BACKGROUND OF THE INVENTION G-protein-coupled receptor kinases (GRK) are enzymes essential in the desensitization of activated G-protein-coupled receptors. Seven members of the family have currently been identified.

Common structural features of the GRK family of polypeptides are the Regulator of G-protein Signaling (RGS) domain, involved in regulating G-proteins such as transducin, and a serine/thereonine protein kinase domain, which has kinase activity. Characteristics and activities of the GRK polypeptide family are described further in Krupnick and Benovic, 1998, Annu. Rev. Pharmacol. Toxicol. 38,289- 319; Hurley et al., 1998 Vision Res. 38,1341-1352, which are incorporated by reference herein in their entirety.

One member of the family, GRK1, has been identified as involved in phototransduction within the mammalian retina. Desensitization in the rod photoreceptor cell of a mammalian retina occurs when rhodopsin is phosphorylated by GRK-1. Failure to achieve desensitization is associated with Oguchi disease (night blindness).

Recently a kinase that phosphorylates cone photoreceptor opsins in a similar manner has been identified in squirrels. This kinase, named GRK7, has been shown to be a possible new member of the GRK family. Weiss et al., 1998, Mol. Vis. 4,27.

In order to develop more effective treatments for conditions and diseases related to cone photoreceptor visual signalings, information is needed about previously unidentified members of the GRK polypeptide family. In particular, there is a need for identification of a substantially complete set of human GRK7 family polypeptides.

SUMMARY OF THE INVENTION The present invention is based upon the discovery of a new human GRK family member, human cone opsin kinase.

The invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of : (a) the amino acid sequences selected from the group consisting of amino acids 1-553 of SEQ ID NO : 2; 55-175 of SEQ ID NO : 2; amino acids 189-455 of SEQ ID NO : 2; or amino acids 481-496 of SEQ ID NO : 2;

(b) fragments of the amino acid sequences of (a) comprising at least 20 contiguous amino acids; (c) fragments of the amino acid sequences of (a) comprising at least 30 contiguous amino acids; (d) fragments of the amino acid sequences of (a) having cone opsin kinase polypeptide activity; (e) fragments of the amino acid sequences of (a) comprising a Regulator of G-Protein Signaling domain; (f) fragments of the amino acid sequences of (a) comprising serine/thereoine protein kinase domain; (g) amino acid sequences comprising at least 20 amino acids and sharing amino acid identity with the amino acid sequences of any of (a)- (f), wherein the percent amino acid identity is selected from the group consisting of : at least 88%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%; (h) an amino acid sequence of (g), wherein a polypeptide comprising said amino acid sequence of (g) binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of (a)- (f) ; and (i) an amino acid sequence of (g) or (h) having cone opsin kinase polypeptide activity.

The instant invention also includes a polypeptide, encoded by the DNA of SEQ ID NO : 1, that has kinase or G-protein modulation activity.

Preferably, such polypeptides are isolated cone opsin kinase polypeptides or isolated polypeptides having cone opsin kinase polypreptide activity. In embodiments of the invention, such polypeptides are isolated cone opsin kinase polypeptides or isolated polypeptides having cone opsin kinase polypeptide activity.

In another embodiment of the invention, polypeptides that inhibit cone opsin kinase polypeptides are provided. Examples of such inhibitory polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO : 4, amino acids 55 through 353 of SEQ ID NO : 4, amino acids 141 through 353 of SEQ ID NO : 4, and amino acids 189 through 353 of SEQ ID NO : 4, and an amino acid sequence of at least 20 amino acids that is 80% identical to the amino acid sequence of SEQ ID NO : 4 and comprises amino acids 351 through 353 of SEQ ID NO : 4.

Nucleotide sequences of the invention include SEQ ID NO : 1 and SEQ ID NO : 3; amino acid sequences of the invention include SEQ ID NO : 2 and SEQ ID NO : 4.

Other aspects of the invention are isolated nucleic acids encoding polypeptides of the invention, and isolated nucleic acids, preferably having a length of at least 15 nucleotides, that hybridize under conditions of moderate stringency to the nucleic acids encoding polypeptides of the invention. In preferred embodiments of the invention, such nucleic acids encode a polypeptide having cone opsin kinase polypeptide activity, or comprise a nucleotide sequence that shares nucleotide sequence identity with the nucleotide sequences of the nucleic acids of the invention, wherein the

percent nucleotide sequence identity is selected from the group consisting of : at least 88%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%.

Further provided by the invention are expression vectors and recombinant host cells comprising at least one nucleic acid of the invention, and preferred recombinant host cells wherein said nucleic acid is integrated into the host cell genome.

Also provided is a process for producing a polypeptide encoded by the nucleic acids of the invention, comprising culturing a recombinant host cell under conditions promoting expression of said polypeptide, wherein the recombinant host cell comprises at least one nucleic acid of the invention. A preferred process provided by the invention further comprises purifying said polypeptide. In another aspect of the invention, the polypeptide produced by said process is provided.

Further aspects of the invention are isolated antibodies that bind to the polypeptides of the invention, preferably peptide antibodies, and more preferably monoclonal antibodies, also preferably humanized antibodies or humanized antibodies, and preferably wherein the antibody inhibits the activity of said polypeptides.

The invention additionally provides a method of designing an inhibitor of the polypeptides of the invention, the method comprising the steps of determining the three-dimensional structure of any such polypeptide, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predicted reactive site, and determining the polypeptide- inhibiting activity of the molecule.

In a further aspect of the invention, a method is provided for identifying compounds that alter cone opsin kinase polypeptide activity comprising (a) mixing a test compound with a polypeptide of the invention; and (b) determining whether the test compound alters the cone opsin kinase polypeptide activity of said polypeptide.

In another aspect of the invention, a method is provided identifying compounds that inhibit the binding activity of cone opsin kinase polypeptides comprising (a) mixing a test compound with a polypeptide of the invention and a binding partner of said polypeptide; and (b) determining whether the test compound inhibits the binding activity of said polypeptide.

The invention also provides a method for increasing kinase activities, comprising providing at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides; with a preferred embodiment of the method further comprising increasing said activities in a patient by administering at least one polypeptide of the invention The invention additionally provides a method for treating a condition or disease related to cone photoreceptor visual signaling or circadian rhythm comprising administering at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides. Because of cone photoreceptor cells'roles in mediation of visual acuity and color vision,

cone opsin kinase polypeptides, also referred to as GRK7 interchangeably throughout this application, are associated with conditions and disease related to cone photoreceptor visual signaling, such as difficulty with color vision, visual sensitivity or visual resolution.

A further embodiment of the invention provides a use for the polypeptides of the invention in the preparation of a medicament for treating a condition or disease related to cone photoreceptor visual signaling or circadian rhythm.

The invention further provides a method for analyzing cone opsin kinase polypeptides comprising identifying a set of one or more polypeptides as cone opsin kinase polypeptides, wherein the set of polypeptides comprises a polypeptide of the invention. In distinct preferred embodiments, the set of polypeptides comprises a polypeptide comprising the amino acid sequence of SEQ ID NO : 2; the method further comprises comparing the amino acid sequence of one polypeptide in the set to the amino acid sequence of another polypeptide in the set; and the method further comprises using an apparatus selected from the group consisting of a computer system, a video display unit, and a computer-readable medium, wherein the apparatus is used to perform a function selected from the group consisting of displaying the amino acid sequence of a polypeptide in the set, comparing the amino acid sequence of one polypeptide in the set to the amino acid sequence of another polypeptide in the set, predicting the structure of a polypeptide in the set, determining the nucleotide sequences of nucleic acids encoding a polypeptide in the set, and identifying a gene corresponding to a polypeptide in the set.

In yet another aspect of the invention, a system for analyzing cone opsin kinase polypeptides is provided comprising a data set representing a set of one or more polypeptides, wherein the polypeptides are identified as cone opsin kinase polypeptides. In distinct preferred embodiments, the set of polypeptides comprises a polypeptide of the invention; one or more members of the set of polypeptides are represented as sequences of amino acids; the set of polypeptides comprising the polypeptide of SEQ ID NO : 2; and the system further comprises an apparatus selected from the group consisting of a computer system, a video display unit, and a computer-readable medium.

DETAILED DESCRIPTION OF THE INVENTION We have identified a novel human cone opsin kinase polypeptide, human GRK7, which was previously an unidentified member of the GRK family of kinase polypeptides. SEQ ID NO : 1 is a cDNA sequence encoding human GRK7, the amino acid sequence of which is reported in SEQ ID NO : 2. SEQ ID NO : 3 is a cDNA sequence encoding a naturally occurring splice variant form of human GRK7, the amino acid sequence of which is reported in SEQ ID NO : 4.

Similarities in Structure of the GRK Family Members The typical structural elements common to members of the GRK family include Regulator of G-protein Signaling (RGS) domain and a serine/thereonine protein kinase domain. In the instant

invention, the RGS domain is found at amino acid residues 55-175 of SEQ ID NO : 2, and is followed by the serine/thereonine protein kinase domain at residues 189-455 of SEQ ID NO : 2. The members of the GRK family also tend to have a particular domain that we have designated"conserved autophosphorolyation site,"which in the instant invention resides between amino acids 481-496 of SEQ ID NO : 2, and in which the phosphorylated residue would be serine 490 of SEQ ID NO : 2. A canonical CaaX box, CLLL, comprises the final four amino acids of the instant invention. By analogy with other GRKs, this box is predicted to be modified by the addition of a geranylgeranyl group and would anchor the protein in a lipid membrane.

Also included in the instant invention is a splice variant of the human cone opsin kinase polypeptide (GRK7). The splice variant (SEQ ID NO : 3) has a deletion of an exon between bases 1050 and 1326 of SEQ ID NO : 1. The splice variant translates to a protein of 353 amino acids as shown in SEQ ID NO : 4. The table below shows how the human GRK7 amino acid sequence (SEQ ID NO : 2) aligns with the kinase subdomain consensus sequences described in Hanks and Hunter, 1995, FASEB J. 9 (8): 576-596. The splice variant of human GRK7 (SEQ ID NO : 4) has an N-terminal region identical to the N-terminal 350 amino acids of SEQ ID NO : 2, followed by a sequence of three C- terminal amino acids (RKV) that is not present in SEQ ID NO : 2. The SEQ ID NO : 4 splice variant therefore has kinase subdomains I-VII as shown in the table below with respect to SEQ ID NO : 2, and part of kinase subdomain VIII, but lacks kinase subdomains IX-XI.

Location of Kinase Subdomains within Human GRK7 Amino Acid Sequence (SEQ ID NO : 2) Sub-III III IV V VIA VIB VII VIII IX X XI domain: GRK7 141-213-232-247-262-286-311-328-345-363-396-419- aaseq. : 212 231 246 261 285 310 327 344 362 395 418 454 The cone opsin kinase is an intracellular protein, predicted to be cytoplasmic or associated with the intracellular face of the plasma membrane. There are certain key residues within the RGS domain/motifs, such that substitutions of those residues are likely be associated with an altered function or lack of that function for the polypeptide. Deletions or substitutions at the following positions in SEQ ID NO : 2 would be deleterious to the RGS function of the protein: amino acids 70,73-74,86-87, 166 and 172. There are also certain key residues within the kinase protein domains/motifs, such that substitutions of those residues are likely be associated with an altered function or lack of that function for the polypeptide. Deletions or substitutions at the following positions in SEQ ID NO : 2 would be deleterious to the kinase function of the protein: amino acids 200,220,239,316,321,334,360,373 and 438. The skilled artisan will recognize that the positions or boundaries of these regions of these polypeptides are approximate and that the precise boundaries of such domains can differ from member to member within the GRK polypeptide family, and more particularly, within the cone opsin kinase polypeptide family. Further, deletions or substitutions at other positions in SEQ ID NO : 2 may also be associated with an altered function or lack of that function for the polypeptide.

Although the GRK7 splice variant of SEQ ID NO : 4 is not predicted to have kinase activity, as it lacks amino acids 351-454 of SEQ ID NO : 2, it appears to be highly expressed in retinal tissue (see Example 2 below). It is likely that the SEQ ID NO : 4 GRK7 splice variant has a reduced or altered binding affinity for opsin substrates, and it may act as a dominant negative inhibitor to negatively regulate opsin phosphorylation. The SEQ ID NO : 4 GRK7 splice variant has an RGS site, but the regulation of its interaction with G-protein is likely to differ from that of the full-length GRK7 and may be unregulated or constitutive. It is also of interest that human GRK family members GRK1 and GRK6 each also have splice variants that vary in C-terminal amino acid sequence (Zhao et a/., 1998, J Biol Chem 273: 5124-5131 ; Premont et al., 1999, JBiol Chem 274: 29382-29389). For each of GRKI, GRK6, and GRK7, one of the splice variants exhibits similarity between its C-terminal amino acid sequence and the consensus sequence identified for binding to PDZ domains (Cowbum, 1997, Ctiii- Opiat Slruct Bol 7 : 835-838). The C-terminal amino acids ofGRKI (also called GRKIa) are DSKT (amino acids 28 through 31 of SEQ ID NO : 8); those of GRK6 (also called GRK6-A) are PTRL (amino acids 573 through 576 of SEQ ID NO : 9); and GRK7 splice variant SEQ ID NO : 4 has an RRKV C- terminal sequence (amino acids 350 through 353 of SEQ ID N0 : 4). For example, all three of these polypeptides have an aliphatic (or aliphatic hydroxyl) C-terminal residue. Expression of these splice variant forms may be involved in regulating GRK activity by producing a form that can interact with a polypeptide containing a PDZ domain. Polypeptides containing PDZ domains have been shown to play a central role in localizing transmembrane signaling complexes in photoreceptor cells (Li and Montell, 2000, J Cell Biol 150 : 1411-1422).

The GRK family is conserved, with an overall protein sequence similarity among kinases of 53-93% (Zhao et al. 1998, Jour. Bio. Chem. 273: 9,5123-5131). The cone opsin kinase subfamily is very conserved, with the instant human family member somewhat similar to chipmunk and squirrel.

Biological Activities and Functions of the Cone Opsin Kinase Family Members Polypeptides of the cone opsin kinase (GRK7) family are expressed in mammalian retinal cone photoreceptor cells and pineal gland cells. Typical biological activities or functions associated with this family of polypeptides are kinase activities. Polypeptides having kinase activity phosphorylate opsin molecules. The kinase activity is associated with the serine/threonine protein kinase domain of cone opsin kinase polypeptides. Thus, for uses requiring kinase activity, preferred cone opsin kinase polypeptides include those having the serine/threonine protein kinase domain and exhibiting kinase biological activity. Preferred cone opsin kinase polypeptides further include oligomers or fusion polypeptides comprising at least the serine/threonine protein kinase domain of one or more cone opsin kinase polypeptides, and fragments of any of these polypeptides that have kinase activity.

The term"kinase activity,"as used herein, includes any one or more of the following: kinase activity which phosphorylates activated opsin as well as the ex vivo, in vivo and in vitro activities of cone opsin kinase family polypeptides. The degree to which individual members of the cone opsin

kinase polypeptide family and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay. Exemplary assays are disclosed herein; those of skill in the art will appreciate that other, similar types of assays can be used to measure cone opsin kinase biological activities.

Another function associated with this family of polypeptides is the switching down or decreasing of G-Protein activity. This desensitizing activity is associated with the RGS domain of the cone opsin kinase polypeptides and involves an increase in the rate at which bound GTP is hydrolyzed by the G-Protein. Thus, for uses requiring G-protein modulation activity, preferred cone photoreceptor opsin kinase polypeptides include those having the RGS domain and exhibiting biological activity which decrease the activity of the interacting G-Proteins. Preferred cone photoreceptor opsin kinase polypeptides further include oligomers or fusion polypeptides comprising at least the RGS domain of one or more cone photoreceptor opsin kinase polypeptides, and fragments of any of these polypeptides that have exhibiting biological activity which decrease the activity of the interacting G-Proteins (by increasing the rate at which bound GTP is hydrolyzed by the G-Protein).

The term"G-protein modulation activity,"as used herein, includes any one or more of the following: biological activity which decrease the activity of the interacting G-Proteins as well as the ex vivo and in vivo activities of cone photoreceptor opsin kinase family polypeptides. The degree to which individual members of the cone photoreceptor opsin kinase polypeptide family and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay.

Exemplary assays are disclosed herein; those of skill in the art will appreciate that other, similar types of assays can be used to measure cone photoreceptor opsin kinase biological activities.

The term"cone photoreceptor opsin kinase polypeptide activity"includes both G-protein modulation activity and kinase activity.

Another aspect of the biological activity of cone photoreceptor opsin kinase polypeptides is the ability of members of this polypeptide family to bind particular binding partners such as the serine/thereonine (ser/thr) protein kinase domain binding to activated opsin. The term"binding partner,"as used herein, includes ligands, receptors, substrates, antibodies, other cone opsin kinase polypeptides, the same cone opsin kinase polypeptide (in the case of homotypic interactions), and any other molecule that interacts with a cone opsin kinase polypeptide through contact or proximity between particular portions of the binding partner and the cone opsin kinase polypeptide. Binding partners for cone opsin kinase polypeptides are also expressed by mammalian retinal and pineal cells, as cone opsin kinase polypeptides bind selectively to these partners. Because the kinase protein domain of cone opsin kinase polypeptides binds to activated opsin or rhodopsin, the ser/thr kinase domain when expressed as a separate fragment from the rest of a cone opsin kinase polypeptide is expected to disrupt the binding of cone opsin kinase polypeptides to their binding partners. By binding to one or more binding partners, the separate ser/thr kinase domain polypeptide likely prevents binding by the native cone opsin kinase polypeptide (s), and so acts in a dominant-negative fashion to inhibit the biological activities, such as phosphorylation, mediated via binding of cone opsin kinase polypeptides

to activated opsin, rhodopsin or substrates that bind to other GRK family members. Particularly suitable assays to detect or measure the binding between cone opsin kinase polypeptides and their binding partners are the affinity binding methods. Additional assays for evaluating the biological activities and partner-binding properties of cone opsin kinase family polypeptides are described below.

Polypeptides of the cone opsin kinase family are involved in diseases or conditions related to cone photoreceptor visual signaling or circadian rhythm that share as a common feature cone opsin kinase polypeptide activity in their etiology. Blocking or inhibiting the interactions between the cone opsin kinase polypeptide and its substrates, ligands, receptors, binding partners, and or other interacting polypeptides is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of cone opsin kinase polypeptide activity.

Preferred methods of administering cone opsin kinase polypeptides to organisms in need of treatment, such as mammals or most preferably humans, include methods of gene therapy and protein transduction.

Additional uses for cone opsin kinase polypeptides is use as a molecular probe to screen for inherited defects in color vision, vision resolution and circadian rhythm and as gene therapy tool for individuals with such disorders.

Polypeptides of the Cone Opsin Kinase Family A cone opsin kinase polypeptide is a polypeptide that shares a sufficient degree of amino acid identity or similarity to the cone opsin kinase polypeptides to (A) be identified by those of skill in the art as a polypeptide likely to share particular structural domains and/or (B) have biological activities in common with the cone opsin kinase polypeptides and/or (C) bind to antibodies that also specifically bind to other cone opsin kinase polypeptides. Cone opsin polypeptides include polypeptides known as rhodopsin kinases that are expressed in cone cells (Weiss et al., 2001, J Neurosci 21: 9175-9184).

Cone opsin kinase polypeptides may be isolated from naturally occurring sources, or have the same structure as naturally occurring cone opsin kinase polypeptides, or may be produced to have structures that differ from naturally occurring cone opsin kinase polypeptides. Polypeptides derived from any human cone opsin kinase polypeptide by any type of alteration (for example, but not limited to, insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; changes in state of prenylation ; changes in state of phosphorylation or any other post-translational modification; and changes to its association with other polypeptides or molecules) are also cone opsin kinase family polypeptides. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those of the cone opsin kinase polypeptides described herein, but into which modifications are naturally provided or deliberately engineered. A polypeptide that shares biological activities in common with the cone opsin kinase polypeptides is a polypeptide having cone opsin kinase polypeptide activity. Examples of biological

activities exhibited by the cone opsin kinase polypeptides include, without limitation, regulation of signaling and kinase activity, as well as other structural or biological functions.

The present invention provides both full-length and mature forms of cone opsin kinase polypeptides. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated. The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame ("ORF") of a cDNA molecule. Several full-length polypeptides may be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites. The"mature form"of a polypeptide refers to a polypeptide that has undergone post- translational processing steps such as cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases that cleave the polypeptide. The mature form (s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a nucleic acid molecule that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites. The cone opsin kinase polypeptides of the invention also include those that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N-or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1-5 terminal amino acids).

Species homologues of cone opsin kinase polypeptides and of nucleic acids encoding them are also provided by the present invention. As used herein, a"species homologue"is a polypeptide or nucleic acid with a different species of origin from that of a given polypeptide or nucleic acid, but with significant sequence similarity to the given polypeptide or nucleic acid, as determined by those of skill in the art. Species homologues may be isolated and identified by making suitable probes or primers from polynucleotides encoding the amino acid sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of cone opsin kinase polypeptides and nucleic acids encoding them; that is, naturally-occurring alternative forms of such polypeptides and nucleic acids in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism (allelic variation among individuals within a population).

Fragments of the cone opsin kinase polypeptides of the present invention are encompassed by the present invention and may be in linear form or cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10,773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,9245-9253 (1992). Polypeptides and polypeptide fragments of the present invention, and nucleic acids encoding them, include polypeptides and nucleic acids with amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, or at least 60%, or at

least 70%, and most preferably at least 80%) of the length of a cone opsin kinase family polypeptide and have at least 88%, (more preferably at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with that cone opsin kinase family polypeptide or encoding nucleic acid, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

Also included in the present invention are polypeptides and polypeptide fragments, and nucleic acids encoding them, that contain or encode a segment preferably comprising at least 8, or at least 10, or preferably at least 15, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids. Such polypeptides and polypeptide fragments may also contain a segment that shares at least 88% sequence identity (more preferably at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with any such segment of any of the cone opsin kinase polypeptides, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two amino acid or two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12: 387,1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl.

Acids Res. 14 : 6745,1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358,1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Other programs used by those skilled in the art of sequence comparison may also be used, such as, for example, the BLASTN program version 2.0.9, available for use via the National Library of Medicine website (ncbi. nlm. nih. gov/gorf/wblast2. cgi), or the UW-BLAST 2.0 algorithm (for standard UW- BLAST 2.0 default parameter settings, see blast. wustl. edu/blast/README. html#References). In addition, the BLASTalgorithm uses the BLOSUM62 amino acid scoring matix, and optional parameters that may be used are as follows : (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993); also see Wootton JC and Federhen S, 1996, Analysis of compositionally biased regions in sequence databases, Methods Enzymol. 266: 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Claverie & States (Computers and Chemistry, 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.);

preferred E-score threshold values are 0.5, or in order of increasing preference, 0.25,0.1,0.05,0.01, 0.001,0.0001, le-5, le-10, le-15, le-20, le-25, le-30, le-40, le-50, le-75, or le-100.

In another aspect of the invention, preferred polypeptides comprise various combinations of cone opsin kinase polypeptide domains, such as the RGS domain and the serine/thereonine protein kinase domain. Accordingly, polypeptides of the present invention and nucleic acids encoding them include those comprising or encoding two or more copies of a domain such as the RGS kinase domain, two or more copies of a domain such as the serine/thereonine protein kinase domain, or at least one copy of each domain, and these domains may be presented in any order within such polypeptides.

Preferably, the RGS domain precedes the ser/thr domain in the sequence.

Further modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the polypeptide sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e. g., U. S.

Pat. No. 4,518,584). Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide or a substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e. g., as described in U. S. Patent No. 5,512,457 and as set forth herein.

Other derivatives include covalent or aggregative conjugates of the polypeptides with other polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions.

Examples of fusion polypeptides are discussed below in connection with oligomers. Further, fusion polypeptides can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U. S. Patent No.

5,011,912 and in Hopp et al., BiolTechnology 6: 1204,1988. One such peptide is the FLAGs peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAGX peptide in the presence of certain divalent metal cations, as described in U. S. Patent 5,011,912. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259.

Monoclonal antibodies that bind the FLAGs peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.

Encompassed by the invention are oligomers or fusion polypeptides that contain a cone opsin kinase polypeptide, one or more fragments of cone opsin kinase polypeptides, or any of the derivative or variant forms of cone opsin kinase polypeptides as disclosed herein. In particular embodiments, the oligomers comprise soluble cone opsin kinase polypeptides. Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers.

In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites. In an alternative embodiment the invention is directed to oligomers comprising multiple cone opsin kinase polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides, such peptides having the property of promoting oligomerization.

Nucleic Acids Encoding the Cone Opsin Kinase Polypeptides Encompassed within the invention are nucleic acids encoding cone opsin kinase polypeptides.

These nucleic acids can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by"back-translation"from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding a cone opsin kinase polypeptide or a desired combination of cone opsin kinase polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5'and 3'primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239: 487 (1988) ; Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols : A Guide to Methods and Applications, Innis et. al., eds., Academic Press, Inc. (1990). An additional method to isolate and amplify a DNA sequence is use of vector recombinant sites. This method is described at www. lifetech. com as the Gateway Cloning Technology instructional manual.

Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof. The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.

An"isolated nucleic acid"is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally occurring sources. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In one preferred embodiment, the invention relates to certain isolated nucleic acids that are substantially free from contaminating endogenous material. The nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd sed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5'or 3'from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

The present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably highly stringent conditions, to nucleic acids encoding cone opsin kinase polypeptides described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., chapters 9 and 11 ; and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about 55 degrees C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees C), and washing conditions of about 60 degrees C, in 0.5 x SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 degrees C, 0.2 x SSC, 0.1% SDS. SSPE (IxSSPE is 0. 15M NaCI, 10 mM NaH. sub. 2 PO. sub. 4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (IxSSC is 0. 15M NaCI and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e. g., Sambrook et al., 1989). When hybridizing a nucleic acid

to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10. degrees C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C) = 2 (# of A + T bases) + 4 (# of #G + C bases). For hybrids above 18 base pairs in length, Tm (degrees C) = 81.5 + 16.6 (log, o [Na+]) + 0.41 (% G + C)- (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC = 0.165M). Preferably, each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at 88%, (more preferably at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above.

The present invention also provides genes corresponding to the nucleic acid sequences disclosed herein."Corresponding genes"are the regions of the genome that are transcribed to produce the mRNAs from which cDNA nucleic acid sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5'and 3'untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. Corresponding genomic nucleic acids can include 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence upstream of the first nucleotide of the genomic sequence corresponding to the initiation codon of the cone opsin kinase polypeptide coding sequence, and 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence downstream of the last nucleotide of the genomic sequence corresponding to the termination codon of the cone opsin kinase polypeptide coding sequence. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.

An"isolated gene"is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.

Methods for Making and Purifying Cone Opsin Kinase Polypeptides Methods for making cone opsin kinase polypeptides are described below. Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods. Suitable host cells include prokaryotes, yeast or higher eukaryotic cells such CHO cells. Preferred host cells for producing recombinant cone opsin kinase polypeptides are mammalian cells, such as COS cells.

The isolated nucleic acid of the invention may be operably linked to an expression control sequence such as the pDC412 or pDC314 vectors, or the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991); and Pouwels et al. Cloning Vectors : A Laboratory Manual, Elsevier, New York, (1985), in order to produce the polypeptide recombinantly.

Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185,537-566 (1990). As used herein"operably linked"means that the nucleic acid of the invention and an expression control sequence are situated within a construct, vector, or cell in such a way that the polypeptide encoded by the nucleic acid is expressed when appropriate molecules (such as polymerases) are present. As one embodiment of the invention, at least one expression control sequence is operably linked to the nucleic acid of the invention in a recombinant host cell or progeny thereof, the nucleic acid and/or expression control sequence having been introduced into the host cell by transformation or transfection, for example, or by any other suitable method. As another embodiment of the invention, at least one expression control sequence is integrated into the genome of a recombinant host cell such that it is operably linked to a nucleic acid sequence encoding a polypeptide of the invention. In a further embodiment of the invention, at least one expression control sequence is operably linked to a nucleic acid of the invention through the action of a trans-acting factor such as a transcription factor, either in vitro or in a recombinant host cell.

Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine- Plus lipid reagent, can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84 : 7413- 7417,1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. (Molecular Cloning : A Laboratory Manual, 2 ed. Vol. 1- 3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185 : 487-511,1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220,1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as

G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.

Alternatively, gene products can be obtained via homologous recombination, or"gene targeting,"techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of the endogenous nucleic acid sequence of interest (see, for example, U. S. Patent No. 5,272,071). The location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene. In a preferred embodiment, the present invention also contemplates the introduction of exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product, again, without the need for isolation of the gene sequence itself from the host cell.

A number of types of cells may act as suitable host cells for expression of the polypeptide.

Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23 : 175,1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al.

(EMBO J. 10: 2821,1991), human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation, glycosylation or prenylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments may be accomplished using known chemical or enzymatic methods. The polypeptide may also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e. g., Invitrogen, San Diego, Calif., U. S. A. (the MaxBac kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, BiolTechnology 6: 47 (1988). As used herein, an insect cell capable of expressing a nucleic acid of the present invention is"transformed."Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein. A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a"recombinant host cell".

The polypeptide of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant polypeptide. The resulting expressed polypeptide may then be purified from such culture (i. e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparin- toyopearl (D or Cibacrom blue 3GA Sepharose@ ; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the polypeptide of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP), glutathione-S-transferase (GST), GFP or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N. J.) and InVitrogen, respectively. The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (FLAG@) is commercially available from Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e. g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the present invention as an"isolated polypeptide" ; such isolated polypeptides of the invention include isolated antibodies that bind to cone opsin kinase polypeptides, fragments, variants, binding partners etc. The polypeptide of the invention may also be expressed as a product of transgenic animals, e. g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide.

It is also possible to utilize an affinity column comprising a polypeptide-binding polypeptide of the invention, such as a monoclonal antibody generated against polypeptides of the invention, or any other binding partner, such as recoverin, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e. g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. In this aspect of the invention, polypeptide-binding polypeptides, such as the anti-polypeptide antibodies of the invention or other polypeptides that can interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding polypeptides of the invention to a solid phase contacting surface can be

accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding polypeptides and held in the incubation vessel through a magnetic field.

Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding polypeptides thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding polypeptide and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non- injurious to the cells and are preferably directed to cleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containing polypeptide-expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding polypeptide of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J. Cell. Biochem., 10D : 239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.

The polypeptide may also be produced by known conventional chemical synthesis. Methods for constructing the polypeptides of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with polypeptides may possess biological properties in common therewith, including polypeptide activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The desired degree of purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.

Agonists of Cone Opsin Kinase Polypeptides In an alternative aspect, the invention further encompasses the use of agonists of cone opsin kinase polypeptide activity to treat or ameliorate the symptoms of a disease for which increased cone opsin kinase polypeptide activity is beneficial. In a preferred aspect, the invention entails

administering compositions comprising an cone opsin kinase nucleic acid or an cone opsin kinase polypeptide to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism such as a vertebrate or mammal. In still another aspect of the invention, the compositions comprise administering a cone opsin kinase polypeptide-encoding nucleic acid for expression of a cone opsin kinase polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant (e. g., decreased) endogenous activity of a cone opsin kinase polypeptide, such as decreased color sensitivity or other cone photoreceptor-mediated diseases. Furthermore, the invention encompasses the administration to cells and/or organisms of compounds found to increase the endogenous activity of cone opsin kinase polypeptides. One example of compounds that increase cone opsin kinase polypeptide activity are agonistic antibodies, preferably monoclonal antibodies, that bind to cone opsin kinase polypeptides or binding partners, which may increase cone opsin kinase polypeptide activity by causing constitutive intracellular signaling (or"ligand mimicking"), or by preventing the binding of a native inhibitor of cone opsin kinase polypeptide activity.

Antibodies to Cone Opsin Kinase Polypeptides Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). In the present invention, specifically binding antibodies are those that will specifically recognize and bind with cone opsin kinase polypeptides, homologues, and variants, but not with other molecules. In one preferred embodiment, the antibodies are specific for the polypeptides of the present invention and do not cross-react with other polypeptides. In this manner, the cone opsin kinase polypeptides, fragments, variants, fusion polypeptides, etc., as set forth above can be employed as"immunogens"in producing antibodies immunoreactive therewith.

More specifically, the polypeptides, fragment, variants, fusion polypeptides, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3: 9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded polypeptides have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the polypeptide and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2: 14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art.

Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as

described in more detail below. Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides, both polyclonal and monoclonal antibodies can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas. A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies : A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988); Kohler and Milstein, (U. S. Pat. No.

4,376,110); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4: 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques.

The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. For the production of antibodies, various host animals may be immunized by injection with one or more of the following: a cone opsin kinase polypeptide, a fragment of a cone opsin kinase polypeptide, a functional equivalent of a cone opsin kinase polypeptide, or a mutant form of a cone opsin kinase polypeptide. Such host animals may include but are not limited to rabbits, mice, and rats. Various adjutants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonal antibodies can be recovered by conventional techniques. Such monoclonal antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

In addition, techniques developed for the production of"chimeric antibodies" (Takeda et al., 1985, Nature, 314: 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a

human immunoglobulin constant region. The monoclonal antibodies of the present invention also include humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332 : 323, 1988), Liu et al. (PNAS 84 : 3439,1987), Larrick et al. (BiolTechnology 7: 934,1989), and Winter and Harris (TIPS 14 : 139, Can, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440, US Patent Nos. 5,569,825 and 5,545,806, and related patents. Preferably, for use in humans, the antibodies are human or humanized; techniques for creating such human or humanized antibodies are also well known and are commercially available from, for example, Medarex Inc.

(Princeton, NJ) and Abgennix Inc. (Fremont, CA).

Antigen-binding antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F (ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the (ab') 2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Techniques described for the production of single chain antibodies (U. S. Pat. No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544- 546) can also be adapted to produce single chain antibodies against cone opsin kinase gene products.

Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. In addition, antibodies to the cone opsin kinase polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that"mimic"the cone opsin kinase polypeptide and that may bind to the cone opsin kinase polypeptide using techniques well known to those skilled in the art. (See, e. g., Greenspan & Bona, 1993, FASEB J 7 (5): 437-444; and Nissinoff, 1991, J. Immunol. 147 (8): 2429-2438).

Screening procedures by which such antibodies can be identified are well known, and can involve immunoaffinity chromatography, for example. Antibodies can be screened for agonistic (i. e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface cone opsin kinase, induce biological effects (e. g., transduction of biological signals) similar to the biological effects induced when the cone opsin kinase binding partner binds to cell surface cone opsin kinase. Agonistic antibodies can be used to induce cone opsin kinase-mediated cell stimulatory pathways or intercellular communication.

Those antibodies that can block binding of the cone opsin kinase polypeptides of the invention to binding partners for cone opsin kinase can be used to inhibit cone opsin kinase-mediated intercellular communication or cell stimulation that results from such binding. Such blocking antibodies can be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of cone opsin kinase binding to certain cells expressing a cone opsin kinase binding partner. Alternatively, blocking antibodies can be identified in assays for the ability to inhibit a biological effect that results from binding of soluble cone opsin kinase to target cells. Antibodies can be assayed for the ability to inhibit cone opsin kinase binding partner-mediated cell stimulatory pathways, for example. Such an antibody can be employed in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of cone opsin kinase with cell surface binding partner receptor thus can be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting cone opsin kinase binding partner- mediated biological activity. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed. Compositions comprising an antibody that is directed against cone opsin kinase, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described below for compositions containing cone opsin kinase polypeptides.

Also provided herein are conjugates comprising a detectable (e. g., diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. The antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.

Antaeonists of GRK7 Polvneptides Any method which neutralizes GRK7 polypeptides or inhibits expression of the GRK7 genes (either transcription or translation) can be used to reduce the biological activities of GRK7 polypeptides. In certain embodiments of the invention, antagonists can be designed to reduce the level of endogenous GRK7 gene expression, e. g., using well-known antisense or ribozyme approaches to inhibit or prevent translation of GRK7 mRNA transcripts; triple helix approaches to inhibit transcription of GRK7 family genes; or targeted homologous recombination to inactivate or"knock out"the GRK7 genes or their endogenous promoters or enhancer elements. Antisense, ribozyme, double-stranded (ds) RNA for RNAi methods, and triple helix antagonists, examples of nucleic acid antagonists, can be designed to reduce or inhibit either unimpaired, or if appropriate, mutant GRK7 gene activity, and can be designed to specifically inhibit the GRK7 splice variant of SEQ ID NO : 4 and related polypeptides. For example, a nucleic acid antagonist can be designed to bind to the junction between exons 2 and 4 which is unique to mRNA and cDNA molecules lacking exon 3 and encoding

SEQ ID NO : 4 and related polypeptides; this junction is located between nucleotides 1049 and 1050 of SEQ ID NO : 3. Nucleic acid antagonists that specifically bind to nucleic acids comprising nucleotides 1048 through 1051, or 1047 through 1052, or 1046 through 1053, or 1045 through 1054 of SEQ ID NO : 3, or to nucleic acids comprising the complement of such sequences, are embodiments of the invention. Similarly, antibodies that specifically bind to the C-terminal sequence unique to SEQ ID NO : 4 (an amino acid sequence comprising amino acids 351 through 353 of SEQ ID NO : 4) are further antagonists of the invention. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a GRK7 mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence "complementary"to a portion of a nucleic acid, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the nucleic acid, forming a stable duplex (or triplex, as appropriate). In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA can thus be tested, or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Preferred oligonucleotides are complementary to the 5'end of the message, e. g., the 5'untranslated sequence up to and including the AUG initiation codon. However, oligonucleotides complementary to the 5'-or 3'- non-translated, non-coding regions of the GRK7 gene transcript, or to the coding regions, could be used in an antisense approach to inhibit translation of endogenous GRK7 mRNA. Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. Chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the"gap"segment of nucleotides is positioned between 5'and 3'"wing"segments of linked nucleosides and a second"open end"type wherein the"gap"segment is located at either the 3'or the 5'terminus of the oligomeric compound (see, e. g., U. S. Pat. No. 5,985,664). Oligonucleotides of the first type are also known in the art as"gapmers"or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as"hemimers"or"wingmers". The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.

The oligonucleotide can include other appended groups such as peptides (e. g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e. g., Letsinger et al., 1989, Proc Natl Acad Sci U. S. A. 86: 6553-6556; Lemaitre et al., 1987, Proc Natl Acad Sci 84: 648- 652; PCT Publication No. W088/09810), or hybridization-triggered cleavage agents or intercalating

agents. (See, e. g., Zon, 1988, Pharm. Res. 5: 539-549). The antisense molecules should be delivered to cells which express the GRK7 transcript in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e. g., antisense molecules can be injected directly into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e. g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs.

Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous GRK7 gene transcripts and thereby prevent translation of the GRK7 mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.

Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.

Ribozyme molecules designed to catalytically cleave GRK7 mRNA transcripts can also be used to prevent translation of GRK7 mRNA and expression of GRK7 polypeptides. (See, e. g., PCT International Publication W090/11364 and US Patent No. 5,824,519). The ribozymes that can be used in the present invention include hairpin ribozymes (US Patent No. 6,221,661), hammerhead ribozymes (Haseloff and Gerlach, 1988, Nature, 334: 585-591), RNA endoribonucleases (International Patent Application No. WO 88/04300; Been and Cech, 1986, Cell, 47: 207-216). As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e. g. for improved stability, targeting, etc.) and should be delivered to cells which express the GRK7 polypeptide in vivo. A preferred method of delivery involves using a DNA construct"encoding"the ribozyme under the control of a strong constitutive pol III or pol 11 promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous GRK7 messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Alternatively, endogenous GRK7 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i. e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target GRK7 gene. (See generally, Helene, 1991, Anticancer Drug Des., 6 (6), 569-584; Helene, et al., 1992, Ann. N. Y. Acad. Sci., 660,27-36; and Maher, 1992, Bioassays 14 (12), 807-815).

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.

Oligonucleotides can be synthesized by standard methods known in the art, e. g. by use of an automated DNA synthesizer (as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., 1988, Nucl. Acids Res. 16: 3209. Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U. S. A. 85: 7448-7451).

Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Endogenous target gene expression can also be reduced by inactivating or"knocking out"the target gene or its promoter using targeted homologous recombination (e. g., see Smithies, et al., 1985, Nature 317,230-234; Thomas and Capecchi, 1987, Cell 51,503-512; Thompson, et al., 1989, Cell 5, 313-321). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e. g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra), or in model organisms such as Caenorhabditis elegans where the"RNA interference" ("RNAi") technique (Grishok, Tabara, and Mello, 2000, Genetic requirements for inheritance of RNAi in C. elegans, Science 287 (5462): 2494-2497), or the introduction of transgenes (Dernburg et al., 2000, Transgene-mediated cosuppression in the C. elegans germ line, Genes Dev. 14 (13): 1578-1583) are used to inhibit the expression of specific target genes.

However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate vectors such as viral vectors.

Organisms that have enhanced, reduced, or modified expression of the gene (s) corresponding to the nucleic acid sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense nucleic acids or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15 (7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62 (1) : 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol.

58: 1-39). Transgenic animals that have multiple copies of the gene (s) corresponding to the nucleic acid sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No.

0 649 464 Bl). In addition, organisms are provided in which the gene (s) corresponding to the nucleic acid sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene (s) or through deletion of all or part of the corresponding gene (s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Proc.

Natl. Acad. Sci. USA 91 (2): 719-722), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U. S. Pat.

Nos. 5,464,764; 5,487,992; 5,627,059; 5, 631, 153; 5,614,396; 5,616,491; and 5,679,523). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals.

Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene (s), and for the development of assay systems for the identification of molecules that interact with the polypeptide product (s) of the corresponding gene (s).

Also encompassed within the invention are GRK7 polypeptide variants with partner binding sites that have been altered in conformation so that (1) the GRK7 variant will still bind to its partner (s), but a specified small molecule will fit into the altered binding site and block that interaction, or (2) the GRK7 variant will no longer bind to its partner (s) unless a specified small molecule is present (see for example Bishop et al., 2000, Nature 407: 395-401). Nucleic acids encoding such altered GRK7 polypeptides can be introduced into organisms according to methods described herein, and can replace the endogenous nucleic acid sequences encoding the corresponding GRK7 polypeptide. Such methods allow for the interaction of a particular GRK7 polypeptide with its binding partners to be regulated by administration of a small molecule compound to an organism, either systemically or in a localized manner.

Rational Design of Compounds that Interact with Cone Opsin Kinase Polypeptides The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e. g., inhibitors, agonists,, etc.

Any of these examples can be used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J (1991) Biotechnology 9: 19-21). In one approach, the three-dimensional structure of a polypeptide of interest, or of a polypeptide-inhibitor complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site (s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous polypeptides. In both cases, relevant structural information is used to design analogous opsin-like molecules, to identify efficient inhibitors, or to identify small molecules that may bind kinases. Useful examples of rational drug design may include molecules which have improved activity or stability as

shown by Braxton S and Wells JA (1992 Biochemistry 31: 7796-7801) or which act as inhibitors, agonists, or of native peptides as shown by Athauda SB et al (1993 J Biochem 113: 742-746). The use of cone opsin kinase polypeptide structural information in molecular modeling software systems to assist in inhibitor design and inhibitor-cone opsin kinase polypeptide interaction is also encompassed by the invention. A particular method of the invention comprises analyzing the three dimensional structure of cone opsin kinase polypeptides for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein.

It is also possible to isolate a target-specific antibody, selected by functional assay, as described further herein, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.

Assays of Activities of Cone Opsin Kinase Polypeptides The purified cone opsin kinase polypeptides of the invention (including polypeptides, polypeptides, fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, the cone opsin kinase molecules of the present invention can be used to identify binding partners of cone opsin kinase polypeptides, which can also be used to modulate intracellular communication or vision signaling. Alternatively, they can be used to identify non-binding-partner molecules or substances that modulate intracellular communication, cell stimulatory pathways, or immune cell activity.

Assays to Identify Binding Partners. Polypeptides of the cone opsin kinase family and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate binding partner in any suitable assay, such as a conventional binding assay.

To illustrate, the cone opsin kinase polypeptide can be labeled with a detectable reagent (e. g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like).

The labeled polypeptide is contacted with cells expressing the candidate binding partner. The cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.

One example of a binding assay procedure is as follows. A recombinant expression vector containing the candidate binding partner cDNA is constructed. CV1-EBNA-1 cells in 10 cm2 dishes are transfected with this recombinant expression vector. CV-l/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV Immediate-early enhancer/promoter. CV1-EBNA-1 was derived from the African Green Monkey kidney cell line CV-1

(ATCC CCL 70), as described by McMahan et al., (EMBO J. 10: 2821,1991). The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4 x 104 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37°C with various concentrations of, for example, a soluble polypeptide/Fc fusion polypeptide made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 25I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37°C. After extensive washing, cells are released via trypsinization. The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc polypeptide that has bound to the cells. In all assays, non-specific binding of 125I-antibody is assayed in the absence of the Fc fusion polypeptide/Fc, as well as in the presence of the Fc fusion polypeptide and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. Cell-bound i25I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N. Y. Acad. Sci.

51: 660,1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.

Binding can also be detected using methods that are well suited for high-throughput screening procedures, such as scintillation proximity assays (Udenfriend et al., 1985, Proc Natl Acad Sci USA 82: 8672-8676), homogeneous time-resolved fluorescence methods (Park et al., 1999, Anal Biochem 269: 94-104), fluorescence resonance energy transfer (FRET) methods (Clegg RM, 1995, Curr Opin Biotechnol 6: 103-110), or methods that measure any changes in surface plasmon resonance when a bound polypeptide is exposed to a potential binding partner, using for example a biosensor such as that supplied by Biacore AB (Uppsala, Sweden). Compounds that can be assayed for binding to cone opsin kinase polypeptides include but are not limited to small organic molecules, such as those that are commerically available-often as part of large combinatorial chemistry compound'libraries'-from companies such as Sigma-Aldrich (St. Louis, MO), Arqule (Woburn, MA), Enzymed (Iowa City, IA), Maybridge Chemical Co. (Trevillett, Cornwall, UK), MDS Panlabs (Bothell, WA), Pharmacopeia (Princeton, NJ), and Trega (San Diego, CA). Preferred small organic molecules for screening using these assyas are usually less than 10K molecular weight and may possess a number of physicochemical and pharmacological properties which enhance cell penetration, resist degradation, and/or prolong their physiological half-lives (Gibbs, J., 1994, Pharmaceutical Research in Molecular Oncology, Cell 79 (2): 193-198). Compounds including natural products, inorganic chemicals, and biologically active materials such as proteins and toxins can also be assayed using these methods for the ability to bind to cone opsin kinase polypeptides.

In another binding assay procedure, recombinant human cone opsin kinase polypeptides are immobolized to serve as a probe of retinal homogenates or other cell types. Binding partners are then

either run out on SDS page gels or typsinized and subjected to Mass Spectrometry. The resulting sequences are compared to a data base of known sequences.

Yeast Two-Hybrid or"Interaction Trap"Assays. Where the cone opsin kinase polypeptide binds or potentially binds to another polypeptide (such as, for example, in a receptor-ligand interaction), the nucleic acid encoding the cone opsin kinase polypeptide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75: 791-803 (1993)) to identify nucleic acids encoding the other polypeptide with which binding occurs or to identify inhibitors of the binding interaction. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Competitive Binding Assays. Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate binding partner.

Competitive binding assays can be performed by conventional methodology. Reagents that can be employed in competitive binding assays include radiolabeled cone opsin kinase and intact cells expressing cone opsin kinase (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble cone opsin kinase fragment can be used to compete with a soluble cone opsin kinase variant for binding to cell surface receptors. Instead of intact cells, one could substitute a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety. Chromatography columns that contain Polypeptide A and Polypeptide G include those available from Pharmacia Biotech, Inc., Piscataway, NJ.

Diagnostic and Other Uses of Cone Opsin Kinase Polypeptides and Nucleic Acids The nucleic acids encoding the cone opsin kinase polypeptides provided by the present invention can be used for numerous diagnostic or other useful purposes. The nucleic acids of the invention can be used to express recombinant polypeptide for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels ; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to"subtract-out"known sequences in the process of discovering other novel nucleic acids; for selecting and making oligomers for attachment to a"gene chip"or other support, including for examination of expression patterns; to raise anti-polypeptide antibodies using DNA immunization techniques; as an antigen to raise anti-DNA antibodies or elicit another immune response, and for gene therapy. Uses of cone opsin kinase polypeptides and fragmented polypeptides include, but are not

limited to, the following: purifying polypeptides and measuring the activity thereof; delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides ; and preparation of antibodies. Any or all nucleic acids suitable for these uses are capable of being developed into reagent grade or kit format for commercialization as products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation"Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F.

Fritsch and T. Maniatis eds., 1989, and"Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987 Probes and Primers. Among the uses of the disclosed cone opsin kinase nucleic acids, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above.

Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e. g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries would include but are not limited to cDNA libraries, genomic libraries, and even electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify non-human cone opsin kinase homologues.

Chromosome Mapping. The nucleic acids encoding cone opsin kinase polypeptides, and the disclosed fragments and combinations of these nucleic acids, can be used by those skilled in the art using well-known techniques to identify the human chromosome to which these nucleic acids map.

The nucleic acids of the present invention map to 3q22, at the telomere-proximal end of that band, near the border of 3q23 of chromosome 3. Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution).

Diagnostics and Gene Therapy. The nucleic acids encoding cone opsin kinase polypeptides, and the disclosed fragments and combinations of these nucleic acids can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with the genes corresponding to these polypeptides. This enables one to distinguish conditions in which this marker is rearranged or deleted. In addition, nucleic acids of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location. The DNA can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes

corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes can be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.

Methods of Screening for Binding Partners. The cone opsin kinase polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners. Cone opsin kinase polypeptide homogenates or fragments may also be used to identify binding partners which are not on the cell surface. For example, the cone opsin kinase polypeptides can be attached to a solid support material and may bind to their binding partners in a manner similar to affinity chromatography. In particular embodiments, a polypeptide is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, NJ). In an alternative, a polypeptide/Fc polypeptide (as discussed above) is attached to Polypeptide A-or Polypeptide G-containing chromatography columns through interaction with the Fc moiety. Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of homogynates of cell mixtures containing potential binding-partner-expressing cells are contacted with the solid phase having the polypeptides thereon. Binding partners of homogynates would bind to the fixed polypeptides, and unbound materials are washed away. Alternatively, cone opsin kinase polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined. In a further alternative, mixtures of cells suspected of expressing the binding partner are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidin-coated beads are known (see Berenson, et al. J. Cell. Biochem., 10D : 239,1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods. In some instances, the above methods for screening for or identifying binding partners may also be used or modified to isolate or purify such binding partner molecules or cells expressing them.

Measuring Biological Activity. Polypeptides also find use in measuring the biological activity of cone opsin kinase-binding polypeptides in terms of their binding affinity. The polypeptides thus can be employed by those conducting"quality assurance"studies, e. g., to monitor shelf life and stability of polypeptide under different conditions. For example, the polypeptides can be employed in a binding affinity study to measure the biological activity of a binding partner polypeptide that has been stored at different temperatures, or produced in different cell types. The polypeptides also can be used to determine whether biological activity is retained after modification of a binding partner polypeptide

(e. g., chemical modification, truncation, mutation, etc.). The binding affinity of the modified polypeptide is compared to that of an unmodified binding polypeptide to detect any adverse impact of the modifications on biological activity of the binding polypeptide. The biological activity of a binding polypeptide thus can be ascertained before it is used in a research study, for example.

Carriers and Delivery Agents. The polypeptides also find use as carriers for delivering agents attached thereto to cells bearing identified binding partners. The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express binding partners on the cell surface) in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application.

Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating polypeptides, mycotoxins such as trichothecenes, and derivatives and fragments (e. g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 1231, 1311, 99'nTc, illIn, and 76Br. Examples of radionuclides suitable for therapeutic use are 3l1, 21lAt, 77Br, 'Re,'Re, 'Pb, Bi,'Pd, Cu, and 67Cu. Such agents can be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the polypeptide or agent can be derivatized to generate or attach a desired reactive functional group. The derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling polypeptides are known.

Radionuclide metals can be attached to polypeptides by using a suitable bifunctional cheating agent, for example. Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.

Treating Diseases with Cone Opsin Kinase Polypeptides Thereof It is anticipated that the cone opsin kinase polypeptides, fragments, variants, agonists, antibodies, and binding partners of the invention will be useful for treating medical conditions and diseases including, but not limited to, conditions related to cone photoreceptor visual signalings and circudian rhythms as described further herein. The therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and variants, fragments, and binding partners of cone opsin kinase polypeptides may have effects similar to or different from cone opsin kinase polypeptides. Therefore, in the following paragraphs"cone opsin kinase"refers to all cone opsin kinase polypeptides, fragments, variants, agonists, antibodies, and binding partners etc. of the invention, including antagonists of inhibitors of cone opsin kinase polypeptides, and it is understood that a specific molecule or molecules can be selected from those

provided as embodiments of the invention by individuals of skill in the art, according to the biological and therapeutic considerations described herein.

Ocular disorders are tratable with the disclosed cone opsin kinase polypeptides, compositions or combination therapies, including rhegmatogenous retinal detachment, retinal degeneration, inflammatory eye disease or any other heriditary disease related to cone or rod phoreceptor cells. Other ocular disorders include retinitous pigmentosa (RP), cone dystrophies, Stargardt's disease, Leber congential dystrophy, macular dystrophy, cone-rod dystrophy, Oguchi disease, fundus albipuctutus, Recessive Usher syndrome, type 2 and type 3, recessive sensorineural deafness without RP, dominant congential stationary night blindness (CSNB), Nougaret type, recessive Bardet-Biedl syndrome dominant spinocerebellar ataxia with MD, dominant RP, dominant CSNB, recessive RP and dominant optic atrophy, Kjer type.

The disclosed cone opsin kinase polypeptides, compositions and combination therapies described herein are useful in treating conditions and diseases related to the Pineal gland. Such conditions include disruptions in internal biological clocks, which may lead to such clinical conditions as depression and anxiety. This chronobiological desynchrony can also contribute to the pathogenesis of migraine, hypertension (high blood pressure), neurologic diseases, myopathy, and premature aging, allergy, arthritis, asthma, cardiovascular disease, depression, and even cancer. Desynchronization of these clocks can cause a variety of mental and physical problems, including depression, mental fogginess, memory loss, headaches, moodiness, short temper, tension, poor appetite, slow reflexes, fatigue, weakness, and off-schedule bowel movements. Furthermore, this cone opsin kinase polypeptide may be used in the treatment or prevention of jet lag.

Administration of Cone Opsin Kinase Polypeptides and Agonists Thereof This invention provides compounds, compositions, and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient, who is suffering from a medical disorder, and in particular a cone opsin kinase-mediated disorder. Such cone opsin kinase-mediated disorders include conditions caused (directly or indirectly) or exacerbated by binding between cone opsin kinase and a binding partner. For purposes of this disclosure, the terms"illness,""disease," "medical condition,""abnormal condition"and the like are used interchangeably with the term "medical disorder."The terms"treat","treating", and"treatment"used herein includes curative, preventative (e. g., prophylactic) and palliative or ameliorative treatment. For such therapeutic uses, cone opsin kinase polypeptides and fragments, cone opsin kinase nucleic acids encoding the cone opsin kinase polypeptides, and/or agonists of the cone opsin kinase polypeptide such as antibodies can be administered to the patient in need through well-known means. Antagonists of inhibitors of cone opsin kinase polypeptides, such as antagonists of the polypeptide of SEQ ID NO : 4, can also be administered.

Compositions of the present invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments.

Polypeptideaceous cone opsin kinase polypeptides may be administered by implanting cultured cells that express the polypeptide, for example, by implanting cells that express cone opsin kinase polypeptides. Cells may also be cultured ex vivo in the presence of polypeptides of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. In another embodiment, the patient's own cells are induced to produce cone opsin kinase polypeptides by transfection in vivo or ex vivo with a DNA that encodes cone opsin kinase polypeptides. This DNA can be introduced into the patient's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes cone opsin kinase polypeptides, or by other means of transfection. Nucleic acids of the invention may also be administered to patients by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). When cone opsin kinase polypeptides are administered in combination with one or more other biologically active compounds, these may be administered by the same or by different routes, and may be administered simultaneously, separately or sequentially.

The sequence of the instant invention may also be administered by the method of protein transduction. In this method, the cone opsin kinase polypeptides linked to a protein-transduction domain (PTD) such as, but not limited to, TAT, Antp or VP22. Schwarze et al., Cell Biology 10: 290- 95 (July 2000). The PTD-linked peptides can then be transduced into cells by adding the peptides to tissue-culture mediums. This method has been widely described in publications such as Schwarze et al., Science 285: 1569 (1999); Lindgren et al., TiPS 21: 99 (2000) and Derossi et al., Cell Biology 8: 84 (1998) as well as published patent applications, WO 00/34308, WO 99/29721 and WO/99/10376.

Manufacture of Medicaments. The present invention also relates to the use cone opsin kinase polypeptides, fragments, and variants; nucleic acids encoding the cone opsin kinase polypeptides, fragments, and variants; agonists of the cone opsin kinase polypeptides such as antibodies; antagonists of inhibitors of cone opsin kinase polypeptides; cone opsin kinase polypeptide binding partners; complexes formed from the cone opsin kinase family polypeptides, fragments, variants, and binding partners, etc, in the manufacture of a medicament for the prevention or therapeutic treatment of each medical disorder disclosed herein.

EXAMPLES The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.

EXAMPLE 1 Identification of New Members of the Human GRK Polypeptide Family A data set was received from Celera Genomics (Rockville, Maryland) containing a listing of amino acid sequences predicted to be encoded by the human genome. This data set was searched with a BLAST algorithm to identify cone opsin kinase family polypeptides. Examination of the nucleotide sequences revealed the appropriate mRNA splice donor/acceptor sites, Kozak consensus sequence and

in-frame termination codon sufficient to assemble a complete open reading frame that can be aligned with the squirrel GRK7 (see Table 1 below, numbering based on amino acid sequence of human GRK7 (SEQ ID NO. : 2)). Embodiments of the invention include cone opsin kinase polypeptides and fragments of cone opsin kinase polypeptides comprising such cone opsin kinase polypeptide domains, as well as polypeptides comprising cone opsin kinase polypeptide domains and having cone opsin kinase polypeptide activities.

Amino acid substitutions and other alterations (deletions, insertions, etc.) to cone opsin kinase amino acid sequences (e. g. SEQ ID NO : 2) are predicted to be more likely to alter or disrupt cone opsin kinase polypeptide activities if they result in changes to the capitalized residues of the amino acid sequences, and particularly if those changes do not substitute an amino acid of similar structure (such as substitution of any one of the aliphatic residues-Ala, Gly, Leu, Ile, or Val-for another aliphatic residue), or a residue present in other cone opsin kinase polypeptides at that conserved position.

Conversely, if a change is made to an cone opsin kinase amino acid sequence resulting in substitution, at a non-capitalized residue, it is less likely that such an alteration will affect the function of the altered cone opsin kinase polypeptide. Embodiments of the invention include cone opsin kinase polypeptides and fragments of cone opsin kinase polypeptides, comprising altered amino acid sequences. Altered cone opsin kinase polypeptide sequences share at least 88%, or more preferably at least 90%, or more preferably at least 95%, or more preferably at least 97.5%, or more preferably at least 99%, or most preferably at least 99.5% amino acid identity with one or more of the cone opsin kinase amino acid sequences shown in Table 1.

TABLE 1 Protein (SEQ ID NO) 1 50 Hs GRK7 (2) mvDMGaLDNL iANTAYLqAR kpsDcDSkEl qRRRRSLA... LPglqgCae St GRK7 (5)-mDMGgLDNL iANTAYLqAR k. tDsDSrEl qRRRRSLA... LPgpqgCae O1 GRKC (6) mcDMGgLDNL vANTAYLkAq... ggDdkEm kkRRRSLs... LpkpeqCva Hs RK (7)-mDfGsLetv vANsAfiaAR gsfDgsSsqp sRdkkyLAkl kLPplskCes consensus--DMG-LDNL-ANTAYL-AR---D-DS-E--RRRRSLA---LP----C-- 51100 Hs GRK7 (2) LRqkLSlnFh SLCEqQPIGr RLFRDFLAtv PtfrkAatFL eDvqnWeLAe St GRK7 (5) LRqSLSphFh SLCEqQPIGr RLFRDFLAtv PkysqAvaFL eDvqnWeLAe O1 GRKC (6) LReSiekdFt lLCErQPIGk RLFRDFLAnt PefklAaeFL delydWdLA. Hs RK (7) LRdSLSleFe SvCleQPIGk kLFqqFLqsa ekhlpAlelw kDiedydtAd consensusLR-SLS--F-SLCE-QPIG-RLFRDFLA--P----A--FL-D---W-LA- 101150 Hs GRK7 (2) EGptKdsAlQ glvAtcasaP apgnpqpFLS qavAtKCqaa tteeervaav St GRK7 (5) EGpaKtstlQ qlaAtcardP... gpqsFLS qdlAtKCraa stdeerktlv O1 GRKC (6) EGaaKdkArQ niinkyck. P dsktfltFLS gepAeKCksv tdatfeevmk Hs RK (7) ndlqpqkA. Q tilAqyl. dP qaklfcsFLd egivaKfkeg pveiqdglfq consensus EG--K--A-Q---A-----P-------FLS---A-KC------------- 151200 Hs GRK7 (2) tlakAeamaF LqeqPFkdfv tSafyDkFLQ WKlfEmQPvS DKYFtEFRVL St GRK7 (5) eqakAetmsF LqeqPFqdfl aSpfyDrFLQ WKlfEmQPvS DKYFtEFRVL O1 GRKC (6) nkvqdgvreF LkgkPFteyq gSqyfDkFLQ WKeyEkQPiS DKYFyEFRtL Hs RK (7) pllqA. tlah LgqaPFqeyl gSlyflrFLQ WKwlEaQPmg edwFldFRVL consensus----A----F L---PF-----S---D-FLQ WK--E-QP-S DKYF-EFRVL 201250 Hs GRK7 (2) GKGGFGEVCA VQVKNTGKMY ACKKLdKKRL KKKGGEKMAL LEKeILEKVs St GRK7 (5) GKGGFGEVCA VQVrNTGKMY ACKKLdKKRL KKKGGEKMAL LEKeILEKVn O1 GRKC (6) GKGGFGEVCA VQVKNTGqMY ACKKLcKKRL KKKGGEKMAL LEKqILEKVn Hs RK (7) GKGGFGEVsA cQmKaTGKlY ACKKLnKKRL KKrkGyqgAm vEKkILmKVh consensus GKGGFGEVCA VQVKNTGKMY ACKKL-KKRL KKKGGEKMAL LEK-ILEKV- 251300 Hs GRK7 (2) SpFIVSLAYA FEsKTHLCLV MsLMNGGDLK fHIYNVG...... trGldMs St GRK7 (5) SpFIVSLAYA FEsKTHLCLV MsLMNGGDLK fHIYNVG...... trGlaMs O1 GRKC (6) SlFlVnLAYA ydtKTHLCLV MtLMNGGDLK yHIYNiGydg kgvdkGieMk Hs RK (7) SrFIVSLAYA FEtKadLCLV MtiMNGGDir yHIYNVneen p.... Gfpep consensus S-FIVSLAYA FE-KTHLCLV M-LMNGGDLK-HIYNVG--------G--M- 301350 Hs GRK7 (2) RvIFYsAQIa CGmLHLHelg IVYRDmKPEN VLLDdlGNCR LSDLGLAVEm St GRK7 (5) RvIFYTAQmt CGvLHLHglg IVYRDlKPEN VLLDdlGNCR LSDLGLAVEv O1 GRKC (6) RiIhYTAQIt tGiLHLHdmd IiYRDmKPEN VLLDsqGqCR LSDLGLAiEi Hs RK (7) RalFYTAQIi CGleHLHqrr IVYRDlKPEN VLLDndGNvR iSDLGLAVEl consensus R-IFYTAQI-CG-LHLH---IVYRD-KPEN VLLD--GNCR LSDLGLAVE- 351400 Hs GRK7 (2) kgGKpiTQ. r AGTnGYMAPE ILmeKvsYsy pVDWFAmGCS IYEMVAGRTP St GRK7 (5) qddKpiTQ. r AGTnGYMAPE ILmdKasYsy pVDWFAmGCS IYEMVAGRTP O1 GRKC (6) apGKtvTQ. m AGTgaYMAPE IL. sKtpYrt sVDWwAlGCS IYEMVAGRTP Hs RK (7) ldGqskTkgy AGTpGfMAPE lLqge. eYdf sVDyFAlGvt lYEMiAaRgP consensus--GK--TQ--AGT-GYMAPE IL--K--Y---VDWFA-GCS IYEMVAGRTP 401450 Hs GRK7 (2) FK... dyKEK VsKEdLKqRt lqdEVKfqHd nFteeaKDiC rlFLaKKPEq St GRK7 (5) FK... dfKEK VsKEdLKeRt mkdEVafhHe nFteetKDiC rlFLaKKPEq 01 GRKC (6) FKgpeskKEK VeKEevqrRi lneEpKweHk cFdaptKDvi qqFLkKKide Hs RK (7) Frarg... EK VenkeLKhRi isepVKyp. d kFsqasKDfC ealLeKdPEk consensus FK-----KEK V-KE-LK-R----EVK--H--F----KD-C--FL-KKPE- 451500 Hs GRK7 (2) RLGsReksdD P. RKHhfFKt iNFPRLEAGL iePPFVPDPs VVYAKDiaeI St GRK7 (5) RLGsRekadD P. RKHpfFqt vNFPRLEAGL vePPFVPDPs VVYAKDvdeI 01 GRKC (6) RLGmRnnmeD P. RKHewFKs iNFPRLEAGL vdPPwVPkPn VVYAKDtgdI Hs RK (7) RLGfRdetcD klRaHplFKd lNwrqLEAGm lmPPFiPDsk tVYAKDiqdv consensus RLG-R----D P-RKH--FK--NFPRLEAGL--PPFVPDP-VVYAKD---I 501550 Hs GRK7 (2) ddFSEVrGVE FDdKDkqFFk nFaTGAVPIa WQEEiIETGL FeELN.... D St GRK7 (5) ddFSEVrGVE FDdKDkqFFq rFsTGAVPva WQEEiIETGL FeELN.... D O1 GRKC (6) aeFSEikGiE FDaKDdkFFk eFsTGAVPIq WQqEmIETGL FdELN.... D Hs RK (7) gaFStVkGVa FDktDteFFq eFaTGncPIp WQEEmIETGi FgELNvwrsD consensus--FSEV-GVE FD-KD--FF--F-TGAVPI-WQEE-IETGL F-ELN----D

551 577 Hs GRK7 (2)... PNRptGc eeG.. nSSKS GvCLLL* St GRK7 (5)... PNRpsGd gkG.. dSSKS GvCLLL- O1 GRKC (6)... PNRkeGa ggG. ddekKS GtCaLL- Hs RK (7) gqmPddmkGi sgGsssSSKS GmCLvs- consensus---PNR--G---G---SSKS G-CLLL- EXAMPLE 2 Analysis of Expression of Alternatively Spliced Form of GRK7 Oligonucleotide primers were designed based on GRK7 nucleotide sequences in the 5'and 3' untranslated (UTR) regions of GRK7 cDNA clones, and these primers were used in PCR experiments to amplify GRK7 cDNA clones from a mammalian retina cDNA library. Two bands were detected in agarose gel electrophoresis of the PCR products, with the band representing the smaller cDNA form being more abundant than the band representing the larger cDNA form. DNA sequencing of three independently isolated cDNA clones corresponding to the smaller cDNA form indicated that these clones were derived from mRNA molecules lacking exon 3 of the GRK7 coding sequence; the coding portion of one of the smaller cDNA clones is presented as SEQ ID NO : 3 and encodes the GRK7 splice variant of SEQ ID NO : 4.

EXAMPLE 3 Monoclonal Antibodies That Bind Polypeptides of the Invention This example illustrates a method for preparing monoclonal antibodies that bind to the new human member of the GRK family. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified cone opsin kinase polypeptide or an immunogenic fragment thereof.

Purified cone opsin kinase polypeptide can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U. S. Patent 4,411,993. Briefly, mice are immunized with cone opsin kinase polypeptide immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 gg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional cone opsin kinase polypeptide emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for cone opsin kinase polypeptide antibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of binding of cone opsin kinase polypeptide to a cone opsin kinase polypeptide binding partner.

Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of cone opsin kinase polypeptide in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e. g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are

plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened by ELISA for reactivity against purified cone opsin kinase polypeptide by adaptations of the techniques disclosed in Engvall et al., (Immunochem. 8 : 871,1971) and in U. S. Patent 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144 : 4212,1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-cone opsin kinase polypeptide monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Polypeptide A or Polypeptide G can also be used, as can affinity chromatography based upon binding to cone opsin kinase polypeptide.

EXAMPLE 4 Measuring Kinase Activity Isolated kinase polypeptides or fusion proteins containing the isolated protein kinase domain can be used in an assay of protein kinase activity. Typically this would be carried out by combining a kinase of the invention with radiolabeled ATP (32P-ATP) and a magnesium (or other divalent cation, such as manganese) salt in buffer solution containing a peptide or protein substrate. Peptide substrates are generally from 8-30 amino acids in length and may terminate at the N-or C-terminus with three or more lysine or arginine residues to facilitate binding of the peptide to phosphocellulose paper. Many such general kinase substrates are known, such as casein, histone H1, myelin basic protein, etc. The substrate may also be a protein known to be phosphorylated readily by a kinase of the invention, such as activated opsin or rhodopsin. After incubation of the reaction mixture at 20-37°C for a suitable time, the transfer of radioactive phosphate from ATP to the substrate protein or substrate peptide may be monitored, by acidifying the reaction mixture then spotting it onto phosphocellulose paper, and subsequent washing of the paper with a dilute solution of phosphoric acid, in the case of a peptide substrate, or by application of the reaction products to a gel electrophoresis system followed by autoradiographic detection in the case of proteins. This last type of assay was used to detect the phosphorylation of rhodopsin by ground squirrel GRK7 (Weiss et al., 1998, Molecular Vision 4: 27).

Preferred polypeptides and polypeptide fragments of the invention have at least about 10% of the kinase activity of ground squirrel GRK7 (SEQ ID NO : 5) or human GRK7 (SEQ ID NO : 2) (more preferably, at least about 25%; still more preferably; at least about 50%; and most preferably, at least about 75%) when phosphorylation of an activated rhodopsin or an activated cone opsin substrate is measured using the phosphorylation assay of Weiss et al.

Another specific example of this type of assay of kinase activiy is the PhosphoSpots assay (Jerini Bio Tools GMBH), in which protein or peptide substrates are attached to a solid support. In one

example, the substrates may be peptides where each is known to be phosphorylated by a particular kinase. When the kinase being tested is added to the substrates in the presence of 32p, any attached proteins or peptides that are suitable subtrates for that kinase will be labeled with the 32P which can be quantitatively detected using a phosphoimager. (See, for example, Tegge et al., 1995, Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper, Biochemistry 34 (33): 10569-10577).

In yet another specific example of an assay of kinase activity, the kinase of the invention may be combined with ATP and a magnesium (or other divalent cation, such as manganese) salt in buffer solution containing a peptide or protein substrate. The membranes will be digested with Endoproteinase Asp N, removing the opsin C-termini. Mass of the separate C-termini may be determined by liquid chromatography-mass spectrometry (LC-MS). As one phosphate group adds 80 daltons to the mass of the opsin C-terminus, it can be determined the kind of opsin phosphorylated, how many opsins are incorporated in response to light, and the sites of phosphorylation (by LC-MS- MS). A similar method is described by Hurley, Spencer and Niemi in Vision Research 1998 May: 38 (10) ; 1341-52.

Kinase activity may also be measured, in vitro or in intact cells, using a fluorescence resonance energy transfer (FRET) assay, in which the transfer of energy between fluorescently tagged kinase and substrate molecules is detected. (For example, see Ng et al., 1999, Imaging protein kinase C alpha activation in cells, Science 283 (5410): 2085-2089.) Other methods are available to conveniently measure the kinase-mediated transfer of phosphate to substrate proteins or peptides, such as the scintillation proximity assay, and the use of monoclonal antibodies that are specific for phosphorylated or non-phosphorylated forms of substrate molecules; these methods are well known to those practiced in the art.

Cell signalling pathways often involve a cascade of phosphorylation events. Over-expression of kinases in cells can activate such signalling pathways, and this activation may be detected by measuring the level of phosphorylation of molecules that are known to be'downstream' phosphorylated recipients of'upstream'kinase activity. Conversely, over-expression of a catalytically inactive, truncated, or otherwise mutated form of a kinase can act as a dominant negative mutation and disrupt or abolish normal signalling events'downstream'of the kinase. Phosphorylation of signalling pathway molecules can be detected in a variety of ways, including incorporation of 32p followed by immunoprecipitation, FRET assays as described above, or the use of phosphorylation-state-specific antibodies in ELISA assays or on Western blots. Additionally, kinase specificity for a particular cell signaling pathway can be assessed by comparing the phosphorylation responses of'downstream' molecules in different pathways to over-expression of that kinase.

Activation of cell signalling pathways by kinases of the invention may also be assayed using reporter gene constructs. In such constructs a reporter gene such as luciferase or galactosidase is placed downstream of a promoter, enhancer, or other transcriptional regulatory element that is known to bind transcription factors as a result of activation of a cell signalling pathway. This transcriptional

regulatory element may be selected on the basis of its known association with a relevant transcription factor, such as AP-1 or NF-kappaB, or on the basis of physical association with a downstream gene known to be regulated by the signalling pathway. An example of the use of such reporter constructs is described in Ling et al., 1998, NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176, Proc Natl Acad Sci USA 95 (7): 3792-3797, which is incorporated by reference herein.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequences Presented in the Sequence Listing r SEQ ID NO Type Description SEQ ID NO: 1 Nucleotide Human cone opsin kinase (Hs GRK7) cDNA sequence SEQ ID NO : 2 Amino acid Human cone opsin kinase (Hs GRK7) amino acid sequence SEQ ID NO : 3 Nucleotide Human cone opsin kinase (Hs GRK7"OK6") splice variant cDNA sequence SEQ ID NO : 4 Amino acid Human cone opsin kinase (Hs GRK7"OK6") splice variant amino acid sequence SEQ ID NO : 5 Amino acid Thirteen-lined ground squirrel (Spermophilus tridecemlineatus) G- protein-coupled receptor kinase 7 (St GRK7) amino acid sequence (GenBank AAC95001) SEQ ID NO : 6 Amino acid Japanese medaka (Oryzias latipes) G-protein-coupled receptor kinase C (Ol GRKC) amino acid sequence (GenBank BAA25670) SEQ ID NO : 7 Amino acid Human rhodopsin kinase (Hs RK) amino acid sequence (Swiss-Prot Q15835) SEQ ID NO : 8 Amino acid C-terminal region of human G-protein-coupled receptor kinase 1 alpha (Hs GRK1) amino acid sequence (GenBank AAC97161) SEQ ID NO : 9 Amino acid Human G-protein-coupled receptor kinase 6, splice variant A (Hs GRK6) amino acid sequence (GenBank NP002073)