increasingly clear that PSD-95/SAP90 is but one of a large family of structurally related proteins. In addition to Dlg-A and ZO-1, which share similar overall domain structures, three additional PSD-95/SAP90 family members, SAP97 [Muller et al., J. Neurosci., 15: 2354-2366 (1995)], SAP102 [Muller et al., Neuron, 17: 255-265 (1996), Lau et al., J. Biol. Chem., 271: 21622-21628 (1996)] and PSD-93/Chapsyn [Brenrnan et al., Cell, 84: 757-767 (1996), Kim et al., Neuron., 17: 103-113 (1996)] have been identified in the mammalian central nervous system.

These homologous proteins may serve functions similar to PSD-95/SAP90 [Ehlers et al., Curr. Opin. in Cell Biol, 8: 490495 (1996), Gomperts, Cell, 84: 659-662 (1996].

For example SAP102 has recently been shown to interact with NMDA receptor complexes in rat brain [Muller et al., Neuron, 17: 255-265 (1996), Lau et al., J. Biol.

Chem., 271: 21622-21628 (1996)]. A common feature of these PSD-95/SAP90 family members is that they localize to specialized sites of cell-cell contact including septate junctions in Drosophila (Dlg-A), vertebrate tight junctions (ZO-1), and synapses in the mammalian nervous system (PSD-95/SAP90, SAP97, SAP102) [Ehlers et al., Curr. Opin. in Cell Biol., 8: 490495 (1996), Kim, Cell Biol., 7: 641-649 (1995), Gomperts, Cell, 84: 659-662 (1996)]. Members of this family appear to be involved in both localizing cellular proteins and assisting in the establishment of cell polarity. That PSD-95/SAP90 family members might be important for synaptic organization in the mammalian CNS is suggested by the fact that mutations in the homologous Drosophila protein, Dlg-A, alter the structure of glutamatergic synapses [Lahey et al., Neuron., 13 (4): 823-35 (1994)].

SUMMARY OF THE INVENTION

The present invention relates to novel GRIP and GRIP-related proteins called (Glutamate Receptor Interacting Proteins). GRIP has been found to bind a specified glutamate receptor. In one aspect, the invention features isolated polynucleotides that encode GRIP or GRIP-related proteins. In another aspect, the invention features isolated GRIP or GRIP-related proteins. Further provided are antibodies that can bind the polypeptides. The present invention has a variety of uses, including detecting and analyzing certain neurological disorders and fertility in a subject.

We have discovered GRIP and GRIP-related proteins. GRIP proteins are new members of the PDZ-containing family of proteins. The GRIP proteins have been found to include multiple PDZ domains and no apparent catalytic domain. GRIP proteins appear to serve as an adapter protein that links AMPA receptors to the synaptic cytoskeleton and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.

For example, we have found that GRIP is expressed in testes. As will be more fully discussed below, GRIP apparently has important functions in the nervous system and in sperm formation (spermatogenesis).

As will be pointed out below, an exemplary GRIP-related protein is GRIP2: a protein that is substantially equivalent to GRIP It is recognized that neurotransmitter receptors mediate signal transduction at the postsynaptic membrane of synaptic connections between neurons in both the central and-peripheral nervous systems. It is further recognized that neurotransmitter receptors are highly concentrated at the synaptic membrane and this clustering of the receptors plays an important role in the efficient transduction of signals at synapses.

This invention comprises a novel family of proteins involved in neurotransmitter receptor functions, wherein this family of proteins are called GRIPs. More specifically, we have identified a synaptic PDZ domain-containing protein designated as GRIP, that specifically interacts with the non-NMDA glutamate receptors and appears to be involved in the synaptic clustering of these receptors. GRIP has been found to be a cytoskeletal-associated protein that directly binds to the C-termini of the GluR2 and 3 subunits of the non-NMDA glutamate receptors via a new protein motif called a PDZ domain. PDZ domains mediate protein-protein interactions in many proteins and appear to be involved in the proper subcellular targeting of membrane

proteins. GRIP contains seven PDZ domains, designated PDZ 1 through PDZ7, which appear to crosslink receptors or link them to other synaptic proteins. GRIP may be a critical component in the formation and maintenance of excitatory synapses in the brains.

For example, we describe the isolation of GRIP, a PDZ domain-containing protein. In one aspect, the GRIP protein specifically interacts with the C-terminus of the GluR2 and GluR3 subunits of AMPA receptors, the major excitatory neurotransmitter receptors in brain. GRIP contains seven PDZ domains and no apparent catalytic domain suggesting that it is a member of a new family of PDZ domain containing-proteins that crosslink AMPA receptors or serve as adapter proteins to link the receptors to other proteins. Moreover, GRIP appears to play an important role in the targeting of AMPA receptors to excitatory synapses.

In one embodiment, the present invention provides a novel family of proteins, called GRIPs (e. g., GRIP), that specifically interact with the non-NMDA glutamate receptors and appear to be involved in the synaptic clustering of these receptors.

The present invention also provides isolated nucleic acid molecules that encode proteins that bind the C-termini of an AMPA glutamate receptor. Preferred are nucleic acid molecules encoding GRIP, GRIP2, or fragments and derivatives thereof.

The present invention also provides different forms of GRIP, each of which function similarly and also have substantial sequence homology. As will be shown below, GRIP and GRIP2 show significant sequence homology at the amino acid level.

The present invention also provides an isolated protein which is GRIP or GRIP2.

The present invention also provides vectors (i. e. recombinant vectors) comprising an isolated nucleic acid molecule encoding GRIP, GRIP2, or a fragment thereof.

This invention further provides vectors such as plasmids comprising a DNA molecule encoding GRIP or GRIP2, adapted for expression in a bacterial cell, a yeast cell, an insect cell or a mammalian cell which additionally comprises the regulatory elements necessary for expression of the DNA in the bacterial, yeast, insect or mammalian cells operatively linked to the DNA encoding GRIP or GRIP2 to permit

expression thereof. In particular, mammalian cells are provided comprising a DNA molecule encoding GRIP or GRIP2.

This invention also provides nucleic acid probes comprising a nucleic acid molecule capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding GRIP or GRIP2.

This invention also provides a method of detecting expression of GRIP by detecting the presence of mRNA coding for GRIP which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding GRIP under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of GRIP by the cell. Related methods can be used to detect GRIP2 expression at the mRNA level.

This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes GRIP or GRIP2 so as to prevent translation of the mRNA molecule. In some embodiments, the antisense oligonucleotide will be capable of specifically binding both GRIP and GRIP2 if desired.

This invention provides a transgenic nonhuman mammal expressing DNA encoding GRIP, GRIP2 or a fragment thereof. Transgenic animals encompass any animal, including, but not limited to, an amphibian, bird, fish, insect, reptile, or mammals, such as mouse, rat, bovine, porcine, and sheep.

This invention further provides a transgenic nonhuman mammal comprising a homologous recombination knockout of native GRIP or GRIP2.

This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding GRIP or GRIP2 so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding GRIP and which hybridizes to mRNA encoding GRIP or GRIP2 thereby reducing its translation. The transgenic nonhuman animal may also include suitable GRIP or GRIP2 fragments if desired.

This invention provides antibodies, both polyclonal and monoclonal, directed to GRIP or GRIP2. In some instances, the antibodies will be capaable of binding both GRIP and GRIP2.

This invention provides a method of determining the physiological effects of expressing varying levels of human GRIP which comprises producing a transgenic nonhuman animal whose levels of GRIP expression are varied by use of an inducible promoter which regulates GRIP expression. Related methods can be used to detect the effects of expressing varying kinds of human GRIP2.

This invention also provides a method of determining the physiological effects of expressing varying levels of human GRIP or GRIP2 which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of GRIP or GRIP2. Related methods can be performed with GRIP or GRIP2 fragments if desired.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific GRIP or GRIP2 allele.

This invention provides a method of preparing isolated GRIP which comprises inducing cells to express GRIP, recovering GRIP from the resultant cells and purifying the protein so recovered. Related methods can be used to prepare isolated GRIP2.

This invention further provides a method of preparing isolated GRIP which comprises inserting nucleic acid encoding GRIP in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the protein produced by the resulting cell, and purifying the protein so recovered. Related methods can be used to prepare isolated GRIP2.

The present invention further relates to methods of treating diseases and disorders, preferably neurological diseases and disorders, in a human or animal through regulating the synaptic targeting of AMPA receptors (e. g., monitoring and controlling excitatory synaptic transmission) by altering the activity and/or the levels of GRIP or GRIP2. For example, altering GRIP or GRIP-related function may involve enhancing or inhibiting excitatory synaptic transmission. Such methods can be used to treat many neurodegenerative diseases, including stroke, ALS, Alzheimer's disease, Parkinson's disease and Huntington's disease. The isolated DNA encoding

GRIP and isolated GRIP will also be useful for the treatment of muscular dystrophies such as Duchenne muscular dystrophy and motility disorders such as irritable bowel syndrome. Related methods can be used to treat the neurological disorders altering levels of GRIP2 or using an isolated DNA encoding GRIP2 or a suitable fragment thereof.

The present invention also provides methods of enhancing or otherwise altering memory by using GRIP-or GRIP2-related technology to regulate neurotransmitter receptor function.

Another embodiment of the invention involves a method of screening for infertility, which comprises detecting the presence of antibodies that bind to a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence from the group consisting of GRIP or GRIP2 in its entirety, specific PDZ binding domains, or any antigenic portion thereof (see Figures 2,3,11A-11C and 12A-12D).

The presence of such antibodies is taken as indicative of infertility.

Polypeptides of the present invention may be used to modulate the behavior of sperm. Agents (antagonists and agonists) may be identified that bind to the sperm, preferably via interaction with a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence from the group consisting of GRIP or GRIP2 in its entirety, specific PDZ binding domains, or any antigenic portion thereof (see Figures 2,3,11A-11C and 12A-12D) of the present invention. Further, the means of affecting fertility, or contribution to fertility, can comprise either stimulating or inhibiting the binding of a particular agent to a polypeptide as define herein, (e. g., a polypeptide encoded by a nucleic acid comprising GRIP as defined herein. Such a method may have a practical application as a means of fertility control (e. g., spermicide use, which prevents sperm form migrating towards or fertilizing an egg), but alternately, as a means of enhancing fertility or contributing to fertility.

Another embodiment of the present invention is directed to an antigenic polypeptide, which is useful as an immunocontraceptive agent or for the diagnosis of infertility, as described above. Preferably, the peptide comprises an antigenic fragment of less than about 30 amino acids in length of a polypeptide comprising an amino acid sequence encoded by a nucleic acid comprising a sequence selected from the group consisting of GRIP or GRIP 2 in its entirety, specific PDZ binding domains,

or any antigenic portion thereof (see Figures 2,3,11A-11C and 12A-12D of the drawings).

The invention also provides a method of screening drugs to identify drugs which specifically interact with, bind to, and/or modify the physiological effects of GRIP or GRIP2. This invention also provides a pharmaceutical composition comprising GRIP or GRIP 2 with or without a drug identified by this method.

Additionally provided are kits that include components for detecting and analyzing GRIP or GRIP2 expression in tissue, e. g., specified neurons and reproductive cells. Further provided are kits that include agents capable of modulating sperm formation and particularly fertility in a subject such as a primate, e. g., a mammal (e. g., a domesticated animal such as a cat, dog, or horse) and more particularly a human patient. The agents can include any of the agents described above, e. g., immunological agents and particularly antibodies capable of specifically binding GRIP, a fragment thereof, GRIP2, or a fragment thereof.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS Figures lA-lF are representations of photomicrographs depicting disruption of synaptic AMPA receptor clustering by over expression of the C-terminus of GluR2 in neurons. Transfections with C-terminal GluRl (Figures 1A-1C). Transfections with C-terminal GluR2 (Figures 1D-IF).

Figure 2 is a schematic drawing showing isolated GRIP cDNAs.

Figure 3 is an illustration of the amino acid sequence of GRIP (SEQ ID NO.

2). The GRIP cDNA encodes a 1112 amino acid protein that contains seven PDZ domains and no obvious catalytic domain. The PDZ domains are indicated by underlining, the GLGF-like repeats and the conserved arginine/lysine residues are indicated by stippling.

Figures 4A-4C depict amino acid sequence alignment of the PDZ domains of GRIP. The PDZ domains of GRIP are aligned with the PDZ domains of dlg, PSD- 95/SAP90 and SAP97. The conserved amino acids in PDZ domains are indicated by stippling and the GLGF-like repeats and the conserved arginine/lysine residues are indicated by the asterisks.

Figures 5A and 5B are drawings depicting structural determinants of GRIP and GluR2 required for interaction.

Figures 6A-6D are representations of Western blots depicting co- immunoprecipitation of GRIP with GluR2 and GluR3.

Figure 7 is a representation of a northern blot of GRIP in rat tissues.

Figures 8A-8E are representations of Western blots showing distribution of GRIP protein in rat tissues. Antibodies were generated against a GRIP fusion protein and a GRIP peptide was used to identify the GRIP protein in the rat tissues. OLF.- olfactory bulb; CTX-cortex; HPC.-hippocampus ; CER.-cerebellum; S. C.-spinal cord.

Figures 9A-9C are representations of photomicrographs showing co- localization of GRIP with AMPA receptors at excitatory synapses. Primary cultures of hippocampal neurons were double-labeled with anti-GRIP and anti-GluR1 antibodies.

Figure 10 is a schematic drawing showing a potential role of GRIP in excitatory synaptic function.

Figures 11A-11C are drawings showing the nucleotide sequence of GRIP (SEQ ID NO : 1). Nucleotides are presented in the 5'to 3'orientation. Numbers represent the nucleotide numbering, starting with the first nucleotide.

Figures 12A-12D are drawings showing the deduced amino acid sequence of GRIP (SEQ ID NO: 2). Deduced amino acid sequence by translation of a long open reading frame is shown, along with the 5'and 3'untranslated regions. Numbers represent the amino acid numbering, starting with the first amino acid.

Figure 13 is a representation of a GRIP Northern blot showing analysis of adult rat testis, isolated seminferous tubules, and germ cells mRNAs.

Figure 14 is a representation of a Western blot showing analysis of GRIP expression in adult rat testis, seminferous tubules and total germ cells.

Figures 15A-15C are representations of photomicrographs showing immunofluorescence analysis with anti-GRIP antibodies on cryosections of adult rat testis. JH226D refers to an anti-grip antibody.

Figuresl6A-16C are representations of photomicrographs showing immunofluorescence analysis with anti-GRIP antibodies on cryosections of adult rat testis. JH2493 refers to an anti-grip antibody.

Figures 17A-17C are representations of photomicrographs showing immunofluorescence analysis with anti-GRIP antibodies on cryosections of adult rat testis. JH2493 and JH2260 refers to an anti-grip antibody"abs + peptide"refers to a control reaction.

Figures 18A and 18B are representations of photomicrographs showing that GRIP is not detectable in immature Sertoli cells prior to organization of blood testis barrier (10 days). JH2493 refers to an anti-GRIP antibody.

Figures 19A and 19B are representations of photomicrographs showing that a GRIP signal appears after tight junctions between Sertoli cells become organized (20 days). JH2493 refers to an anti-GRIP antibody.

Figure 20A and 20B are representations of photomicrographs showing that a GRIP expression in Sertoli cells (20 days). JH2260 refers to an anti-GRIP antibody.

Figure 21 is a representation of a Western blot showing GRIP expression in germ cells and other cell types.

Figures 22A-22I are drawings showing a GRIP 2 nucleotide sequence (SEQ ID NO. 47). Numbers above the sequence refer to nucleotide numbers.

Figures 23A-23I are drawings showing GRIP 2 nucleotide sequence (SEQ ID NO. 48) and conceptual translation (SEQ ID NO. 49) of the GRIP 2 nucleotide sequence.

Figures 24A-24B are drawings showing GRIP 2 protein seqeunce (SEQ ID NO. 50).

Figures 25A-25C are diagrams illustrating protein sequence comparison between GRIP and GRIP 2. The diagram highlights substantial amino acid equivalency between GRIP and GRIP 2. Stippled boxes indicate amino acid sequence identity and hatched boxes show amino acid seqeunce similarity. Gaps have been introduced to optimize alignment.

DETAILED DESCRIPTION OF THE INVENTION As discussed, the present invention relates to GRIP and fragments thereof, and GRIP2 and fragments thereof. Additionally provided are polynucleotides including sequence encoding GRIP or GRIP fragments, and polynucleotides including sequence encoding GRIP 2 and fragments thereof.

In one aspect, the present invention relates to polynucleotides including sequence that is substantially equivalent to the sequence of Figures 1 lA-11C (SEQ ID NO: 1). In another aspect, the invention relates to polynucleotides including sequence that is substantially equivalent to the sequence of Figures 22A-22E (SEQ ID NO. 47).

In another aspect, the invention features amino acid sequences that are substantially equivalent to the GRIP sequence shown in SEQ ID NO: 2. In another aspect, the invention provides amino acid sequences that are substantially equivalent to the GRIP 2 polypeptide sequence shown in SEQ ID. NO. 50. Further provided are molecules capable of detecting or modulating GRIP or GRIP 2 expression in the nervous or reproductive system of a subject.

It has been reported that synaptic clustering of postsynaptic neurotransmitter receptors is important for the efficiency of synaptic transmission. Recent studies on synaptic plasticity have suggested that the regulation of the synaptic targeting of AMPA receptors may be important for the modulation of synaptic function [Liao et al., Nature, 375 (6530): 400-4 (1995), Isaac et al., Neuron., 15 (2): 427-34 (1995), Durand et al., Nature, 381 (6577): 71-5 (1996)]. Although peripheral membrane proteins involved in the synaptic aggregation of the nicotinic acetylcholine receptor, the glycine receptor, and the NMDA receptor have recently been characterized [Froehner, Annul Rev. Neurosci., 16: 347-368 (1993), Hall et al., CelllNeuron, 72: 99- 121 (1993), Gautam et al., Nature, 377: 232-236 (1995), Kirsch et al., Nature, 266: 745-748 (1993), Meyer et al., Neuron, 15: 563-572 (1995), Kirsch et al., Neuroscience, 15: 41484156 (1995), Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996)], the proteins involved in the clustering of AMPA receptors have yet to be identified.

As noted, the present invention discloses the identification of GRIP, a novel protein that specifically interacts with the GluR2 and GluR3 subunits of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family and contains seven PDZ domains and no apparent catalytic domain. The 4th and 5th PDZ domains are sufficient to interact with the GluR2 subunit. In contrast, the 1 st, 2nd, 3rd and 7th PDZ domains do not appear to interact with the GluR2 subunit. These results indicate the different PDZ domains in GRIP may have distinct specificities for peptide substrates. The C-terminal seven amino acids of the GluR2 subunit appear to

be critical for the interaction of GluR2 with GRIP. In addition, although the sequence of the GluR2 C-terminus does not contain the T/SXV consensus sequence that has been shown to be important for the NMDA receptor-PSD95/SAP90 interaction, a serine residue in this region of GluR2 is also important for its interaction with GRIP.

The importance of this serine residue in the interaction of GluR2 with GRIP may indicate a potential role for protein phosphorylation in the regulation of GRIP-GluR2 interaction. Interestingly, the C-terminus of the GluR2 subunit has recently been shown to be alternatively spliced to give a variant of GluR2 that has a longer C- terminus with a sequence similar to GluR1 and GluR4 [Kohler et al., J. Biol. Chem.

269 (26): 17367-17370 (1994)]. In addition, the C-terminus of GluR4 has also been shown to be alternatively spliced to give both a long form, like GluRl, and a short for (GluR4c), like GluR2 and GluR3 [llo et al., J. Neuro., 12 (3): 1010-1023 (1992)]. This suggests that alternative splicing of the C-terminus of AMPA receptor subunits may play a regulatory role in the clustering of AMPA receptors similar to that seen with the NMDA receptor [Ehlers et al., Science, 269: 1734-1737 (1995)].

Figure 10 is a schematic showing that GRIP contains seven PDZ domains that are involved in protein-protein interactions at the synapse. The 4th and 5th PDZ domains of GRIP interact with the GluR2 and GluR3 subunits and may crosslink AMPA receptors to form synaptic clusters. In addition, GRIP contains five other PDZ domains that may interact with many different synaptic cytoskeletal proteins or proteins involved in synaptic signal transduction.

In one aspect of the present invention, GRIP mRNA was found to be present in brain suggesting that it plays a role in neuronal function. Immunoblotting of various rat tissues demonstrates that the cytoskeleton associated 130 kDa GRIP protein is also exclusively expressed in brain including the cerebral cortex, olfactory bulb, hippocampus, cerebellum, and spinal cord. Interestingly, an immunologically related soluble protein with an apparent molecular weight of 90 kDa is present in most tissues examined. This protein may be a proteolytic breakdown product of the 130 kDa protein. However, the fact that the mRNA for GRIP is not observed in many non-neuronal tissues suggests that the 90kDa protein may be the product of a GRIP- related gene. Immunocytochemical staining of hippocampal neurons with the GRIP fusion protein antibody demonstrated that GRIP is localized to neuronal dendrites and

clusters at excitatory synapses. Although clustering of GRIP was seen in some neurons, not all AMPA receptor clusters contained high levels of GRIP staining. This may be due to a limited accessibility of the antibody to GRIP in the postsynaptic density, or alternatively, GRIP may be selectively localized at certain excitatory synapses. Antibodies against SAP 102, a member of the PSD95/SAP90 family, also selectively label excitatory synapses [Muller et al., Neuron, 17: 255-265 (1996)]. By analogy with SAP 102, it is possible that additional members of the GRIP protein family which also may be involved in excitatory synaptic function.

As will be described in the discussion and examples which follow, over expression of the C-terminal region of GluR2 which interacts with GRIP dramatically decreased the number of AMPA receptor clusters in neurons without significantly effecting the number of synaptophysin positive synapses. Moreover, over expression of the C-terminal truncation mutant GluR2 AC, which does not interact with GRIP, did not disrupt AMPA receptor clusters. These results strongly suggest that the interaction of GluR2 with GRIP may be important in AMPA receptor clustering.

The exact role of GRIP in AMPA receptor clustering, however, is not completely understood. Studies on the NMDA receptor and K+-channels have suggested that the three PDZ repeats in the PSD-95/SAP90 family members could provide a mechanism for neurotransmitter receptor clustering by crosslinking receptors or anchoring them to the cytoskeleton [Kornau et al., Science, 269: 1737- 1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996), Kim et al., Nature, 378: 85-88 (1995)]. PSD95/SAP90 family members have also been shown to be involved in co-localizing cellular signal transduction machinery with NMDA receptors near the postsynaptic membrane [Brenrnan et al., Cell, 84: 757-767 (1996)].

Neuronal nitric oxide synthase (nNOS) possesses a PDZ domain and can interact with PSD-95/SAP90 and PSD-93 through a PDZ-PDZ domain interaction [Brenrnan et al., Cell, 84: 757-767 (1996)]. As nNOS is activated by Ca2+ influx through NMDA receptors, their coupling via PSD-95/SAP90 could provide a possible mechanism for localized intracellular signaling upon activation of NMDA receptors. GRIP contains seven PDZ domains which may play a similar role to the PDZ domains in the PSD- 95/SAP90 family by crosslinking AMPA receptors or linking them to the cytoskeleton or to signal transducing enzymes (Figure 10). Consistent with these

ideas, preliminary results have indicated that PDZ7 specifically interacts with a novel brain-specific cytoskeletal protein. The multivalent nature of GRIP with its seven PDZ domains allows for a large diversity of potential interactions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

One embodiment of the invention comprises isolated nucleic acid molecules which encode a GRIP polypeptide, allelic variant, or analog, including fragments or derivatives thereof. Thus, one aspect of the present invention is isolated polynucleotide operably encoding GRIP, preferably mammalian GRIP, more preferably human. Examples of such an isolated nucleic acid molecule are an RNA, cDNA or isolated genomic DNA molecule encoding GRIP.

Additionally provided are isolated nucleic acid molecules which encode GRIP 2 polypeptide, allelic variant, or analog, including fragments or derivatives thereof. In one aspect, the isolated polynucleotide operably encodes GRIP 2, preferably mammalian GRIP 2, more preferably human GRIP 2. Examples of such an isolated nucleic acid molecule are an RNA, cDNA or isolated genomic DNA molecule encoding GRIP 2.

A"fragment"or"derivative"of GRIP or GRIP2 refers to herein 1) a peptide in which one or more amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) a peptide in which one or more of the amino acid residues includes a substituent group, or (iii) a peptide in which the mature protein is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Thus, a fragment or derivative for use in accordance with the methods of the invention includes a proprotein, which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Moreover, GRIP or GRIP2 may have potential for N-linked glycosylation in some settings. Such glycosyl

groups, if present, can be partially or completely removed or otherwise modified to provide a GRIP derivative or fragment; or GRIP 2 derivative or fragment.

The GRIP and GRIP2 fragments and derivatives of the invention are of a sufficient length to uniquely identify a region of GRIP or GRIP2. GRIP and GRIP2 fragments thus preferably comprise at least 8 amino acids, usually at least about 12 amino acids, more usually at least about 15 amino acids, still more typically at least about 30 amino acids, even more typically at least about 50 or 70 amino acids.

Preferred fragments or derivatives for use in the methods of the invention include those that have at least about 70 percent homology (sequence identity) to SEQ ID N0 : 2 (amino acid sequence shown in Figure 3 of the drawings) or SEQ ID NO. 50 (amino acid sequence in Figures 24A-24B). More preferably about 80 percent or more homology to SEQ ID N0 : 2 or to the polypeptide shown in the SEQ ID NO : 50, still more preferably about 85 to 90 percent or more homology to SEQ ID N0 : 2 or the polypeptide shown in the SEQ ID NO : 50. Sequence identity or homology with respect to GRIP or GRIP2 refers to herein as the percentage of amino acid sequences of a GRIP protein or fragment or derivative thereof (or GRIP2 protein or fragment or derivative thereof) that are identical with SEQ ID N0 : 2 or the polypeptide shown in SEQ ID NO : 50, after introducing any gaps necessary to achieve the maximum percent homology.

For example, Figures 25A-C provide an illustration of suitable gap alignments that serve to highlight substantial sequence equivalency between GRIP and GRIP 2 GRIP, GRIP2, and fragments and derivatives thereof of the invention are "isolated", meaning the protein or peptide constitutes at least about 70%, preferably at least about 85%, more preferably at least about 90% and still more preferably at least about 95% by weight of the total protein in a given sample. A protein or peptide of the invention preferably is also at least 70% free of immunoglobulin contaminants, more preferably at least 85% free, still more preferably at least 90% free and even more preferably at least 95% free of immunoglobulin contaminants. GRIP, GRIP2, and fragments and derivatives thereof may be present in a free state or bound to other components, e. g. blocking groups to chemically insulate reactive groups (e. g. amines, carboxyls, etc.) of the peptide, or fusion peptides or polypeptides (i. e. the peptide may be present as a portion of a larger molecule).

The term"complementary sequence"as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.

Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99 to about 100%. Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software.

A"substantially equivalent"amino acid sequence is a sequence that varies from a GRIP sequence (SEQ ID NO 2) or the GRIP 2 sequence (SEQ ID NO 50) by one or more substitutions, deletions, or additions, the effect of which may or may not result in a difference in the sequence at the amino acid level, and does not result in an undesirable functional dissimilarity between the two sequences, i. e., the polypeptide derived from the substantially equivalent sequence has the functional activity characteristic of unmodified GRIP or GRIP 2.

In particular, the term"substantially equivalent"is meant to point out relationship between two nucleic acid molecules or two polypeptides and generally refers to subunit sequence similarity between two molecules. For example, when the two molecules are polypeptides, and a subunit position in both of the molecules is occupied by the same monomeric subunit, i. e. an amino acid residue, then the molecules are homologous or equivalent at that position. Equivalency between the two sequences is a direct function of the number of matching or homologous positions, egg., if 50% of the subunit positions in the two polypeptide sequences are equivalent then the two sequences are 50% equivalent. By"substantially equivalency"is meant largely but not wholly equivalent.

In particular, the term"substantial equivalency"is meant to denote at least about 60%, 70%, 80%, 90%, 95% or greater equivalency up to about 99% equivalency with respect to amino acid sequences shown in Figure 3 (SEQ ID NO. 2) or amino acid sequence shown in SEQ ID NO 50. Additionally, the term is meant to define the same or closely related percent of equivalency with respect to a polynucleotide of interest and the GRIP nucleotide sequence shown in Figure 11A-C (SEQ ID NO. 1) or the nucleotide sequence shown in Figure 22A-22I (SEQ ID NO 47).

Additionally, a difference in a nucleotide sequence, which does not result in a difference at the amino acid level, can include modifications that result in conservative amino acid substitutions, as well as DNA sequence differences due to the degeneracy of the genetic code, i. e., the fact that different nucleic acid sequences can code for the same protein or peptide. A difference in sequence at the amino acid level, i. e., due to a difference in the nucleotide sequence, can include single amino acid substitution (preferably, a conservative substitution), deletion, and/or insertion, or a plurality of amino acid substitutions, deletions, and/or insertions, wherein the resulting polypeptide is still recognizable as related to GRIP as defined by functional activity. Further, a difference in sequence at the amino acid level also can include those amino acid sequence differences that result in a polypeptide of altered size, such as a larger protein, or a truncated protein.

Preferred DNA molecules according to the present invention encode a GRIP polypeptide its entirety and PDZ binding domains having an amino acid sequence as set out in (amino acid positions made in reference to SEQ ID NO : 2, Figures 12A- 12C): a) amino acids 1 through 1112 (SEQ ID NO : 3); b) amino acids 52 through 135 (SEQ ID NO : 4); c) amino acids 152 through 249 (SEQ ID NO : 5) ; d) amino acids 252 through 335 (SEQ ID NO : 6); e) amino acids 471 through 558 (SEQ ID NO : 7); f) amino acids 572 through 655 (SEQ ID NO : 8); g) amino acids 672 through 753 (SEQ ID NO : 9); h) amino acids 988 through 1069 (SEQ ID NO : 10);

i) amino acids 52 through 249 (SEQ ID NO: 11); j) amino acids 52 through 335 (SEQ ID NO : 12); k) amino acids 52 through 558 (SEQ ID NO : 13); 1) amino acids 52 through 655 (SEQ ID NO : 14); m) amino acids 52 through 753 (SEQ ID NO: 15); n) amino acids 52 through 1069 (SEQ ID NO : 16); o) amino acids 152 through 335 (SEQ ID NO : 17); p) amino acids 152 through 558 (SEQ ID NO : 18); amino acids 152 through 655 (SEQ ID NO : 19); r) amino acids 152 through 753 (SEQ ID NO : 20); s) amino acids 152 through 753 (SEQ ID NO : 21); t) amino acids 252 through 558 (SEQ ID NO : 22); u) amino acids 252 through 655 (SEQ ID NO : 23); v) amino acids 252 through 753 (SEQ ID NO : 24); w) amino acids 252 through 1069 (SEQ ID NO : 25); x) amino acids 471 through 655 (SEQ ID NO : 26); y) amino acids 471 through 753 (SEQ ID NO : 27); z) amino acids 471 through 1069 (SEQ ID NO : 28); aa) amino acids 572 through 753 (SEQ ID NO : 29); bb) amino acids 572 through 1069 (SEQ ID NO : 30); cc) amino acids 672 through 1069 (SEQ ID NO : 31) ; dd) amino acids 52 through 1112 (SEQ ID NO : 32); ee) amino acids 152 through 1112 (SEQ ID NO : 33); ff) amino acids 252 through 1112 (SEQ ID NO : 34); gg) amino acids 471 through 1112 (SEQ ID NO : 35); hh) amino acids 572 through 1112 (SEQ ID NO : 36); ii) amino acids 672 through 1112 (SEQ ID NO : 37); and jj) amino acids 988 through 1112 (SEQ ID N0 : 38).

Most preferred DNA molecules according to the present invention encode a GRIP polypeptide in its entirety and PDZ binding domains having an amino acid sequence as set out in a) (SEQ ID NO : 3), b) (SEQ ID NO : 4), c) (SEQ ID NO : 5), d)

(SEQ ID NO : 6), e) (SEQ ID NO : 7), f) (SEQ ID NO : 8), g) (SEQ ID NO : 9), h) (SEQ ID NO : 10), j) (SEQ ID NO : 11), and x) (SEQ ID NO : 26) as disclosed above.

The enriched or isolated nucleic acid, which comprises the complementary sequence, or the substantially equivalent sequence, can be identified by hybridization under relatively highly stringent conditions to a probe comprising a region GRIP, or a segment thereof, for example, either the entire sequence or a portion of the nucleic acid sequence of Figure 11 A-C. A"probe"is a molecule, such as a DNA fragment, cDNA fragment or oligonucleotide, that is labeled in some fashion, e. g., with a radioactive isotope, and used to identify or isolate a gene or cDNA, or a fragment thereof. When only a portion of any of these nucleic acid sequences is employed as a probe, preferably that portion comprises a sequence of from about 15 to 500 base pairs, more preferably from about 15 to 100 base pairs, and most preferably from about 15 to about 50 base pairs.

Highly stringent hybridization conditions are known in the field. For example, a stringent hybridization can be performed by use of a hybridization buffer comprising 30% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a temperature of 45°C and remaining bound when subject to washing twice with that SSC buffer at 45°C. Additionally, use of a suitable GRIP or GRIP2 probe can be performed under moderately stringent hybridization conditions if desired. For example, a moderately stringent hybridization condition would includes use of a hybridization buffer comprising 20% formamide in 0.8M saline/0. 08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.

Preferred GRIP and GRIP2 nucleic acid fragments and derivatives of the invention will bind to the sequence of SEQ ID NO : 2 or SEQ ID NO 47 under the moderately stringent conditions and more preferably the highly stringent conditions discussed above (referred to herein as"high stringency"conditions).

The GRIP or GRIP2 nucleic acid fragments and derivatives preferably should comprise at least about 20 base pairs, more preferably at least about 50 base pairs, and still more preferably a nucleic acid fragment or derivative of the invention comprises at least about 100,200,300 or 400 base pairs. In some preferred embodiments, the

nucleic acid fragment or derivative is bound to some moiety which permits ready identification such as a radionucleotide, fluorescent or other chemical identifier.

Particularly preferred GRIP, GRIP2, and fragments and derivatives thereof of the invention have substantial equivalency (sequence identity) to SEQ ID NO: 1 (nucleic acid sequence shown in Figure 3 of the drawings), or to SEQ ID NO: 47 (nucleic acid sequence shown in Figure 22A-22I of the drawings), preferably at least about 70 percent equivalent (sequence identity) to SEQ ID NO : 1 are to SEQ ID NO : 47, more preferably about 80 percent or more equivalent to SEQ ID NO : 1 or to SEQ ID NO: 47, still more preferably at least about 85,90 or 95 percent equivalent to SEQ ID NO : 1 or to SEQ ID NO: 47.

As noted, substantial sequence equivalency or identity with respect to the nucleic acid sequence of GRIP shown in Figure 3 of the drawings or to the GRIP2 sequence shown in Figure 22A-22I refers to herein as the percentage of base sequences of a GRIP or GRIP2 nucleic acid fragment or derivative thereof that are identical with SEQ ID NO: 1 or SEQ ID NO : 47, after introducing any gaps necessary to achieve the maximum percent equivalency.

Additionally preferred GRIP and GRIP2 nucleic acid fragments encode polypeptides that are capable of specifically binding the C-terminal end of GluR2. By the term"C-terminal end of GluR2"is meant nearly any amino acid sequence near the C-terminus of the GluR2 with about the final 50 amino acids being generally preferred. Specific binding between the GRIP or GRIP2 polypeptide and the C- terminal end of GluR2 can be determined by a variety of means, e. g., Western blotting, ELISA, RIA, gel mobility shift assay, enzyme immunoassay, competitive assays, saturation assays or other suitable protein binding assays known in the field.

By the term"specific binding"or similar term is generally meant polypeptide disclosed herein which binds another polypeptide, thereby forming a specific binding pair, but which does not recognize and bind to other molecules.

A particularly preferred assay for detecting specific binding between the polypeptide encoded by the GRIP or GRIP2 nucleic acid fragment and the C-terminal end of a the GluR2 is what is oftentimes referred to in the field as the two-hybrid system. Typically, the assay is conducted in a suitable yeast strain such as those discussed below. The two-hybrid system registers the specific binding by formation

of specific yeast colonies that can be readily detected by inspection of growth plates that include a detectable chromophore. Incidence of the specific yeast colonies with respect to a suitable control is indicative of specific binding between the GRIP or GRIP2 nucleic acid fragment and the C-terminal end of the GluR2. The yeast colonies can be isolated and propagated according to standard techniques to obtain the fragment. Suitable controls generally include performing the same or related two- hybrid system test without the GRIP or GRIP2 nucleic acid fragment. In general, specific binding is readily detectable in the two-hybrid system if the number of "positive"yeast colonies exceeds non-positive control colonies by at least about 5% to about 10% and preferably at least about 20% to about 50%, up to about 100% or more. Preferred yeast two-hybrid systems are discussed more fully in the examples and discussion which follows.

A particularly preferred GRIP nucleic acid fragment encodes at least the following GRIP regions: 1) about 30 amino acids on the N-terminal side of PDZ4 ; 2) PDZ4 and 3) PDZ5. The preferred GRIP fragment can be readily identified by the yeast two-hybrid system described below in the examples. A related fragment is preferred for the GRIP2 polypeptide.

In addition, GRIP can be identified either from previously unidentified sources or from different species by using a probe under highly or moderately stringent conditions, as desired, using either the entirety or a portion of the nucleic acid sequence of Figure 1 lA-C. Ideally, such a probe is derived from regions of GRIP nucleic acid sequences that demonstrate commonality with GRIP at the amino acid level. A specific example of such a nucleic acid molecule is an isolated nucleic acid molecule having substantially the same nucleotide sequence as the nucleotide sequence shown in SEQ ID NO: 1. One means of isolating nucleic acid molecules encoding GRIP is to probe an organism's genomic library with a natural or artificially designated DNA probe, using methods well known in the art. DNA probes derived from the nucleotides of SEQ ID NO: 1 are particularly useful probes for this purpose.

DNA and cDNA molecules which encode GRIP may be used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, using methods well known to those skilled in the art.

Transciptional regulatory elements from the 5'untranslated region of the isolated clones, and other stability, processing, transcription, translation, and tissue specificity- determining regions from the 3'and 5'untranslated regions of the isolated genes are thereby obtained. Alternatively, the polynucleotide sequence can be an mRNA transcript of SEQ ID NO: 1. Moreover, the invention includes an antisense mRNA sequence to GRIP. The use of antisense technology and gene modification are well known to those skilled in the art. The present invention also provides an enriched or isolated nucleic acid comprising a sequence, which is complementary to, or substantially equivalent to, a sequence selected from the group consisting of Figure 11 A-C.

GRIP and GRIP2 nucleic acids used in the methods of the invention are typically isolated, meaning the nucleic acids comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome and usually constitute at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid present in a given fraction.

Constructing and screening libraries of polypeptide sequences that specifically bind to a given protein are techniques well known to the skilled artisan and as depicted in an article by Scott et al [Scott et al., Science, 249: 386-390 (1990)], for example.

Another embodiment of the invention comprises purified GRIP, preferably mammalian GRIP, and more preferably human GRIP. As used herein, the term GRIP encompasses any amino acid sequence, polypeptide or protein having substantially the same properties as the polypeptide or protein that binds to the C-termini of an AMPA glutamate receptor, described herein. As used herein, the term"isolated protein"means a protein molecule essentially free of other cellular components. An example of such a protein is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2. One means for

obtaining isolated GRIP is to express DNA encoding GRIP in a suitable host, such as bacterial, yeast, insect or mammalian cell, using methods well known in the art, and recovering the protein after it has been expressed in such a host, using methods well known in the art.

It is generally preferred that the polypeptides of the present invention, including GRIP and GRIP2, be substantially pure. That is, the polypeptides have been isolated from cell substituents that naturally accompany it so that the polypeptides are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Polypeptides having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the polypeptide should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the polypeptides can be used therapeutically, or in performing a desired assay. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.

This invention also encompasses different forms of GRIP, wherein the different forms of GRIP have a similar function and substantial sequence homology.

By the use of the term"substantial sequence homology"it is intended that DNA, RNA, and amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are within the scope of the present invention. In this regard, the"slight and non-consequential" sequence variations mean that the homologous sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein. As one of ordinary skill in the art is aware, conservative substitutions may be made in the amino acid sequence or the disclosed peptides without losing functionality. These substitutions are well known and are based upon the charge and structural properties of each amino acid. Such"functionally equivalent"peptides are also encompassed in the present invention.

Another embodiment of this invention includes vectors comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA encoding GRIP. Nucleic acid molecules are inserted into vector genomes by methods well known in the art. For

example, vectors comprise a nucleic acid molecule encoding GRIP, adapted for expression in a bacterial cell, a yeast cell, insect or a mammalian cell which additionally comprises the regulatory elements necessary for expression of the nucleic acid molecule in a bacterial cell, a yeast cell, insect or a mammalian cell operatively linked to the nucleic acid molecule coding GRIP to permit expression thereof. Any appropriate expression vector (e. g., [Pouwels et al., Cloning Vectors : A Laboratory Manual (Elsevior, NY 1985)]) and corresponding suitable host can be employed for production of recombinant proteins. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomas, Salmonella, yeast hosts include, Saccharomyces, Pichia, Candida, Hansenula, and Torulopsis, mammalian, or insect host cell systems including baculovirus systems [Luckow et al., BiolTechnology, 6: 47 (1988)]. Preferred host cells include bacteria, yeast, mammalian, plant, and insect cells and human cells in tissue culture. Illustratively, such cells are selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, Rl. 1, B-W, L-M, COS-1, BSC1, BSC40, BMT10, COS 7, C127, 3T3, HeLa, BHK, and Sf9 cells. The choice of expression host has a direct bearing on the type of protein produced. The ordinary skilled artisan is aware, for example, that the glycosylation of proteins produced in yeast or mammalian cells, such as COS-7 cells, will differ from those proteins produced in bacterial cells, such as Escherichia coli. As noted. the same or related vectors can be employed to manipulate and express GRIP 2.

The term"vector"or"recombinant vector"as used herein means any nucleic acid sequence of interest capable of being incorporated into a host cell and resulting in the expression of a nucleic acid sequence of interest. Vectors can include, e. g., linear nucleic acid sequences, plasmids, cosmids, phagemids, and extrachromosomal DNA.

Specifically, the vector can be a recombinant DNA. Also used herein, the term "expression"or"gene expression", is meant to refer to the production of the protein product of the nucleic acid sequence of interest, including transcription of the DNA and translation of the RNA transcript. Most recombinant vectors will include a "cloning site"which as used herein is intended to encompass at least one restriction endonuclease site. Typically, multiple different restriction endonuclease sites (e. g., a polylinker) are contained within the vector to facilitate cloning. See generally

Sambrook et al., Molecular Cloning (2d ed. 1989), and Ausubel et al., Current Protocols in Molecular Biology, (1989) John Wiley & Sons, New York for examples of suitable vectors and host cells for practicing the present invention.

Also provided in the present invention are mammalian cells containing a GRIP polypeptide encoding DNA sequence and modified in vitro to permit higher expression of GRIP polypeptides by means of a homologous recombinational event consisting of inserting an expression regulating sequence in functional proximity to the GRIP polypeptide encoding sequence. The expression regulating sequence can be a GRIP polypeptide expression sequence or not and can replace a mutant GRIP polypeptide regulating sequence in the cell. Related cells can be made comprising the GRIP 2 polypeptide disclosed herein.

In another embodiment, transgenic nonhuman mammals are provided that express nucleic acid molecules encoding GRIP. In still another embodiment, transgenic nonhuman mammals are provided that express antisense DNA complementary to DNA encoding GRIP. As noted, related mammals can be made that include GRIP 2.

In another embodiment, transgenic nonhuman mammals comprise a homologous recombination knockout of native GRIP. The term"knockout"refers to partial or complete reduction of the expression of at least a portion of a polypeptide encoded by an endogenous gene of a single cell, selected cells, or of all of the cells of a mammal. Included within the scope of this invention is a mammal in which two or more genes have been knocked out. Such mammals can be generated by repeating the procedures set forth in U. S. Patent Nos. 5,532,158 or 5,557,032, both incorporated by reference herein. Alternatively, the knockout mammals can be produced by breeding mammals, each with a single gene knocked out, to each other, and screening for those mammals with the double knockout genotype. A related non-human animal including GRIP 2 can also be made by these methods.

Conventional abbreviations for amino acids, peptides, and their derivatives are used as generally accepted in the peptide art and as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature [European J. Biochem., 138: 9-37 (1984)].

A conservative amino acid substitution is defined as a given amino acid is substituted by another amino acid of similar size, charge density, hydrophobicity/hydrophilicity,

and/or configuration, without significantly altering the biological or chemical activity of the variant peptide as compared to the original peptide. A nonconservative amino acid substitution is an amino acid substituted by an alternative amino acid of differing charge density, hydrophobicity/hydrophilicity, and/or configuration.

Purified proteins of the invention are also useful as antigens to produce monoclonal or polyclonal antibodies against the protein or specific peptide sequence, using standard methods which are well known to the person skilled in the art.

Generally, antibody preparation involves a) conjugating a polypeptide, such as GRIP, to a carrier; b) immunizing a host animal with the polypeptide fragment-carrier protein conjugate and adjuvant; and c) obtaining antibody from the immunized host animal. In particular, anti-peptide and anti-fusion antibodies were generated against GRIP. A GST-fusion protein corresponding to amino acids 798-904 of GRIP was used to produce antiserum. This antisera was then affinity purified on a fusion protein affinity column. In addition a 20 a. a. peptide (KEDLVKLKIRKDEDNSDEQE ; SEQ ID NO. 39) corresponding to amino acids 646-664 of GRIP was synthesized, crosslinked to carrier protein and injected into rabbits to generate antiserum. The resulting antiserum was then affinity purified on a peptide affinity column.

Peptides can be synthesized using standard peptide synthesizing techniques well known to the ordinary artisan, (e. g., [Bodanszky, Principles of Peptide Synthesis (1984), (Springer-Verlag, Heidelberg), Merrifield, Am. Chem. Soc., 85 : 2149-54 (1963), Barany et al., Int. J Peptide Protein Res., 30: 705-739 (1987)] and U. S. Patent No. 5,424,398). Synthesized peptides may be further purified (e. g., HPLC). Also, it may be preferable to produce fusion proteins using the peptides. Peptides may also be modified by well known chemical and genetic manipulation methods, such as glycosylation, amidation, carboxylation, or phosphorylation, or by the addition of salts, amides, esters, and the like. The may be desirable to manipulate peptides to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. For example, covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N-or C-terminus.

Peptides, variant peptides, or molecules comprising a peptide or a variant peptide of the invention may be conjugated to a reporter group, including, but not

limited to, a radiolabel (e. g., 32p), a fluorescent label, an enzyme, a substrate, a solid matrix, or a carrier (e. g., biotin or avidin) for use in the detection of specific levels of molecules or the specific binding activity of particular molecules.

Derivatives of a GRIP polypeptide or polynucleotide according to the invention have 1 or more chemical moieties attached thereto including, but not limited to, proteins such as serum albumin, heparin, or immunoglobulin, polymers such as natural polymers (dextran), modified natural polymers (carboxymethyl cellulose) and synthetic polymers (Ficoll, polyvinyl alchol, and polyacrylamide). The carrier is preferably a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regiment. Preferably, the carrier is relatively stable in the circulatory system with an acceptable plasma half life for clearance. Such carriers may be varied in physical structure from the highly cross-linked Ficoll, the branched dextran, to the linear polyacrylamide, carboxymethyl cellulose and polyvinyl alcohol. Polymers such as polyethylene glycol or polyoxyethylated polyols or proteins may be modified to reduce antigenicity by, for example, derivitizing with polyethylene glycol.

The present invention also includes a method of regulating the activity or levels of GRIP in an animal, such as a mammal, but particularly a human, which method comprises administering to a mammal in need of modulation of AMPA receptors a therapeutically effective amount, including a prophylactically-effective amount, of a polypeptide or nucleic acid of the present invention. The polypeptides and nucleic acids of the present invention can be administered as part of a pharmaceutical composition. In addition, the present inventive nucleic acids can be included in a vector and/or a host cell prior to such administration.

The method of the present invention has particular usefulness in the treatment of any disease, disorder, or condition involving AMPA receptors and/or the regulation of GRIP and GRIP-related activity including GRIP 2. Thus, the following disease states and conditions can be treated in accordance with the present invention: Alzheimer's disease, dementia including Alzheimer type dementia and senility, Schziophrenia, Huntington's disease, hypoglycemia, anoxia/hypoxia, modulation of neurotransmitters, (e. g. acetylcholine function), epilepsy, ischemia, ischemia

reperfusion, cerebral ischemia (all ischemia-related disorders, including stroke), neurofibromatosis, Parkinson's disease, dystonia, dyskinesia, chores, tics, memory degeneration, cerebral vasospasm, myasthenia gravis and other neuromuscular disorders, including drug-induced neuromuscular disorders and other drug-induced dementia. The polypeptides and the nucleic acids may be acutely administered, (e. g., within about a few minutes to about an hour of the onset or realization of symptoms.

The polypeptides and nucleic acids of the present invention also may be used in the treatment of chronic disease states and conditions. In particular, those conditions and disease states wherein chronic polypeptides and nucleic acids of the present invention will treat the disease, prevent the onset of symptoms, or will reduce recovery time.

All the methods of this invention can be used in any animal, including, but not limited to, an amphibian, bird, fish, insect, reptile, or mammal. One or more of the methods may have particular utility in humans, domestic animals, such as cow, pigs, sheep, dogs, cats, or horses, and in common pests, such as insects or rodents.

The GRIP protein or nucleic acid encoding GRIP may be administered, e. g., to a patient. It is generally more preferred that the GRIP polypeptide be administered to a patient. Nucleic acid coding for GRIP preferably is at least partially pure, i. e. the GRIP nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of nucleic acid present in a given fraction. More typically the GRIP nucleic acid will be substantially pure, i. e. the nucleic acid constitutes at least about 80%, more preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid in a given fraction.

Related administration methods can be employed with GRIP2 if desired.

The invention further includes a method of detecting a molecule that enhances or inhibits the binding activity of GRIP polypeptides and nucleic acids or GRIP- related polypeptides and nucleic acids using a yeast two-hybrid assay system. The yeast two-hybrid system is generally known in the art. See e. g., Fields, S and Song, O-K (1989) Nature 340: 245-246; and Chevray, P. M and Nathans, D. (1992) PNAS (USA) 89: 5789-5793. Yeast two-hybrid screening [Fields et al., supra,] was performed using a random primed cDNA library from rat hippocampus subcloned into the Sal I/Not I sites of the pPC86 vector [Chevray et al., supra,], which contains the GAL4 activation domain and a tryptophan selection marker. The final 50 amino

acids of GluR2 or mutant forms of the GluR2 C-terminus were subcloned in-frame into the Sal I/Not I sites of the pPC97 vector, which contains the GAL4 DNA binding domain and a leucine selection marker. The plasmid containing the GAL4 binding domain-GluR2 C-terminus fusion protein (bait vector) was transformed into Y190 yeast cells (Staudinger, J. et al. (1995) J. Cell. Biol. 128 (3) 263-271.) by the lithium acetate method and the transformants selected on the basis of leucine auxotrophy, and subsequently transformed with plasmids 5 containing the GALA activation domain- cDNA library fusion proteins. Positive clones were selected on triple minus plates (Leu-, Trp-, His-) and assayed for p-galactosidase activity. Clones that grew on triple minus plates and turned blue in the presence of X-gal were rescued from yeast and sequenced. Positive clones were also cotransformed with either the bait vector or the original pPC97 vector (backbone) into yeast to confirm the interaction. All the constructs that were used in other mating tests were from PCR products subcloned in- frame into pPC97 or pPC86 vectors and were confirmed by sequencing.

The compositions of the present invention are directed to the use of nucleic acid molecules of the invention, as well as antisense nucleic acid molecules hybridizable to a nucleic acid encoding a GRIP or GRIP-related polypeptide, including GRIP 2, or a polypeptide of the present invention that binds to the C-termini of an AMPA glutamate receptor, or a polypeptide of the present invention that is capable of enhancing or inhibiting excitatory synaptic transmission by interacting with the C-termini of an AMPA glutamate receptor, for the manufacture and administration of a medicament for treatment, (e. g., gene therapy). Preferably, critical regulatory sequences, or binding sequences (e. g., seven PDZ binding domains of the present invention, individual PDZ binding domains, (e. g., PDZ binding domain 6 or 7) or a combination of PDZ binding domains, preferably PDZ binding domains 1,2, and 3, and PDZ binding domains 4 and 5, which enhance (e. g., agonists) or inhibit (e. g., antagonists) excitatory synaptic transmission involving the C-termini of an AMPA receptor, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents. If appropriate, pharmaceutical compositions may be formulated into preparations including, but not limited to, solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, in the usual

ways for their respective route of administration. Methods known in the art can be utilized to prevent release or absorption of the composition until it reaches the target organ or to ensure time-release of the composition. A pharmaceutically-acceptable form should be employed which does not in effectuate the compositions of the present invention. In pharmaceutical dosage forms, the compositions may be used alone or in appropriate association, as well as in combination with, other pharmaceutically-active compounds. For example, in applying the method of the present invention for delivery of a nucleic acid comprising a sequence that encodes a GRIP or GRIP-related protein and/or gene-related elements, such delivery may be employed in conjunction with other means of treatment of neurological diseases and disorders, for example.

Accordingly, the pharmaceutical compositions of the present invention can be delivered via various routes and to various sites in an animal body to achieve a particular effect. Local or system delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation (e. g. nasal delivery), or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraarterial, peritoneal, subcutaneous, intradermal, oral, or pulmonary delivery systems, as well as topical administration.

The composition of the present invention can be provided in unit dosage form, wherein each dosage unit, e. g., a teaspoon, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other pharmaceutically active agents. The term"unit dosage form"refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically- acceptable diluent, carrier (e. g., liquid carrier such as a saline solution, a buffer solution, or other physiological aqueous solution), or vehicle, where appropriate. The specifications for the novel unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host. Related administration protocols can be used with GRIP 2 polypeptides or polynucleotides as needed.

Additionally, the present invention specifically provides a method of transferring nucleic acids to a host, which comprises administering the composition of the present invention using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for the particular application. The"effective amount"of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art. For example, one desired effect might comprise effective nucleic acid transfer to a host cell. Such transfer could be monitored in terms of a therapeutic effect, e. g., alleviation of some symptom associated with the disease being treated, or further evidence of the transferred gene or expression of the gene within the host, e. g. using PCR, Northern or Southern hybridization techniques, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, or particularized assays, as described in the examples, to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted level or function due to such transfer. These methods described are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan.

Furthermore, the amounts of each active agent included in the compositions employed in the examples described herein, i. e., add range, provide general guidance of the range of each component to be utilized by the practitioner upon optimizing the method of the present invention for practice either in vitro or in vivo. Moreover, such ranges by no means preclude use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts may vary in vitro applications depending on the particular cell line utilized, e. g., the ability of the plasmid employed for nucleic acid transfer to replicate in that cell line. Furthermore, the amount of nucleic acid to be added per cell or treatment will likely vary with the length and stability of the nucleic acid, as well as the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and may be altered due to factors not inherent to the method of the

present invention, e. g., the cost associated with synthesis, for instance. One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.

It will be apparent from the foregoing that the present invention has a variety of other uses and advantages. For example, the invention can be used to modulate AMPA receptor clustering in primary or immortalized cells of interest. Illustrative of such cells include cultured neurons, e. g., serotonergic or cholinergic neurons, as well as neuronal precursor cells, e. g., chromaffin cells propagated under appropriate conditions. In this example, the invention can be used to increase or decrease AMPA receptor clustering as needed. Thus, synaptic clustering of the receptors can be substantially formed or broken as needed. Accordingly, the present invention is particularly useful for analyzing and modulating synapses in tissue culture. This aspect of the invention lends itself to the design of screens that can be used to detect compounds (agonists or antagonists) that interact with excititory synapses.

As noted, the invention also provides a kit for detecting GRIP or GRIP2 expression. In one embodiment, the kit includes at least one container means which means includes a system for detecting or modulating the expression. In this embodiment, the system comprises at least one of 1) an antibody (preferably a monoclonal antibody) capable of specifically binding GRIP (SEQ ID NO. 2) or a suitable fragment thereof 2) an antibody (preferably a monoclonal antibody) capable of specifically binding GRIP2 (SEQ ID NO : 50) or a suitable fragment thereof ; 3) a polynucleotide sequence or fragment of same comprising sequence substantially equivalent or identical to the sequence shown in Figure 11 A-11C (SEQ ID NO 2) or the complement thereof 4) a polynucleotide sequence or the sequence of SEQ ID NO 47 or the complement thereof; and 3) a GRIP polypeptide or suitable fragment substantially equivalent or identical to the sequence shown in Figure 3 (SEQ ID NO 1) or a GRIP2 polypeptide or suitable fragment substantially equivalent or identical to the sequence shown in SEQ ID NO 50.

Additionally contemplated is a kit to modulate GRIP or GRIP2 expression. In one embodiment, the kit can be used to monitor or to treat fertility in a subject and particularly a human patient. In a particular embodiment, the kit can be used to monitor or treat spermatogenesis.

The kits of this invention can include any of the components mentioned above, including additional components, as needed, such as suitable buffers, indicators (e. g., fluorophores, chromophores or enzymes providing same), controls (e. g., a suitable polynucleotide or GRIP (or GRIP2) polypeptide of this invention) and directions for using the kit. Kit components can be provided in nearly any acceptable form, including a liquid or solid, e. g, as a lyophilized powder.

Further provided is cDNA library comprising sequence substantially equivalent or identical to the DNA sequence of shown in Figure 11 A-11C (SEQ ID NO. 1) or SEQ ID NO. 47.

The invention is not intended to be limited to the specific terminology so selected, and it is to be understood that specific elements and specific examples include all technical equivalents which operate in a similar manner to accomplish a similar purpose and aid in the understanding of the invention, and should not be construed in any way limiting its scope.

The following non-limiting examples are illustrative of the invention.

Example 1: Involvement of the C-Terminus of the GluR Subunit in AMPA.

1. Receptor Clustering Neurons A variety of studies have demonstrated that the C-terminal region of glutamate receptors is intracellular and appears to be involved in the modulation of glutamate receptor function [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J.

Neurosci., 16: 2157-2163 (1996), Ehlers et al., Curr. Opin. in Cell Biol., 8: 490495 (1996), Tingley et al., Nature, 364: 70-73 91993), Roche et al., Neuron, 16: 1179-1188 (1996), Ehlers et al., Science, 269: 1734-1737 (1995), Muller et al., Neuron, 17: 255- 265 (1996), Lau et al., J. Biol. Chem., 271: 21622-21628 (1996)]. The C-terminal seven amino acids of the NR2A and NR2B subunits of the NMDA receptor have been shown to directly interact with the synapse associated PSD-95/SAP90 protein and this interaction has been implicated in the clustering of NMDA receptors at excitatory synapses [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J.

Neurosci., 16: 2157-2163 (1996), Ehlers et al., Curr. Opin. in Cell Biol., 8: 490495 (1996)]. Although the C-termini of AMPA receptors do not have the consensus site (T/SXV) that has been shown to be important in the interaction of NMDA receptors with the PDZ domains of PSD-95/SAP90 [Kornau et al., Science, 269: 1737-1740

(1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996)], the GluR2 and GluR3 subunits do have weak homology to the C-terminus of NMDA receptor subunits (see Table 1), suggesting that the C-terminus of GluR2 or GluR3 may directly interact with PDZ domain containing proteins.

Table I-Amino Acid Sequence of the C-termini of Glutamate Receptors and K+-Channels PROTEIN SEQUENCE SEO ID NO NR2A PSIESDV 40 NR2B SSIESDV 41 SHAKER A/B VSIETDV 42 GLUR2 GIESVKI 43 GLUR3 GTESVKI 44 GLUR1 PLGATGL 45 GLUR4 VIASDLP 46 To determine whether the C-terminus of the GluR2 subunit is involved in the clustering of AMPA receptors at excitatory synapses, primary neuronal cultures were transfected with constructs containing only the C-terminal region of the GluR2 subunit. Neurons were transfected with myc-tagged cDNA constructs encoding the last 50-226 amino acids of the C-termini of the GluR2 (GluR2C) or GluR1 (GluRIC) subunits of the AMPA receptor or the NR1 subunit of the NMDA receptor (NR1C).

The cultures were then fixed and triple stained with: 1) an anti-myc antibody to identify transfected neurons; 2) anti-GluRl antibodies to identify AMPA receptor clusters within the transfected neurons; and 3) the synapse specific protein synaptophysin to identify synapses. Neurons transfected with GluRIC or NR1C have multiple synaptic AMPA receptor clusters which co-localize with the synaptic protein synaptophysin (Figure 1 and Table II) similar to that seen in non-transfected neurons.

Table II is shown below.

Table II-Disruption of Synaptic AMPA Receptor Clustering by the C- Terminus of the GluR2 Subunit GluRl Synaptophysin Clusters Clusters R2 C-Terminal (n = 34) 1.2 +/-0.29* 6.9 +/-0.8 R1 C-Terminal (n = 19) 3.6 +/-0.53 7.8 +/-0.9 NR1 C-Terminal (n = 48) 3.5 +/-0.38 R2 C-Term. Mutant (n = 10) 2.9 +/-0.48 'Values are expressed +/-SEM.

*P<. 01 compared with each of the other C-terminal fragments.

Neurons transfected with C-terminal constructs were either fixed and stained with a CY3 labeled C-terminal GluRl antibody or incubated with CY3 labeled Fab fragment against an extracellular epitope of GluRl for 1 hour prior to fixation.

Synaptophysin clusters were identified with a mouse monoclonal antibody.

In contrast, when GluR2C was transfected into neurons, the number of synaptic AMPA receptor clusters dramatically decreased (see Figure 1 and Table II).

Expression of GluR9C, however, had no effect on the number of synaptic contacts detected with the anti-synaptophysin antibody (Table II) or the presence of diffuse surface AMPA receptor staining (Figure 1). To determine whether this disruption of AMPA receptor clustering by the GluR2 C-terminus was dependent on the C-terminal seven amino acids homologous to the NMDA receptor subunits, we deleted this region of the GluR2 subunit. Transfection of the GluR2 construct of the C-terminus which lacked the last seven amino acids (GluR2C A7) did not disrupt clustering of the AMPA receptor (Table II). These results suggest that expression of the C-terminal tail of GluR2 disrupts the interaction of the GluR2 subunit with a protein important in clustering of AMPA receptors and that the last seven amino acids of GluR2 are required for this interaction.

Figure 1 is explained in more detail as follows. In an attempt to disrupt the interactions of GluR2 with potential PDZ containing proteins we transfected a myc- tagged construct of the last fifty amino acids of GluR2 (or as a control the C-terminus

of GluRl) into primary cultures of spinal cord neurons. The clustering of AMPA receptors was then analyzed by immunocytochemical techniques. The neurons were triple-labeled with: 1) the anti-myc antibody to detect transfected cells; 2) an antibody to synaptophysin to detect synapses; and 3) a Cy3-labeled FAB fragment of an antibody generated against an extracellular epitope of the GluRl subunit to analyze AMPA receptor clustering. Synapses associated with GluRl clusters are indicated with arrows while synapses without GluRl clusters are indicated with arrowheads.

Asterisks in Figure 1 refer to nearby untransfected neurons. Clustering of AMPA receptors was dramatically disrupted by the C-terminus of GluR2 but not GluRl.

Example 2: Isolation of GRIP, a Protein Which Specifically Interacts with the C-Terminus of the GluR2 and GluR3 Subunits of AMPA Receptors.

In order to identify proteins that interact with the C-terminus of the GluR2 subunit, the C-terminal region of GluR2 was used as a probe to screen a rat hippocampal cDNA library using the yeast two-hybrid technique [Fields et al., Nature, 340: 245-246 (1989), Chevray et al., Natl. Acad. Sci USA, 89: 5789-5793 (1992)]. The carboxyl terminal 50 amino acids of the rat GluR2 subunit was fused to the DNA binding domain of the transcription factor Gal4 and used as bait to screen a cDNA library of rat hippocampus fused to the Gal4 activation domain. Eight overlapping clones of a GRIP protein were isolated (see Figure 2 showing three of such clones). The interaction between GRIP and the GluR2 subunit in the yeast two- hybrid system was specific, since co-transformations of the isolated GRIP cDNAs with the Gal4 DNA binding domain alone, or with the C-terminal tail of GluRl were not positive. The longest GRIP cDNA encoded a protein that contained four separate regions that were homologous to PDZ domains (Figure 2).

In particular, Figure 2 shows three overlapping cDNA clones of a novel protein (GRIP) that was isolated in a yeast two-hybrid screen using the C-terminus of GluR2 as bait. These cDNAs encoded protein that contained three to four PDZ domains. Using these cDNAs a full length cDNA was isolated which encoded seven PDZ domains. The numbers in parentheses indicate how many times each clone was isolated.

With these cDNAs as probes, we obtained a full length GRIP cDNA after repetitive screening of an oligo dT-primed k-ZAP cDNA library generated from rat

hippocampus. This cDNA is nearly 6 kb with a large open reading frame of 3336 bp.

The sequence encodes a 1112 amino acid protein with a predicted molecular weight of 120,350 Da (Figure 3). The start codon was identified by the presence of a Kozak consensus sequence and several upstream in-frame stop codons. In addition to the PDZ domains mentioned above (PDZ3, PDZ4, PDZ5, PDZ6), the full-length GRIP cDNA encodes three additional PDZ domains (PDZ1, PDZ2 and PDZ7). Sequence alignment of GRIP with other proteins that include one or more PDZ domains indicated that each PDZ domain in GRIP contains GLGF-like repeats, a characteristic of PDZ domains, as well as other amino acids that are conserved in PDZ domains (Figure 3 and Figure 4).

Standard computer-assisted database searches showed that GRIP is homologous to many PDZ containing proteins, including members of the PSD- 95/SAP90 family, protein tyrosine phosphatases and syntrophin. The regions of GRIP that are homologous to other proteins are limited to the PDZ domains, with the amino acid sequence identity between the GRIP PDZ domains and other PDZ- containing proteins ranging from 35-45%. However, GRIP is a novel PDZ domain- containing protein which, in contrast to PSD-95/SAP90, NOS and the protein tyrosine phosphatases, does not contain any apparent catalytic domains in its C-terminus.

Analysis of the translated protein sequence shows no obvious hydrophobic putative transmembrane segments suggesting that GRIP is not a transmembrane protein.

These results suggest that GRIP may cross link AMPA receptors or serve as an adaptor protein to attach AMPA receptors to other proteins.

Example 3: Characterization of the Structural Domains Necessary for the Interaction of GRIP with the GluR2 Subunit.

The yeast two-hybrid system was used to examine the domains of GRIP and GluR2 that are responsible for the GRIP GluR2 interaction. Various constructs of GRIP were made that contained different combinations of PDZ domains 4,5 and 6 in the yeast pPC86 vector (see Figure 5A). The shortest construct that was positive when paired with the GluR2 C-terminus contained PDZ4, PDZ5 and about 30 amino acids on the N-terminal side of PDZ4, suggesting that PDZ6 is not necessary for the interaction. Interestingly, PDZ123 and PDZ7 do not interact with the C-terminus of GluR2 in yeast (data not shown), suggesting that the various PDZ domains in GRIP

may have different specificities for peptide substrates. GRIP does not interact with the C-terminus of the GluRl subunit of the AMPA receptor or the C-terminus of the NR2A or NR2B subunit of NMDA receptors in the yeast two-hybrid system (Figure 5B). These results suggested that the C-terminus of GluR2 may directly interact with the PDZ domains in GRIP with a unique specificity. To analyze whether the C- terminal amino acids of GluR2 were involved in the interaction of GluR2 with GRIP we used the truncation mutant GluR2C A7, which deleted the last 7 amino acids (GIESVKI; SEQ ID NO. 42) of the GluR2 C-terminal construct, and two point mutants GluR2C I878R and GluR2C E879R. Deletion of the last seven amino acids of GluR2 completely disrupted the association of GRIP with GluR2 in this system, while mutating 1878 and E879 had no effect (Figure 5B).

Figure 5A-5B is explained in more detail as follows. The yeast two hybrid system was used to test the domains of GRIP (Figure 5A) or GluR2 (Figure 5B) that are required for interaction. Positive selection on His plates and P-Gal activity of each construct is indicated. (GluR2C = the last 50 amino acids of GluR2 ; GluRIC = the last 82 amino acids of GluRl ; NR2A/BC= the last 221 or 226 amino acids, respectively, of the NR2A/B subunits).

Example 4: Interaction GRIP with the GluR2 and GluR3 Subunits in Transfected HEK-293 Cells.

To examine the interaction between the GluR2 subunit and GRIP in systems other than yeast, we subcloned the cDNA encoding the 4th, 5th and 6th PDZ domains of GRIP into a mammalian expression vector with a myc-tag in the N-terminus (myc- GRIP456) and cotransfected the myc-GRIP456 with the full length GluR2 subunit into HEK-293 cells. These transfected cells were then solubilized with 1% Triton X- 100 and the soluble cell lysate was immunoprecipitated with an anti-myc antibody (anti-myc). The resulting immunoprecipitates were then immunoblotted with an amenity purified antiGluR2/3 antibody that recognizes both GluR2 and GluR3. As shown in Fig. 6A, GluR2 could be immunoprecipitated by the anti-myc antibody only when it was co-transfected with GRIP demonstrating that GluR2 associates with GRIP in this system. Interestingly, this co-immunoprecipitation could be inhibited when the extracts were preincubated with a synthetic peptide corresponding to the last 20 amino acids of the C-terminus of GluR2.

Since the GluR3 subunit has a similar carboxyl terminal amino acid sequence to GluR2 (see Table I), we examined whether GluR3 would also associate with GRIP in this system. Co-transfection of GluR3 with myc-GRIP456 also resulted in the coimmunoprecipitation of GluR3 with GRIP (see Figure 6A). In contrast, the GluR1 subunit which does not have a similar C-terminal amino acid sequence, does not associate with GRIP under these conditions (Figure 6B).

We also examined the interaction of GRIP with C-terminal mutants of the GluR2 subunit in transfected HEK-293 cells. The same mutants described above, GluR2 I878R, GluR2 E879R and GluR2 A7, were generated in the GluR2 subunit cDNA and cotransfected with GRIP into HEK-293T cells. In addition, a mutant in which serine 880 was mutated to an alanine was also tested. All four mutants expressed well and were recognized by the GluR2/3 antibody. As described above for the yeast two-hybrid system. Deletion of the last seven amino acids eliminated the interaction of GRIP with the GluR2 subunit in the HEK-293 cells (Figure 6C), while mutation of I878 and E879 did not disrupt the GRIP-GluR2 interaction (Figure 6C).

Interestingly, mutation of serine 880 completely eliminated the GRIP-GluR2 interaction (Figure 6C), suggesting that similar to the interaction between the PDZ domains of PSD-95/SAP90 and substrate peptides [Kornau et al., Science, 269: 1737- 1740 (1995), Niethammer et al., J. Neurosci., 16: 2157-2163 (1996), Doyle et al., Cell, 85: 1067-1076 (1996)], a serine (or threonine) residue is important for the interaction of GRIP PDZ domains with their substrate peptides.

Figures 6A-6D are explained in more detail as follows. Figure 6A and 6B show that full-length GluR2, GluR3 or GluRl subunits were expressed with or without myc-GRIP456 (as indicated) in QT-6 cells. The cells were then solubilized with Triton X-100 and the myc-GRIP456 immunoprecipitated with an anti-myc antibody. The immunoprecipitates were then analyzed by immunoblot techniques using an antibody that recognizes both GluR2 and GluR3 or GluRl. Figures 6C and 6D show various mutants of GluR2 that were also analyzed.

Example 5: Tissue Distribution of GRIP Northern blot analysis was used to examine the expression of GRIP mRNA in various rat tissues. The GRIP mRNA was 6 kb, consistent with the isolated full length cDNA, and was only detected in brain and testes (see Figure 7). Level of

expression in testes was higher than that seen in the brain suggesting an important function for GRIP in testes. The Northern blot shows expression of GRIP a Northern blot of mRNA in various rat tissues that were probed with the GRIP cDNA. The 6kb GRIP mRNA was only detected in brain and in testes. The results suggest GRIP involvement in localization of receptors in sperm that are required for fertility. The degree of GRIP expression may enhance or prevent fertility.

To analyze the expression of the GRIP protein, an anti-fusion protein (amino acids 798-904) antibody and an antipeptide (amino acids 646-664) antibody were generated against GRIP. The affinity purified anti-fusion protein antibody recognized a single 130 kDa protein in cells transfected with the full length GRIP cDNA (see Figure 8A). The specificity of this antibody was confirmed by blocking the immunoreactivity with the immunogenic fusion protein (Figure 8A). Interestingly, this antibody specifically recognized a 130 kDa protein as well as an additional protein with an apparent molecular weight of 90 kDa in rat brain (see Figure 8A). The recognition of both of these proteins by the antibody was blocked by preincubation with the fusion protein (Figure 8A). These results suggest that the anti-GRIP fusion protein antibody cross reacts with another protein, possibly a GRIP-related protein, or alternatively, it recognizes a proteolytic breakdown product of GRIP. The anti- peptide antibody also recognized the 130 kDa protein in cells transfected with full length GRIP cDNA (data not shown) and in rat brain but did not recognize the 90kDa protein in brain (Figure 8A). The specificity of this antibody was also confirmed by blocking the immunoreactivity with the immunogenic peptide.

The 130 kDa protein appears to be primarily associated with the membrane, while the 90kDa is a soluble protein (Figure 8B). In addition, the 130 kDa protein was resistant to solubilization in non-denaturing detergents in brain and in transfected cells suggesting that it may be associated with the cytoskeleton. Attempts to coimmunoprecipitate GRIP with AMPA receptors from detergent extracts of rat brain have so far been unsuccessful due to the lack of solubility of GRIP in non-denaturing detergents.

Using the anti-GRIP fusion protein antibody, the distribution of GRIP in neuronal and non-neuronal tissues was examined. The 130 kDa protein seems to be brain specific and was widely expressed in various brain regions including the

olfactory bulb, cerebral cortex, hippocampus, cerebellum and spinal cord (Figure 8C).

In addition, the 130 kDa protein was observed in testes. In contrast, the 90kDa protein was expressed in most tissues examined.

Figures 8A-8E are explained in more detail as follows. In Figure 8A, both the anti-fusion protein and the antipeptide antibody recognized a 130 kDa protein in HEK-293 T cells transfected with the full length GRIP cDNA and in rat hippocampus.

The anti-fusion protein also specifically recognized a 90 kDa protein in hippocampus.

Immunorecognition of these proteins by the antibodies was blocked by the immunogen (+F. P., +Peptide). In Figure 8C, the 130 kDa protein was mostly associated with the particulate fraction (P), while the 90 kDa protein is present in the soluble fraction (S).

Figure 8B-8C show an immunoblot of various brain regions and tissues with the antifusion protein antibody. Figures 8D and 8E are Western blots using anti-GRIP antibody, showing that the 130 kDa protein is only expressed in brain and testes while the 90kDa protein is expressed in most tissues examined.

Example 6: Co-localization of GRIP with AMPA Receptors in Neurons To examine the subcellular distribution of GRIP in neurons, primary cultures of rat hippocampal neurons were stained with the anti-GRIP fusion protein antibody and visualized with a FITC-coupled secondary antibody. GRIP was expressed throughout the dendrites of hippocampal neurons and was clustered near the cell surface in many but not all neurons (Figure 9A). AMPA receptors in hippocampal cultures consist of heteromeric complexes of the GluRl, GluR2 and GluR3 subunits which cluster at excitatory synapses. See Craig, A. M. et al. (1993) Neuron 10: 1055- 1068; and Craig, A. M. et al. (1994) PNAS (USA) 91: 12373-12377. Double labeling of the neurons with an antibody to the GluRl subunit demonstrated that in many cases the clusters of GRIP co-localized with AMPA receptors associated with excitatory synapses (Figures 9B and 9C). These results provide supporting evidence that GRIP interacts with AMPA receptors in situ and combined with the results presented in Figure 1 strongly suggest that GRIP-GluR2 interactions are important for AMPA receptor clustering.

In Figure 9A, GRIP is present throughout neuronal dendrites and is clustered on dendritic processes. A high power magnification of a neuronal dendrite is shown

in Figures 9B and 9C with characteristic synaptic AMPA receptor clusters that colocalize with GRIP.

Example 7: GRIP is expressed in rat Sertoli Cells and Germ Cells The following results show high expression of the GRIP protein in the testis.

The results point to a significant role for GRIP in spermatogenesis.

1. Northern and Western blot analysis For Northern blot analysis on mRNA of adult rat testes, tubules and isolated total germ cells a cDNA probe of 919 bp was generated by PCR using specific primers for GRIP. Proteins of total adult rat testis, seminiferous tubules and isolated germ cells were spearated by SDS-PAGE and analyzed in Western blot analysis by using two affinity purified polyclonal antibodies specific for GRIP (JH2260, JH2493).

Furthermore, cryosections of adult rat testis were fixed in 4% paraformaldehyde, as well as in acetone/ethanol, and immunostained with three different anti-GRIP antibodies (JH2260, JH2493, D7).

Figure 13 shows results of Northern blot analysis of mRNA derived from total testes, seminiferous tubules, or enriched germ cell preparations demonstrated that several different transcripts are present in seminiferous tubule cells.

Western blot analysis with three different antibodies showed that a 130kDa protein is present in both somatic and germ cells, together with a lower MW protein at 46 kDa (Figure 14).

2. Immunofluorescence localization of GRIP The presence of GRIP in seminiferous epithelium cells was confirmed by immunofluorescence localization. Selected antibodies recognized GRIP antigens at the basal region of Sertoli cells and at Sertoli cell-germ contacts, as well as in maturing germ cells (Figure 15A-B ; 16A-B, 17A-B).

The data demonstrate that the signal transduction scaffold protein GRIP is expressed in somatic and germ cells of rat testis. The localization observed at the basal compartment of the Sertoli cells suggests that this protein plays a role in the polarization of these cells, as well as in localizing signaling proteins at their interface with the extratubular environment. FSH receptor have been shown to be localized in the same subcellular compartment. Without wishing to be bound to any particular

theory, GRIP may be involved in compartmetnalization of the FSH-dependent signaling.

Example 8: Immunofluorescence localization of GRIP in developing testis Immunofluorescence localization experiments were performed with adult testis sections using anti-GRIP antibodies described above. It was found that GRIP is localized in the basal membrane of somatic Sertoli cells facing the basal lamina, as well as in specialized junctions that are reported to anchor developing spermatids to these cells. Using Northern blot analysis and reverse transcriptase-polymerase chain reactions (RT-PCR) using GRIP oligonucleotide primers in Sertoli cell cultures essentially devoid of any germ cells, a GRIP signal can be obtained.

Additional studies were performed on the developing testis using selected antibodies. Figures 18A-18B; 19A-19B show that GRIP is not expressed in the immature Sertoli cell prior to the organization of the blood testis barrier at 10 days (Figure 18A-B). The signal appears after the tight junctions between Serrtoli cells become organized at 20 days (Figure 19A-B). The data indicate that GRIP expression is coordinated with the polarization of the Sertoli cell.

Oligonucleotide primers have been designed to detect the GluR2 and to determine whether the receptors are expressed in the Sertoli cells. An amplified band (ie. PCR amplified) was seen in brain but not in the testis or Sertoli cells.

Example 9: Isolation of PDZ containing proteins that interact with EPH- related RTKs The (EPH-related RTKs) are thought to be involved in regulating cell movement, axonal pathfinding, typographical neural projections, pattern formation, and have been locallized to regions of cell-cell contact. Different studies have converged on a domain (PDZ) that interacts with transmembrane proteins. PDZ- containing proteins are emerging at protein-protein interaction modules that are thought to bring specific multimeric complexes to specialized cell surface sites. PDZ domains were previously shown to interact directly with the cytoplasmic carboxy terminal sequences-T/SXV (SEQ ID NO. 41) of transmembrane receptors. Since EPH-family members have a related VXH sequence at their carboxy termini, it was believed that PDZ-containing proteins that interact with the carboxy termini of the EPH-RTKs could be found.

To isolate VXH-interacting proteins, the carboxy terminis of EPH members was employed as bait to screen mouse E I I and E 17 libraries in the yeast two-hybrid system. Several PDZ-containing proteins were isolated that specifically interact with the C-terminal tail of EPH receptors.

The following methods were used as needed in the preceeding examples.

1. Immunoprecipitation Coimmunoprecipitation of GRIP and GluR2/3 the cDNA containing the 4th, 5th and 6th PDZ domains (amino acids 435-969) of GRIP (myc-GRIP456) were directly subcloned into the Sal I/Not I sites of a myc-tagged pRK5 vector. HEK-293 T cells cotransfected with GluR2 (15ng) and myc-GRIP456 zig were solubilized with 1% Triton X-100 in I. P. buffer and the supernatant used for immunoprecipitation. Anti-myc antibody was added to about 100 pl protein A Sepharose beads. The cell lysate supernatant was then added to the mixture and rotated slowly at 4°C for 1-2 hours. After the incubation, the protein A beads were pelleted, and washed once in I. P. buffer containing 1% Triton X-100, twice in I. P. buffer containing 1% Triton X-100 and 0. 5M NaCl, and three times in I. P. buffer.

The immunoprecipitated proteins were eluted with sample buffer, separated using SDS-PAGE, transferred to PVDF and subjected to immunoblot analysis with anti- GluR2/3 antibodies.

2. Transfection of Spinal Cord Cultures The C-terminal 50 amino acids of the GluRl subunit (GluRIC), the GluR2 subunit (GluR2C), the NR1 subunit (NR1C) or a mutant of the GluR2 C-terminus lacking last 7 amino acids (GluR2C A7) were subcloned into the Sal I/Not I sites of the myc-tagged pRK5 vector, amplified and the plasmid purified with a Qiagen column (Qiagen Co.). Spinal cord neurons were taken from the ventral lumbar spinal cord of E 19 rat embryos using papain digestion. The cells were grown at low density (40 neurons/mm2) on a feeder layer of spinal cord glia. The media was 75% MEM, 25% Neurobasal Media (Gibco) supplemented with N2 supplements [Banker, G and Goslin K. (1991). Culturing Nerve Cells. MIT Press, Cambridge, MA.], 2% horse serum, and 15 ug/ml chick leg extract. On day 2 in culture the neurons were pretreated for 30 minutes with DMEM adjusted to an Osm of 280 and supplemented with 1mM kynurenate. Neuronal cell transfection was performed using a modification

of a previously published method [Xia et al. J. Neuro. 16 (17): 5425-5436 (1996)].

The cDNA precipitate was prepared by adding 8pg of DNA to 85.5, , 1H20 and then mixed with 12.5p1 of 2.0 M CaCl2 and 2gl Cal-Phos Maximizer (Clontech). The above solution was gently vortexed and then added 10gel at a time to a 1001 solution of 2X HBS (pH 7.0) with vortexing after every addition. After the entire solution was added it was sucked up and down several times through a yellow tip and allowed to stand for 25 minutes. The coverslips were added to a 12 well dish with 2ml of the DMEM solution and 100 i I of the DNA solution was added. The neurons were incubated with DNA for two hours and then the coverslips were washed for two hours with multiple changes of DMEM alternating with standard growth media. This procedure transfected from 1 to 2% of the neurons (and glia) and maximal expression appeared at 72 hours but was still strong at 96 hours. The neurons were triple labeled with: 1) anti-myc antibody to detect neurons transfecte with the myc-tagged proteins; 2) anti-synaptophysin antibodies to label synapses ; and 3) anti-GluR1 antibodies to identify endogenous AMPA receptor clusters. Surface staining of GluRl was performed using a Cy3 labeled FAB fragment of an antibody raised against a synthetic peptide corresponding to amino acids 251-269 in the N-terminal region of GluRl. Live neurons were incubated for one hour with lOpg/ml Cy3 labeled FAB fragment in growth media, rinsed with MEM three times and fixed in 4% paraformaldehyde. Neurons were then labeled with a mouse monoclonal synaptophysin antibody (Boehringer Mannheim) followed by FITC coupled anti- mouse secondary antibody. Finally, the cells were labeled with the anti-myc antibody and AMCA coupled anti-mouse secondary antibody.

3. HEK-293 Cell Transfection HEK-293 T cells were maintained in MEM medium (Gibco) with 10% fetal bovine serum (FBS, Gibco), 0.5% sodium pyrophosphate (Gibco), 0.5% streptomycin-penicillin (Gibco) and 0.5% L-glutamine. cDNAs subcloned into the pBKCMV vector or in the pRK5 vector with or without a N-terminal 16 amino acid myc-tag were used. cDNA (20 ug) was transfected into one 10 cm culture dish of HEK-293 cells using calcium phosphate co-precipitation as described (Ehlers, MD (1995) Science 269: 1743-1737). After transfection (36-48 hours), HEK-293 cells were harvested in immunoprecipitation (I. P.) buffer (25mM TRISHC1 buffered saline

(pH7.4), containing 10 units/ml Trasylol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5mM EDTA and SmM EGTA) and 1% Triton X100. Cell lysates were either centrifuged at 14,000 rpm for 10 min at 4°C and the supernatant used for immunoprecipitation, or the whole lysate was diluted into sample buffer (125 mM Tris (pH6.8), 5.0% (3-mercaptoethanol. 2.0% SDS, with pyronin Y dye) for SDS- PAGE-.

4. Northern Blot Hybridization One example of a northern blot hybridation was performed as follows. A nitrocellulose blot containing total RNA (10 ng) from various tissues (Clontech) was prehybridized in hybridization buffer (5x SSPE, Sx Denhards'solution, 50% formamide, 100-200pg/ml denatured salmon sperm DNA (ssDNA), 0.2% SDS) at 42°C overnight. The blot was then incubated in fresh hybridization buffer with denatured cDNA probe (5-10 x 106 cpm/ml buffer) at 42°C for 2 days. Blots were then subjected to wash in lx SSC, 1% SDS at room temperature for 30-60 minutes, with 2-3 changes of washing buffer, and then further washed in more stringent conditions (0. 5x SSC, 2% SDS, 55°C) for 1-4 hours, with 2-3 changes of washing buffer and subjected to autoradiography at-70 °C.

5. GRIP Immunocytochemistry Cultured Hippocampal neurons were fixed in 4% paraformaldehyde, 4% sucrose for 30'at room temperature, permeabilized for 5'with 0.5% triton and blocked with 10% normal goat serum (NGS). Affinity purified anti-GRIP fusion protein antibody (0.2, ug/ml) was added overnight in 3% NGS followed by incubation for 1 hour with FITC anti-rabbit antibody at room temperature. Cells were then rinsed for 1 hour in PBS and incubated in 5% normal rabbit serum for 30'. Cy3 labeled anti- GluRl antibody in 5% normal rabbit serum was then incubated with the neurons overnight. Cultures were then rinsed, mounted in Permafluor (Immunon) with 20mg/ml Dabco and viewed at 100X Images were collected with a SIT Camera and digitized using Image 1 (Universal Imaging). Appropriate controls using fusion protein and peptide blocking of the anti-GRIP or anti-GluRl antibodies showed no staining of the neurons.

Example 10: Isolation of GRIP 2 cDNA

A blast search of rat GRIP DNA sequence yielded one mouse homologue AA003555. The clone AA003555 was purchased from the Genome system, and sequenced. A 300bp region which shows the least homology with GRIP was amplified by PCR, and used as a probe to screen the lambda zap cDNA library constructed from the rat hippocampus. After high stringency screening, a 4.5 Kb clone was obtained. Nucleic acid sequences and amino acid sequences were analyzed using the MacVector program, and the homology with GRIP was analyzed by the Blast search and the MACAW program.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention has been described in detail with particular reference to the preferred embodiments thereof. However, it will be appreciated that modifications and improvements within the spirit and teachings of the invention may be made by those in the art upon considering the present disclosure.