Polypeptide Derived From a Popamine Receptor, and Compositions and Methods Thereof

The present invention relates to compounds, compositions and methods involving synthetic, isolated and/or recombinant G-protein coupled receptor polypeptides that comprise fragments and/or consensus peptides of G-protein coupled receptors.

The membrane protein gene superfamily of G-protein coupled receptors (GPRs) has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane .alpha.-helices connected by extracellular or cytoplasmic loops. Of the 74 sequenced members of this G-protein receptor superfamily, the shortest sequence of 324 amino acids represents the rat mas oncogene and the longest, of 744 amino acids, represents the human thyroid-stimulating hormone (TSH) receptor. GPRs thus include a wide range of biologically active receptors, such as hormone-, viral-, growth factor- and neuroreceptors.

G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches of about 20-30 amino acids, connecting at least 8 divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind in a noncovalent but high affinity manner to neuroleptic drugs used for treating psychotic and neurological disorders. For example, the dopamine D.sub.2 receptor includes these transmembrane domains, two of which (TM III and TM V; see below) have been implicated by site-selective mutagenesis to demonstrate functional, association with D.sub.2 ligands.

Transmembrane domains of G-protein coupled receptors are designated TM1, TM2, TM3, TM4, TMS, TM6 and TM7. TM4, TM5, TM6 and TM7 are the most highly conserved and are postulated to provide sequences which impart biological activity to GPRs. Most GPRs have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. TM3 is also implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPRs. Most GPRs contain potential phosphorylation sites (e.g., serine or theronine residues) within the third cytoplasmic loop and/or the carboxy terminus. For several GPRs, such as the .beta.-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.

Non-limiting examples of GPRs include cAMP receptors, adenosine receptors, .beta.-adrenergic receptors, muscarinic acetylcholine receptors, .alpha.-adrenergic receptors, serotonin receptors (5-HT), histamine H2 receptors, thrombin receptors, kinin receptors, follicle stimulating hormone receptors, opsins and rhodopsins, odorant receptors, cytomegalovirus receptor, etc. See e.g., Probst et al DNA and Cell Biology 11:1-20(1992), which is entirely incorporated herein by reference.

The ligand binding sites of GPRs are believed to comprise a hydrophilic socket formed by several GPR transmembrane domains, which socket is surrounded by hydrophobic residues of the GPRs. The hydrophilic side of each GPR transmembrane helix is postulated to face inward and form the polar ligand binding site. TM3 has been implicated in several GPRs as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.

GPRs can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters. See, e.g., Johnson et al Endoc. Rev. 10:317-331(1989) ; and Birnbaumer et al Blochem. Biophys. Acta 1031:163-224(1990) which references are incorporated entirely herein by reference. GPR agonist binding catalyzes the exchange of GTP for GDP on the .alpha.-subunit of the G-protein. Different G-protein .alpha.-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPRs has been identified as an important mechanism for the regulation of G-protein coupling of some GPRs.

As a non-limiting example of a GPR ligand, dopamine (3,4-dihydroxyphenethylamine) is a critical neurotransmitter in the central nervous system (e.g., in the substantial nigra, midbrain, and hypothalamus). Since the elucidation of the ascending mesolimbic and nigrostriatal pathways, these pathways have been found to be critical in the control of both motor initiation (nigrostriatal) behavior and affectire (mesolimbic) behavior. The clinical efficacy of the major neuroleptic antipsychotic medications has been found to correlate with the respective affinities of these agents for the dopamine D.sub.2 receptor in the brain. A dopaminergic role in the symptomatology of the major psychoses has thus been hypothesized, although it is unclear if dopamine alone is etiological, (see, e.g., Davis et al. Am. J. Psych. 148:1474-1476 (1991)). Nonetheless, this hypothesis has served as a stimulus for current research in this area.

One model for studying possible interactions of G-protein coupled receptors with their ligands has emerged from site-directed mutagenesis and biochemical analysis of the .beta.-adrenergic receptor, as well as from biophysical analysis of the interaction of retinal with opsin.

According to such a model, the binding of a GPR ligand to a G-protein coupled receptor involves multiple interactions between functional groups on the GPR ligand and residues within the hydrophophilic binding site of the receptor.

While a number of the amino acid residues in the dopamine D.sub.2 receptor have been postulated to participate in D.sub.2 ligand binding, based on results obtained from site-directed mutagenesis studies and photoaffinity labeling studies performed on the .beta.-adrenergic receptor, such studies have failed to specifically determine which residues are actually involved in binding in the D.sub.2 system. Sibley et al. Soc. Neurosci. Abs. 17:36.10, 324.5, 324.6 (1991).

The clinical use of neuroleptics has provided a means for treating patients suffering from psychotic disorders. Short-term use of neuroleptics is indicated in several types of psychotic disorders, e.g., acute psychotic episodes, regardless of type; exacerbations of schizophrenia; acute manic excitement while deferring use of lithium or awaiting onset of its effects; adjunctive therapy for major depression with prominent psychotic symptoms, or when an antidepressant or ECT alone is not successful; for agitation in delirium, dementia, or severe mental retardation while seeking to identify and treat the primary basis of the problem; in certain chronic, degenerative, or idiopathic neuropsychiatric disorders with dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome; or for ballism or hemiballism; childhood psychoses or apparently allied conditions marked by severe agitation or aggressive behavior; miscellaneous medical indications, notably nausea and vomiting, or intractable hiccups.

Additionally, continuous long-term use of neuroleptics is indicated in many psychotic disorders, such as (for more than six months) (i) primary indications such as Schizophrenia, Paranoia.sup.a,b, Childhood psychoses, some degenerative or idiopathic neuropsychiatric disorders (notably, Huntington's disease and Gilles de la Tourette's syndrome); (ii) secondary indications such as extremely unstable manic-depressive or other episodic psychoses (unusual), otherwise unmanageable behavior symptoms in dementia, amentia, or other brain syndromes; and (iii) questionable indications such as chronic characterological disorders with schizoid, "borderline," or neurotic characteristics; substance abuse; or antisocial behavior, recurrent mood disorders. See, e.g., Baldessarini, Chemotherapy in Psychiatry, Revised and Enlarged Edition, Harvard University Press, Cambridge, Mass., (1985), the contents of which is entirely incorporated herein by reference.

Neuroleptics are also referred to as neuroplegics, psychoplegics, psycholeptics, antipsychotics and major tranquilizers, but are sometimes distinguished from non-neuroleptic anti-psychotics. Neuroleptics have recently been characterized as an agent that produces sedative or tranquilizing effects, and which also produces motor side effects, such as catalepsy or extrapyramidal symptomatology. Nonlimiting representative examples of neuroleptics include phenothiazine derivatives (e.g., chlorpromazine); thioxanthine derivatives (e.g., thiothixene); butyrophenone derivatives e.g., haloperidol); dihydroindolone (e.g., molindone); dibenzoxazepine derivatives (e.g., loxapine); and "atypical" neuroleptics (e.g., sulpiride, remoxipiride pimozide and clozapine). See Berstein Clinical Pharmacology Littleton, Mass. :PSG Publishing (1978); Usdin et al Clinical Pharmacology in Psychiatry New York:Elsevier North-Holland (1981); and Baldessarini, supra, (1985); and , which references are herein entirely incorporated by reference.

The term "atypical neuroleptics" has been used to describe antipsychotic neuroleptics that produce few or no extrapyramidal side effects and which do not cause catalepsy in animals (See, e.g., Picket et al, Arch. Gen. Psychiatry 49:345 (May 1992). Alternatively, atypical neuroleptics, such as clozapine, have been described as those neuroleptics which have a higher affinity for D.sub.4 and D.sub.1 sites than for D.sub.2 sites (See, e.g., Davis et al Amer. J. Psych. 148:1474, 1476 (November 1991).

The long term use of all known anti-psychotics, such as neuroleptics or non-neuroleptic antipsychotics, has resulted in serious side effects, as present in Table I, such as persistent and poorly reversible motoric dysfunctions (e.g., tardive dyskinesia) in a significant number of patients. These side effects are especially prevalent in geriatric populations, and adequate pharmacological treatment of these debilitating morotic dysfunctions is not currently available. This problem has severely limited the long-term, clinical administration of these agents.

In addition, clozapine, although apparently capable of producing less motor side effects, can cause irreversible, potentially fatal agranulocytosis in a minority of patients administered the drug. Such serious side effects limit the use of clozapine to patients who are resistant to treatment with other neuroleptics.

Antipsychotics have a variety of significant pharmacological effects, e.g., as presented in the following Tables II and III.

See Ebadi, PHARMACOLOGY, Little, Brown and Co., Boston, 61-65 (1985); Cattabeni et al Adv. Biochem. Psychopharmacology 24:275 (1980). Baldessarini, supra, which references are herein incorporated entirely by reference.

However, despite the face that thousands of neurolepticor antipsychotic-type compounds have been synthesized and reported in the literature, such compounds which lack serious side effects and which have sufficient pharmacological activity, have not been disclosed.

Alternative to dopamine receptor GPRs, as presented above, other neuroreceptor GPRs are involved in neurological pathologies, and drugs such as neuroreceptor GPR binding agents, presently used for treating these pathologies, also suffer from similar side effects as those of neuroleptics, as presented above.

Other GPRs are also involved in receptor-related pathologies, such as hormone related GPRs involved in endocrine related pathologies,

Accordingly, there is a need to provide G-protein coupled receptor binding agents, including neuroreceptor and endocrine receptor GPRs, which do not produce such deleterious and debilitating side effects as those produced by known agents, such as neuroleptics, which can be used for therapy or diagnosis of GPR related pathologies.

Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentabilty of the claims of the present application. All statements as to the date or representations as to the contents of these documents are based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

It is therefore an object of the present invention to overcome one or more deficiencies found in the related art.

It is another object of the present invention to provide non-naturally occurring synthetic, isolated and/or recombinant GPR polypeptides which are fragments, consensus fragments and/or sequences having conservative amino acid substitutions, of at least one transmembrane domain of at least one G-protein coupled receptor, which polypeptides have been discovered to have receptor-like functional binding sites of neuroreceptor and endocrine GPRs, such that GPR polypeptides of the present invention may bind GPR ligands, or which may also modulate, quantitatively or qualitatively, GPR ligand binding to GPRs.

In is still another object of the present invention to provide GPR polypeptides and compositions that have only partially helical structures, in contrast to known characterized transmembrane domains of GPRs, such as, but not limited to, GPR transmembrane domains I-VII.

it is yet another object of the present invention to provide synthetic or recombinant GPR polypeptides, conservative substitution derivatives thereof, antibodies, anti-idiotype antibodies, compositions and methods that can be used as potential modulators of G-protein coupled receptor function, by binding to GPR ligands or modulate GPR ligand binding, due to their expected biological properties, which may be used in diagnostic, therapeutic and/or research applications.

It is a further object of the present invention is to provide synthetic, isolated or recombinant polypeptides which are designed to inhibit or mimic various GPRs or fragments thereof, as receptor types and subtypes.

According to one aspect of the present invention, a synthetic or recombinant GPR polypeptide is provided that comprises a GPR amino acid sequence of, e.g., at least 5, 10, 15 or 20 amino acids, substantially corresponding to at least one transmembrane domain, or fragment and/or consensus peptide thereof, of a G-protein coupled receptor, wherein at least 20 amino acids are preferred. In a preferred embodiment, the polypeptide is (a) chemically synthesized and/or (b) obtained from a recombinant host cell or organism which expresses a recombinant nucleic acid encoding a GPR polypeptide, as defined herein.

In another preferred embodiment, the transmembrane domain is selected from at least one of TM1, TM2, TM3, TM4, TM5, TM6 or TM7, corresponding to transmembrane domains I, II, III, IV, V, VI and VII, respectively, of a GPR. In another preferred embodiment, the transmembrane domain is a dopamine receptor transmembrane domain selected from the group consisting of at least one of a D.sub.1, D.sub.2, D.sub.3, D.sub.4 and D.sub.5 dopamine receptor transmembrane domain. The transmembrane domain, e.g., may be selected from at least one of D.sub.2 receptor transmembrane domains III or V. In still another preferred embodiment, the GPR polypeptide amino acid sequence substantially corresponding to an amino acid sequence contained in at least one of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:3) or FIG. 5 (SEQ ID NO:5).

In another aspect of the present invention, a GPR composition is provided, comprising a GPR polypeptide, or a pharmaceutically acceptable ester, ether, sulfate, carbonate, malate, glucuronide or salt thereof, the composition further comprising a pharmaceutically acceptable carrier and/or diluent.

In still another aspect of the present invention, a method is provided for treating a subject suffering from a disease state involving a qualitative or quantitative pathological abnormality of a GPR protein or a biological molecule functionally associated therewith. Such biological molecule may be a membrane cytoplasmic protein, lipid, carbohydrate, saccharide, nucleoside or nucleotide mono-, di-, or tri-phosphate, an enzyme, a cofactor, a nucleic acid, a neurotransmitter, an ion, a carrier, a cell receptor, or any combination thereof.

In a preferred embodiment, the GPR protein is a dopamine receptor and the abnormality involves a dopamine related pathology, wherein the method comprises administering an effective dopamine receptor modulating amount of a GPR polypeptide of the present invention. In another preferred embodiment, the transmembrane domain is a D.sub.2 dopamine receptor domain and the disease state is a psychiatric disorder, such as schizophrenia or schiz affective disorder (see American Psychiatric Association, Revised Manual of Diagnostic and Statistical Criteria for Psychiatric Disorders (DSM-III-R), American Psychiatric Assoc. Press, Washington, DC (1989)).

In another preferred embodiment, the GPR composition is administered as a pharmaceutical composition to provide a GPR polypeptide in an amount ranging from about 0.01 .mu.g to 100 mg/kg, and also preferably, about 10 .mu.g to 10 mg/kg. In another preferred embodiment, the administering is by oraal intravenous, intramuscular, parenteral or topical administration, including mucosal administration to the nasal mucosa or the oral mucosa, by aerosol, nebulizer or drop administration as non-limiting examples.

Other objects of the invention will be apparent to skilled practitioners from the following detailed description and examples relating to the present invention.

FIG. 1 is the amino acid sequence of a control peptide (SEQ ID NO:1), which is hydrophobic in its properties, but does not correspond to a known GPR transmembrane domain.

FIG. 2 represents the amino acid sequence of a GPR transmembrane polypeptide, polypeptide II (SEQ ID NO:2), which corresponds to a portion of the dopamine D.sub.2 receptor transmembrane segment III.

FIG. 3 represents the amino acid sequence of a transmembrane polypeptide, polypeptide III (SEQ ID NO:3), norresponding to a consensus peptide of the dopamine D.sub.2 receptor transmembrane domains I-VII.

FIG. 4 represents the amino acid sequence of a consensus sequence of transmembrane domains that is shortened to be less than the length required to span a lipid bilayer.

FIG. 5 represents a consensus amino acid sequence of transmembrane domain as a consensus peptide between dopamine receptors D.sub.1 and D.sub.2,

FIG. 6 is a representation of a circular dichroism spectrum of a solution of the consensus polypeptide III (SEQ ID NO: 3) of FIG. 3.

FIG. 7 is a graphical representation of radioligand binding assay data comparing control polypeptide II (SEQ ID NO:1) of FIG. 1, labeled as "II" and consensus polypeptide I (SEQ ID NO: 3 of FIG. 3, labeled as "I".

FIG. 8A-G are a comparison listing of amino acid sequences of transmembrane domains and adjacent amino acid sequences of representative GPRs (SEQ ID NOS:6-79).

The present invention relates to G-protein coupled receptor (GPR) polypeptides which can be used to mimic naturally occurring or isolated GPRs, or to modulate the binding of GPR ligands to GPRs, such as inhibition or enhancement of binding. GPR polypeptides of the present invention can include GPR transmembrane domain fragments and/or consensus peptides thereof, of at lease 4-10 amino acids in length, and/or corresponding sequences having conservative amino acid substitutions as "substitution peptides", wherein the GPR polypeptide binds a GPR ligand or modulates the binding of a GPR ligand to a GPR in vitro, in vivo or in situ.

GPR polypeptides of the present invention can be synthesized or recombinantly produced, or optionally purified, to provide commercially useful amounts of GPR polypeptides for use in therapeutic, diagnostic or research applications, according to known method steps, see, e.g., Ausubel et al, eds. Current Protocols in Molecular Biology, Wiley Interscience, N.Y., (1987, 11992); and Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd edition, Vols. 1-3, Cold Spring Harbor Press, (1989), which references are herein entirely incorporated by reference.

Additionally, GPR polypeptides according to the present invention can be used to generate polyclonal and/or monoclonal antibodies, anti-idiotype antibodies thereto, or fragments thereof, which may used for diagnostic and/or therapeutic applications, according to known method steps, see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988), which is herein entirely incorporated by reference.

GPR polypeptides, anti-GPR antibodies or anti-idiotype antibodies (or fragments thereof) to GPR polypeptides have been unexpectedly discovered to quantitatively or qualitatively modulate G-protein coupled receptors, such that binding of GPR polypeptides or anti-idiotype antibodies (or fragments thereof) to G-protein coupled receptor ligands may be used for diagnostic research or therapeutic applications of the present invention. Such GPR polypeptides, antibodies or anti-idiotype antibodies of the present invention may therefore be used as modulators of G-protein coupled receptors, such as neuroreceptors or endocrine receptors, as non-limiting examples.

Binding of such GPR polypeptides, (including GPR fragments, consensus peptides, substitution derivatives and anti-idiotype antibody fragments) of the present invention may be used to treat symptoms of, and provide diagnosis and treatment for, pathologies related to GPRs. Such pathologies have been found to correlate with symptoms occurring in neurological. viral or endocrine pathologies. D.sub.2 receptor-related psychotic disorders, including schizophrenia, now treated with neuroleptics, is a non-limiting example thereof.

The use of synthetic or recombinant GPR polypeptides of the present invention can be preferable to the use of known drugs that bind G-protein coupled receptors, such as neuroleptics that bind or inhibit the biological effect of binding to neuroreceptors as a non-limiting example. Such polypeptides are expected to have significantly less side effects than presently used drugs presently used for inhibiting such receptor binding including neuroleptics, as they would structurally mimic naturally occuring GPRs and/or modulate ligand binding. Thus, GPR polypeptides are expected to have reduced side effects attributable to known foreign compound drugs, with less immunogenicity, and reduced potential for motoric side effects (e.g., extrapyramidal symptoms and/or tardive dyskinesia).

The present invention is also related to the production, by chemical synthesis or recombinant DNA technology, of GPR polypeptides, preferably as small as possible while still retaining sufficiently high affinity or interaction with G-protein coupled receptors to modulate, such as to inhibit or to enhance, binding to such receptors by GPR ligands.

GPR polypeptides of the present invention may include 5-10 to 50-150 amino acid fragments, consensus sequences or substitution sequences of GPRs, e.g., as presented in FIG. 8A-G (SEQ ID NOS:6-79) including, but not limited to, multiple dopamine receptors, cAMP receptors, adenosine receptors, .beta.-adrenergic receptors, muscarinic acetylcholine receptors, .alpha.-adrenergic recepnors, serotonin receptors (5-HT), histamine H2 receptors, thrombin receptors, kinin receptors, follicle stimulating hormone receptors, opsins and rhodopsins, odorant receptors, cytomegalovirus GPRs, adenosine A2 receptors, dopamine receptor, histamine H2 receptors, octopanmine receptors, N-formyl receptors, anaphylatoxin receptors, thromboxane receptors, IL-8 receptors, platelet activating factor receptors, endothelin receptors, bombesin gastrin releasing peptide receptor, neuromedin B preferring bombesin receptors, vasoactive intestinal peptides, neurotensin receptors, bradykinin receptors, thyrotropin-releasing hormone receptors, substance Preceptors, neuromedin K receptors, drenal angiotensen II type I receptors, mas oncogene (angiotensin) receptors lutropin-choriogonadotropin receptors, thyrotropin receptors, follicle stimulating hormone receptors, cannabinoid receptors, glucocorticoid-induced receptors, endothelial cell GPRs, testis GPRs, and thoracic aorta GPRs, and homologs thereof having a homology of at least 80% with at least one of transmembrane domains 1-7, as described herein. See, e.g., Probst et al DNA and Cell Biology 11:1-20(1992), which is entirely incorporated herein by reference.

Accordingly, a "G-protein coupled receptor polypeptide" or "GPR polypeptide" of the present invention includes polypeptides having a "GPR amino acid sequence" which substantially corresponds to at least one 10 to 50 amino acid fragment and/or consensus sequence of a known GPR or group of GPRs, wherein the GPR polypeptide has homology of at least 80%, such as 81, 82, 83, 84, 85, 86, 87, 88, 89 90 91, 92 93, 94, 95, 96, 97, 98, 99 or 100% homology, while maintaining GPR modulating activity, wherein a GPR polypeptide of the present invention is not naturally occurring or is naturally occurring but is in a purified or isolated form which does not occur in nature. Preferably, a GPR polypeptide of the present invention substantially corresponds to a transmembrane domain of a GPR or group of GPRs as a consensus sequence.

Also preferred are GPR polypeptides wherein the GPR amino acid sequence is 4-10 to 50 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids, or any range therein.

An amino acid or nucleic acid sequence of a GPR polypeptide of the present invention is said to "substantially correspond" to another amino acid or nucleic acid sequence, respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one GPR transmembrane domain, or which may be synergistic when two or more transmembrane domains, consensus sequences or homologs thereof are present.

Additionally or alternatively, such "substantially corresponding" sequences of GPR polypeptides include conservative amino acid or nucleotide substitutions, or degenerate nucleotide nodon substitutions wherein individual amino acid or nucleotide substitutions are well known in the art.

Alternatively or additionally, substantially corresponding refers to GPR polypeptides having amino acid sequences having at least 80% homology or identity to an amino acid sequence of SEQ ID NO:l, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology or identity.

Accordingly, GPR polypeptides of the present invention, or nucleic acid encoding therefor, include a finite set of substantially corresponding sequences as substituuion peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al, supra, at .sctn..sctn.A.1.1-A.1.24, and Sambrook et al, supra, at Appendices C and D.

Conservative substitutions of a GPR polypeptide of the present invention includes a variant wherein at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid. Such substitutions pruferably are made in accordance with the following list as presented in Table IV, which substitutions may be determined by routine experimentation provide modified structural and functional properties of a synthesized polypeptide molecule, while maintaining the receptor binding, inhibiting or mimicking biological activity, as determined by known GPR receptor activity assays.

Alternatively, another group of substitutions of GPR polypeptides of the present invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue inserted in its place according to the following Table V. The types of substitutions which may be made in the protein or peptide molecule of the present invention may be based on nalysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al., .Supra and FIGS. 3-9 of Creighton, supra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than .alpha.-helical. Pro, because of its unusual geometry, tightly constrains the chain. It generally tends to promote .beta.-turn-like structures, although in some cases Cys can be capable of participating in disulfide bond formation which is important in protein folding. Note the Schulz et al. would merge Groups 1 and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. .alpha.-helix or .beta.-sheet, as well as changes in physiological activity, e.g. in receptor binding assays.

However, when the exact effect of the substitution, deletion, or insertion is to be confirmed one skilled in the art will appreciate that the effect of the substitution or substitutions will be evaluated by routine screening assays, either immunoassays or bioassays to confirm biological activity, such as receptor binding or modulation of ligand binding to the corresponding GPR. See, e.g., Maranges et al., eds., for example, a substituted polypeptide mypically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a chemically derivatized column or immobilized membranes or hollow fibers (to absorb the mutant by binding to at least one epitope).

A preferred use of this invention is the production, by chemical or recombinant DNA technology, of GPR polypeptides, preferably as small as possible while still retaining sufficiently high affinity for binding to, or association with, GPRs. By production of GPR polypeptides including smaller fragments or variants of such transmembrane domains, one skilled in the art, using known binding and inhibition assays, can readily identify the GPR polypeptides capable of binding minimizing or modulating G-protein coupled receptors using known methods. Non-limiting examples of fragments of GPRs to be used as GPR polypeptides or as a basis for consensus sequences thereof for GPR polypeptides, are presented in FIGS. 2-5 and FIG. 8A-G, wherein fragments or consensus sequences of 10 to 50 amino acids of at least one sequence of FIGS. 2-5 or corresponding to at least one transmembrane domain or domains 1-7 listed in FIG. 8A-G (SEQ ID NOS:6-79) are encompassed by the present invention, such as at least one transmembrane domain of one or more GPRs, such as a cAMP receptor (1), adenosine receptors (2-3); muscarinic acetylcholine receptors (4- 8); human adrenergic receptors (9-11, 14-16, 19-25, 28); adrenergic receptors (9-28); human thrombin receptor (31); endothelin receptors (35-36), bombesin receptors (37-38), endocrine receptors (48-50), rhodopsin (51) . opsins (52-54), odorant receptors (55-64), and cytomegalovirus GPRs (72-54), as non-limiting examples, wherein ("#") refers to the listed sequences in FIG. 8A-G.

Accordingly, GPR polypeptides may include consensus sequences and/or fragments of at least one of transmembrane domain 1-7 of one or more GPRs as presented in FIGS. 2-5 (SEQ ID NO:2-5) or FIG. 8A-G. (SEQ ID NOS:6-79) or homologs thereof, which GPR polypeptides do not occur naturally, and/or which are provided in an isolated and/or purified form not found in nature.

Consensus peptides of GPR polypeptides of the present invention may include peptides which are distinct from known GPR sequences in critical structural features, but which are derived from consensus sequences of homologous GPR transmembrane domains 1-7, e.g., as presented in FIG. 8A-G (SEQ ID NOS:6-79). Such consensus peptides may be derived by molecular modeling, optionally combined with hydrophobicity analysis and/or fitting to model helices, as non-limiting examples. Such modeling can be accomplished according to known method steps using known modeling algorithms, such as, but not limited to, ECEPP, INSIGHT, DISCOVER, CHEM-DRAW, AMBER, FRODO and CHEM-X. Such algorithms compare transmembrane domains between related G-protein coupled receptors, determine probable energy-miminized structures and define alternative consensus polypeptide fragments.

Such consensus peptides or fragments of GPRs may then be synthesized or produced recombinantly, in order to provide GPR polypepnides according to the present invention which mimic, modulate or inhibit binding of ligands to G-protein coupled receptors. GPR ligands, in the context of the present invention, refer to biological molecules that bind GPRs in vitro, in situ or in vivo, and may include hormones, neurotransmitters, viruses or receptor binding domains, thereof, opsins, rhodopsins, nucleosides, nucleotides, coagulation cascade factors, odorants or pheremones, toxins, colony stimulating factors, platelet activating factors, neuroactive peptides, neurohumors, or any biologically active compounds, such as drugs or synthetic or naturally occurring compounds.

The following non-limiting examples of consensus peptides of GPRs of the present invention are provided by way of guidance and not by way of limitation. In GPR polypeptides of the present invention, one or more, preferably 4-10, Asp and/or Lys residues may additionally be incorporated at the carboxy and/or amino terminal ends in order to provide expected helix forming effects of the helix dipole effect, e.g., as described in Baldwin et al Biochem. 28:2130 (1989); Baldwin et al Proc. Nat'l Acad. Sci. USA 84:8898 (1987); and Baldwin et al Proc. Nat'l Acad. Sci. USA 86:5286 (1989), which references are entirely incorporated herein by reference.

As a non-limiting example of GPR polypeptide of the present invention, dopamine receptor transmembrane fragments of D.sub.2 transmembrane domain (e.g., domain III) as presented in FIG. 2 (SEQ ID NO:2) or a consensus sequence as presented in FIG. 3 (SEQ ID NO:3), e.g., of D.sub.2 domains I-VII. Additionally or alternatively a consensus sequence may include less than 20 amino acids, such as 15 amino acids corresponding to a transmembrane domain, such as a D.sub.2 receptor domain, as presented in FIG. 4 (SEQ ID NO:4) as polypeptide IV, which is smaller than the length required by spanning an average lipid bilayer of a cell membrane.

However, in the context of the present invention, GPR polypeptides of greater than 15-20 amino acids are preferred such that the GPR polypeptides are able to span the lipid bilayer.

Another non-limiting example of a GPR polypeptide using dopamine receptor transmembrane domains is a consensus sequence of two or more GPR receptors, such as the dopamine D.sub.1 and D.sub.2 receptors. A non-limiting example of such a consensus GPR polypeptide is presented in FIG. 5 (SEQ ID NO:5).

Additionally, modified amino acids or chemical derivatives of amino acids of consensus or fragments of GPRs proteins, according to the present invention may be provided, which polypeptides contain additional chemical moieties or modified amino acids not normally a part of the protein. Covalent modifications of the peptide are thus included within the scope of the present invention. Such modifications may be introduced into a GPR polypeptide by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- or L-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylainines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, non-acidic amino acids, of C1--C20.

Acidic amino acids call be substituted with non-carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfated (e.g., -SO.sub.3 H) threonine, serine, tyrosine.

Other substitutions may include unnatural hyroxylated amino acids may made by combining "alkyl" (as defined and exemplified herein) with any natural amino acid. Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is define as above. Nitrile derivatives (e.g., containing the CN-moiety in place of COOH) may also be substituted for asparagine or glutamine, and methionine sulfoxide may be substituted for methionine. Methods of preparation of such peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage in any of the GPR polypeptides can be replaced by a ketomethylene moiety, e.g. (--C(.dbd.O)-- CH.sub.2 --) for (--(C.dbd.O)--NH--). Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

In addition, any amino acid representing a component of the said peptides can be replaced by the same amino acid but of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D- amino acid but which can additionally be referred to as the R- or the S-, depending upon its composition and chemical configuration. Such derivatives have the property of greatly increased stability co degradation by enzymes, and therefore are advantageous in the formulation of compounds which may have longer in vivo half lives, when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

Additional amino acid modifications of amino acids of GPR polypeptides of to the present invention may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se is well-known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.

Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers, according to known method steps. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidateyieldphotoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 (which are herein incorporated entirely by reference), may be employed for protein immobilization.

Other modifications of GPR polypeptides of the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.

Such derivatized moleties may improve the solubility, absorption, permeability across the blood brain barrier biological half life, and the like. Such moleties or modifications of GPR polypeptides may alternatively eliminate or attenuate any possible undesirable side effect of the protein and the like. Moleties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

Such chemical derivatives of GPR polypeptides also may provide attachment to solid supports, including but not limited to, agarose, cellulose, hollow fibers, or other polymeric carbohydrates such as agarose, cellulose, such as for purification, generation of antibodies or cloning; or to provide altered physical properties, such as resistance to enzymatic degradation or increased binding affinity or modulation for GPRs, which is desired for therapeutic compositions comprising GPR polypeptides, antibodies thereto or fragments thereof. Such peptide derivatives are well-known in the art, as well as method steps for making such derivatives using carbodiimides active esters of N-hydroxy succinimmide, or mixed anhydrides, as non-limiting examples.

Variation upon consensus peptide sequences of GPR polypeptide of the present invention may also include: the addition of one, two, three, four, or five lysine, arginine or other basic residues added to the --COOH terminal end of the peptide; and/or one, two, three, four, or five glutamate or aspartate or other acidic residues added to the amino terminal end of the peptide, where "acidic" and "basic" are as defined herein. Such modifications are well known to increase the .alpha.-helical content of the peptide by the "helix dipole effect". They also can provide enhanced aqueous solubility of the peptide. See, e.g., Baldwin et al., supra.

As another non-limiting example of a GPR polypeptide of the present invention, serotonergic receptors (5-HT) consensus sequences may be determined using presently known 5-HT sequences and include, e.g., as consensus peptides of TM3, TM5 and TM7, respectively:

Such non-naturally occurring consensus sequences may also be further modified according to known method steps to provide additional consensus peptides with substituted amino acids to increase or decrease .alpha.-helical propensity and/or solubility (e.g., hydrophilicity). As a non-limiting example, 5-HT consensus peptide (1) above may be modified according to the present invention to have increase helical propensity and increased aqueous solubility as follows:

wherein, e.g., smaller, non-polar residues replace either larger, more polar residues (e.g., Ala for Ile or Val) or larger aromatic residues (e.g., Ala for Phe).

Another non-limiting, illustrative example of consensus GPR polypeptides of the present invention are those for adrenergic receptors, are the following:

An example of the consensus GPR polypeptide for domain VII across all presently known adrenergic receptors is as follows:

An example of a consensus GPR polypeptide for domain V across all adrenergic receptors is as follows:

Examples of a consensus GPR polypeptide for domain III across all adrenergic receptors are as follows:

An example of a consensus GPR polypeptide for domains III, V, and VII of all adrenergic receptors is as follows:

wherein variations and substitutions of amino acids may be made as described herein.

Non-limiting examples of consensus GPR polypeptides for transmembrane domain III across several or many, such as 1-500, or any range or value therein, G-protein receptors are as follows:

TM3-(1) YAIFVLYASAWLSFLNCPFIVTLNI (SEQ ID N0:96)

TM3-(2) YAIFVLYATAWLSFLNCPFIVTLNI(SEQ ID NO:97)

TM3-(3) YAIFVLYATAWLTFLNCPFIVTLNI(SEQ ID NO:98)

TM3-(4) YAIFVLYASAWLTFLNCPFIVTLNI(SEQ ID NO:99)

TM3-(5) WAIFVLYASAWLSFLNCPFIVTLNI(SEQ ID NO:100)

TM3-(6) WAIFVLYATAWLSFLNCPFIVTLNI (SEQ ID NO: 101)

TM3-(7) WAIFVLYATAWLTFLNCPFIVTLNI(SEQ ID NO:102)

TM3-(8) WAIFVLYASAWLTFLNCPFIVTLNI (SEQ ID NO: 103)

TM3-(9) YAVFVLYASAWLSFLNMPFIVTLNI(SEQ ID NO: 104)

TM3-(10) YAVFVLYATAWLSFLNMPFIVTLNI (SEQ ID NO: 105)

TM3-(11) YAVFVLYATAWLTFLNMPFIVTLNI (SEQ ID NO: 106)

TM3-(12) YAVFVLYASAWLTFLNMPFIVTLNI (SEQ ID NO: 107)

TM3-(13) YAIFVLYASAWLSFLNCVTASIPFIVTLNI (SEQ ID NO: 108)

TM3-(14) YAIFVLYASAWLSFLNCTSSIVVTASIVTLNI (SEQ ID NO: 109)

TM3-(15) YAIFVLYASAWLSFLNVTLNICTSSIV (SEQ ID NO: 110)

TM3-(16) YAIFVLYASAWLSFLNTASILNLMFIVTLNI (SEQ ID NO: 111)

TM3-(17) YAIFVLYASAWLSFLNMASILNLPFIVTLNI (SEQ ID NO: 112)

TM3-(18) YAIFVLYASAWLSFLNSGILLLAPFIVTLNI (SEQ ID NO: 113)

TM3-(19) YAIFVLYASAWLSFLNMSGILLLAPFIVTLNI (SEQ ID NO: 114)

TM3-(20) YAIFVLYASAWLSFLNSELSVYTLTVCPFIVTLNI (SEQ ID NO: 115)

TM3-(21) YAIFVTYASAWLS FLNMSELSVYTLTVPFIVTLNI (SEQ ID NO: 116)

TM3-(22) YAIFVLYASAWLASELSVYTLTVSFLNCPFIVTLNI (SEQ ID NO: 117)

TM3-(23) YAIFVLYASAWLASELSVYTLTVPFIVTLNI (SEQ ID NO: 118)

TM3-(24) YAIFVLYASAWLSFLASELSVYASELSSTLTTVNMPFIVTLNI (SEQ ID NO: 119)

TM3-(25) YAIFVLYASAWLSFLNGGEIALWSLCPFIVTLNI (SEQ ID NO: 120)

TM3-(26) YAIFVLYASAWLSFLNGGEIALWSLIVTLNI (SEQ ID NO: 121)

TM3-(27) YAIFVLYASAWLGGEIALWSLNCPFIVTLNI (SEQ ID NO: 122)

TM3-(28) YAIFVLYAGGEIALWSLSFLNCPFIVTLNI (SEQ ID NO: 123)

TM3-(29) YAIFVLYASAWLSFFFLLFGYLGNFLLNCPFIVTLNI (SEQ ID NO: 124)

TM3-(30) YAIFVLYASAWLFFFLLFGYLGNFLLPFIVTLNI (SEQ ID NO: 125)

TM3-(31) YAIFVLYASAWLSFLNTACFYVAITASLCFITEIALIPFIVTLNI (SEQ ID NO: 126)

TM3-(32) YAIFVLYASAWLTACFYVAITASLCFITEIALICPFIVTLNI (SEQ ID NO: 127)

TM3-(33) YAIFVLYATACFYVAITASLCFITEIALISFLNCPFIVTLNI (SEQ ID NO: 128)

TM3-(34) YAITACFYVAITASLCFITEIALIASAWLSFLNCPFIVTLNI (SEQ ID NO: 129)

TM3-(35) YAIFVLYATACFYVAIITEIALISAWLSFLNCPFIVTLNI (SEQ ID NO: 130)

TM3-(36) YAIFVLYASAWLSFLNACFYICLFAGVCFLIPFIVTLNI (SEQ ID NO: 131)

TM3-(37) YAIFVLYASAWNACFYICLFAGVMFLILSFLNCPFIVTLNI (SEQ ID NO:132)

TM3-(38) YAIFVLYFYICLFAGVCFLIASAWLSFLNCPFIVTLNI (SEQ ID NO: 133)

TM3-(39) YAIFVLYASVDAVNMFTSAWLSFLNCPFIVTLNI (SEQ ID NO: 134)

TM3-(40) YAIFSVDAVNMFTVLYASAWLSFLNCPFIVTLNI (SEQ ID NO: 135)

TM3-(41) YAIFVLYASAWLSVDAVNMFTSFLNCPFIVTLNI (SEQ ID NO: 136)

TM3-(42) YAIFVLYASAWLSFLNSVDAVNMFTPFIVTLNI (SEQ ID NO: 137)

TM3-(43) YAIFVLYASAWLSFLNCPFIVSVDAVNMFTTLNI (SEQ ID NO: 138)

TM3-(44) YAIFVLYASAWLSVDMFTSFLNCPFIVTLNI (SEQ ID NO: 139)

TM3-(45) YAISVDAVNMFTFVLYASAWLSFLNCPFIVTLNI (SEQ ID NO: 140)

TM3-(46) YAIFSLSVFSLLAIVLYASAWLSFLNCPFIVTLNI (SEQ ID NO: 141)

TM3-(47) YAIFVLYASLSVFSLLAISAWLSFLNCPFIVTLNI (SEQ ID NO: 142 )

TM3-(48) YAIFVLYASAWLSLSVFSLLAISFLNCPFIVTLNI (SEQ ID NO: 143 )

TM3-(49) YAIFVLYASAWLSFLSLSVFSLLAINCPFIVTLNI (SEQ ID NO: 144 )

TM3-(50) YAIFVLYASAWLSFLNPFSLSVFSLLAIIVTLNI (SEQ ID NO: 145 l )

TM3-(51) YAIFVLYATAWLTFLNCVTATIPFIVTLNI (SEQ ID NO: 146)

TM3-(52) YAIFVLYATAWLSFLNCTSSIVVTATIVTLNI (SEQ ID NO: 147)

TM3-(53) YAIFVLYATAWLSFLNVTLNICTTTIV (SEQ ID NO: 148)

TM3-(54) YAI FVLYATAWLTFLNTATILNLMFIVTLNI (SEQ ID NO: 149)

TM3-(55) YAIFVLYATAWLSFLNMATILNLPFIVTLNI (SEQ ID NO: 150)

TM3-(56) YAIFVLYATAWLTFLNSGILLLAPFIVTLNI (SEQ ID NO: 151)

TM3-(57) YAIFVLYASAWLTFLNMTGILLLAPFIVTLNI (SEQ ID NO: 152)

TM3-(58) YAIFVLYASAWLTFLNTELTVYTLTVCPFIVTLNI (SEQ ID NO: 153)

TM3-(59) YAIFVLYASAWLTFLNMTELTVYTLTVPFIVTLNI (SEQ ID NO: 154)

TM3-(60) YAIFVLYATAWLATELTVYTLTVTFLNCPFIVTLNI (SEQ ID NO: 155)

TM3-(61) YAIFVLYASAWLATELSVYTLTVPFIVTLNI (SEQ ID NO: 156)

TM3-(62) YAIFVLYATAWLSFLATELSVYASELSTTLTTVNMPFILNI (SEQ ID NO: 157)

TM3-(63) YAIFVLYATAWLSFLNGGEIALWTLCPFIVTLNI (SEQ ID NO: 158)

TM3-(64) YAIFVLYASAWLTFLNGGEIALWTLIVTLNI (SEQ ID NO: 159)

TM3-(65) YAIFVLYASAWLGGEIALWTLNCPFIVTLNI (SEQ ID NO: 160)

TM3-(66) YAIFVLYAGGEIALWTLSFLNCPFIVTLNI (SEQ ID NO: 161)

TM3-(67) YAIFVLYATAWLSFFFLLFGYLGNFLLNCPFIVTLNI (SEQ ID NO: 162)

TM3-(68) YAIFVLYATAWLFFFLLFGYLGNFLLPFIVTLNI (SEQ ID NO: 163)

TM3-(69) YAIFVLYATAWLTFLNTACFYVAITASLCFITEIALIPFIVTLNI (SEQ ID NO: 164)

TM3-(70) YAIFVLYATAWLTACFYVAITATLCFITEIALICPFIVTLNI (SEQ ID NO: 165)

TM3-(71) YAIFVLYATACFYVAITATLCFITEIALISFLNCPFIVTLNI (SEQ ID NO: 166)

TM3-(72) YAITACFYVAITASLCFITEIALIATAWLTFLNCPFIVTLNI (SEQ ID NO: 167)

TM3-(73) YAIFVLYATACFYVAIITEIALITAWLTFLNCPFIVTLNI (SEQ ID NO. 168)

TM3-(74) YAIFVLYASAWLTFLNACFYICLFAGVCFLIPFIVTLNI (SEQ ID NO: 169)

TM3-(75) YAIFVLYASAWNACFYICLFAGVMFLILTFLNCPFIVTLNI (SEQ ID NO: 170)

TM3-(76) YAIFVLYFYICLFAGVCFLIATAWLTFLNCPFIVTLNI (SEQ ID NO: 171)

TM3-(77) YAIFVLYATVDAVNMFTTAWLTFLNCPFIVTLNI (SEQ ID NO: 172)

TM3-(78) YAIFTVDAVNMFTVLYATAWLTFLNCPFIVTLNI (SEQ ID NO: 173)

TM3-(79) YAIFVLYATAWLTVDAVNMFTSFLNCPFIVTLNI (SEQ ID NO: 174)

TM3-(80) YAIFVLYATAWLSFLNTVDAVgMFTPFIVTLNI (SEQ ID NO: 175)

TM3-(81) YAIFVLYASAWLTFLNCPFIVSVDAVNMFTTLNI (SEQ ID NO: 176)

TM3-(82) YAIFVLYATAWLSVDMFTTFLNCPFIVTLNI (SEQ ID NO: 177)

TM3-(83) YAISVDAVNMFTFVLYATAWLSFLNCPFIVTLNI (SEQ ID NO: 178)

TM3-(84) YAIFVLYASLTVFSLLAISAWLTFLNCPFIVTLNI (SEQ ID NO: 179)

TM3-(85) YAIFVLYASAWLTLSVFTLLAISFLNCPFIVTLNI (SEQ ID NO: 180)

TM3-(86) YAIFVLYASAWLTFLSLSVFTLLAINCPFIVTLNI (SEQ ID NO: 181)

TM3-(87) YAIFVLYASAWLTFLNPFSLSVFSLLAIIVTLNI (SEQ ID NO: 182)

TM3-(88) YAIFVLYASAWLSFLNLGGVTASFTASVGPFIVTLNI (SEQ ID NO: 183)

TM3-(89) YAIFVLYASAWLSFLNLGGVTASFTASVGVTLNI (SEQ ID NO: 184)

TM3-(90) YAIFVLLGGVTASFTASVNYASAWLSFLNCPFIVTLNI (SEQ ID NO: 185)

TM3-(91) YAIFVLYAIFFFLLFSAWLSFLNCPFIVTLNI (SEQ ID NO: 186)

TM3-(92) YAIFVLYASAWLSFLNCPFIVTLNIIFFFLLFIVTLNI (SEQ ID NO: 187)

TM3-(93) YAIFVLYASAWIFFFLLFLSFLNCPFIVTLNI (SEQ ID NO: 188)

TM3-(94) YAIFVLYASAWLFFTVLASELSVYTLTVSFLNCPFIVTLNI (SEQ ID NO: 189)

TM3-(95) YAIFVLYASAWLSFLFATLGGEIALCPFIVTLNI (SEQ ID NO: 190)

TM3-96) YAIFVLYAFATLGGEIkLSAWLSFLNCPFIVTLNI (SEQ ID NO: 191)

TM3-(97) YAIFFTVLASELSVYTLTVYASAWLSFLNCPFIVTLNI (SEQ ID NO: 192)

TM3-(98) YAIFFPIAALFAIASAWLSFLNCPFIVTILNI (SEQ ID NO: 193)

TM3-(99) YAIFVLYASAWLSFFPIAALFASIPFIVTLNI (SEQ ID NO: 194)

TM3-(100) YAIFVLYASAWLSFLNCPFFPIAALFASILNI (SEQ ID NO: 195)

TM3-(101) YAIFVLYASAWLSLDVLFSTASIMHLSFLNGGEIALWSLIVTLNI (SEQ ID NO: 196)

TM3-(102) YAIFVLYASLDVLFSTASIMHLIALWSLNCPFIVTLNI (SEQ ID NO: 197)

TM3-(103) YAIFVLYAGGEIALWSLSFLNSLDVLFSTASIMLPFIVTLNI (SEQ ID NO: 198)

TM3-(104) YAIFVLYASAWLSFFDVLFSTASIMLFGYLGNFLLNCPFIVTLNI (SEQ ID NO: 199)

TM3-(105) YAIFVLYASAWLFFFLLFGYLSLDVLFSTASIMHLGNFLLPFIVTLNI(SEQ ID NO: 200)

TM3-(106) YAIFVLYASAWLSFLNTACFYVAITASLSLMHLFITEIALIPFIVTLNI (SEQ ID NO: 201)

TM3-(107) YASLDVLFSTAIMHLSAWLTACFYVAITASLCFITEIALICPFIVTLNI (SEQ ID NO: 202)

TM3-(108) YAIFVLYATACFYVAITASLSFLNCPFIVTLNISLDVLFSTASIMHL(SEQ ID NO:203)

TM3-(109) YAITACFYVAITASLCFITEIALIASAWLSFLNCPFIVTLNI (SEQ ID NO: 204)

TM3-(110) YAIFVLYATACFYSTASILNLIMHLCAISLVAIITEIALISAWLSFLN(SEQ ID NO:205)

TM3-(111) YAIFVLYASAWLSFLNACFYICLFASILNLIMHLGVCFLIPFIVTLNI (SEQ ID NO:206)

TM3-(112) YAIFVLYASAWNASILNLIMHLCFYICLFAGVMLILSFLNCPFIVTLNI(SEQ ID NO:207)

TM3-(113) YAI FPFVQCWS IFSLVLIAVVLYFYIAGVCFLIASAWLS FLNCPFIVTI (SEQ ID NO: 208)

TM3-(114) PFVQCVSITVSIFSLVLIAVYAIFVLYASVDAVNMFTSAWCPFIVTLNI (SEQ ID NO:209)

TM3-(115) YAIFGDWSSVDAVNMFTVLYASAWLSFLNCPFIVTLNI (SEQ ID NO:210)

TM3-(116) YAIFVLYAGDWSSAWLSVDAVNMFTSFLNCPFIVTLNI (SEQ ID NO: 211)

TM3-(117) YAIFVLYASAWLGDWSSFLNSVDAVLGMFTPFIVTLNI (SEQ ID NO:212)

TM3-(118) YAIFVLYASAWLSFLNCPFIVGDWSSVDAVNMFTTLNI (SEQ ID NO: 213)

TM3-(119) YAIFVLYASAWLGYLGSVDMFTSFLNCPFIVTGDWSLNI(SEQ ID NO:214)

TM3-(120) YAISVDAVNMFTFVLYAGYLGSAWLSFLNCPFIVTLNI (SEQ ID NO:215)

TM3-(121) YAIFSLSVFSLLAIVLAYASAWLGYLGSFLNCPFIVTLNI (SEQ ID NO: 216)

TM3-(122) YAIFVLYAGYLGAGNMDSLSVFSLLAISAWLSFLNCPFIVTLNI (SEQ ID NO:217)

TM3-(123) YAIFVLYASAWLSLSVFGNMSLLAISFLNCPFIVTLNI (SEQ ID NO:218)

TM3-(124) YAIFVLYASAWLSFLSLSVFGGSLLAINCPFIVTLNI (SEQ ID NO: 219)

TM3-(125) YAIFVLYASAWLSFLNPFSLSVFGSLLAIIVTLNI(SEQ ID NO:220)

TM3-(126) YAIFVLYATAWLTFLSLANCVTATIPFIVTLNI (SEQ ID NO: 221)

TM3-(127) YAIFVLYATAWLSFLNCTSLASSIVVTATIVTLNI (SEQ ID NO: 222)

TM3-(128) YAIFVLYATAWLSFLNVTLNISLACTTTIV(SEQ ID NO: 223)

TM3-(129) YAIFVLYATAWLTFLNTATILSLANLMFIVTLNI(SEQ ID NO:224)

TM3-(130) YAIFVLYATAWLSFLNMATILNLPFSVDAVIVTLNI (SEQ ID NO:225)

Recently discovered G-proteins also can be used according to the presently claimed invention to provide GPR polypeptides of the present invention, based on the teaching and guidance presented herein. Exampled of such GPR polypeptides of the present invention may include, as non-limiting examples, GPR polypeptides corresponding to transmembrane domain III, e.go, as follows:

TM3-(131) ISTMYTVTGRWTLGQVVCDFWLSSDITCCTASILHLCVIAL (SEQ ID NO: 226)

TM3-(132) ILYGYRWPLPSKLCAVWIYLDVLFSTASIMHLCAISL (SEQ ID NO: 227 )

TM3-(133) IIYIVMDRWKLGYFLCEVWLSVDMTCCTCSILHLCVIAL (SEQ ID NO :228)

TM3-(134) IADKTVRVAMGAENDLGYNFRSDDVCGHCWQWYCSL (SEQ ID NO: 229)

TM3-(135) ILNYWPFGLALCHFVNYSQAVSVLVSAYTLVAISI (SEQ ID NO:230)

TM3-(136) ILGRWEFGIHLCKLWLTCDVLCCTSSILNLCAIALD (SEQ ID NO: 231)

TM3-(137) IMASVMHRHCLPLIGICLSSERHCLVSIFVELGAL (SEQ ID NO:232)

Further non-limiting examples of consensus GPR polypeptides for transmembrane domain III of several or many, such as 1-500, or any range or value therein, more recently discovered G-protein receptors are as follows:

TM3-(138) YAIFVLYASAWLSFLNCPFISILHLCVIALVTLNI (SEQ ID NO:233)

TM3-(139) YAIFVLYATAWLSFLNCPFISILNLCAIALDVTLNI (SEQ ID NO:234)

TM3-(140) YAIFVLYATAWLTFLNCPFISIFVELGALVTLNI (SEQ ID NO: 235 )

TM3-(141) YAIFVLYASAWLTFLNCPFISIFVELSIMHLCAISLGALVTLNI (SEQ ID NO: 236)

TM3-(142) WAIFVLYAILGRWEFGIHLCKLWLTSAWLSIMHLCAISLSFLNCPFIVTLNI (SEQ ID NO:237)

TM3-(143) WAIFVLYAILGRWEFGIHLCKLWLTTAWLSIMHLCAISLSFLNCPFIVTLNI (SEQ ID NO: 238)

TM3-(144) WAIFVLYATAWLTFLNCPFSIMHLCAISLIVTLNI (SEQ ID N0:239)

TM3-(145) WAIFVLYASAWLTFLNCPFISIMHLCAISLVTLNI (SEQ ID NO: 240)

TM3-(146) YAVFVLYASAWLSFLNMSIMHLCAISLPFIVTLNI (SEQ ID NO: 241)

TM3-(147) YAVFVLYATAWLSFLBTMPFSILNLCAIALDIVTLNI(SEQ ID NO:242)

TM3-(148) YAVFVLYATAWLSILNLCAIALDTFLNMPFIVTLNI (SEQ ID NO:243)

TM3-(149) YAVFVLYASILNLCAIALDSAWLTFLNMPFIVTLNI (SEQ ID NO:244)

TM3-(150) YAFVLYASAWLSFLNCVTASIPFCLVSIFVELGALIVTLNI (SEQ ID NO: 245 )

TM3-(151) YAIFVLYASAWLSFLNCLVSIFVELGALIVVTASIVTLNI (SEQ ID NO: 246)

TM3-(152) YAIFVLYASAWLSFLNVTLNCLVSIFVELGALII (SEQ ID NO: 247)

TM3-(153) YAIFVLYASAWLSFLNTASILNLMFICLVSIFVELGALVTLNI (SEQ ID NO: 248)

TM3-(154) YAIFVLYASAWLSFLNMASILNLPFCLVSIFVELGALVTLNI (SEQ ID NO:249)

TM3-(155) YAIFVLYASAWLSFLNILGRWEFGIHLCKLWLTCDVLCCTSSGILLLAPFIVTLNI (SEQ ID NO:250)

TM3-(156) YAIFVLYASAWLSFLNMILGRWEFGIHLCKLWLTCDVLCCTSSGILLLAPFIVTLNI (SEQ ID NO:251)

TM3-(157) YAIFVLYASAWLILGRWEFGIHLCKLWLTCDVLCCTSSFLNSELSVYTLTVCPFIVTLNI (SEQ ID NO: 252 )

TM3-(158) YAIFVLYAILGRWEFGIHLCKLWLTCDVLCCTSSAWLSFLNMSELSVYTLTVPFIVTLNI (SEQ ID NO:253)

TM3-(159) YAIFVLYASAWLASRWPLPLSVYTLTVSFLNCPFIVTLNI (SEQ ID NO: 254 )

TM3-(160 ) YAIFVLYASAWLASELILYYWRWPLPCLHDLVWLCTCSILHLCVIALSVYTLTVPFIVTLNI (SEQ ID NO:255)

TM3-(161) YAIFVLYASAWLSFLASELSVYASELSSTLHDLVWLWLDVFCVIALTTVNMPFIVTLNI (SEQ ID NO:256)

TM3-(162) YAIFVLYASAWLSFLNGGEIALWSLCPFIILYYWRWPLPCLHDLVSILHCVIALVTLNI (SEQ ID NO:257)

TM3-(163) YVWLWLDVFCCTCSILHLCVIALFVLYASAWLSFLNGGEIALWSLIVTLNI (SEQ ID NO: 258)

TM3-(164) YAIFVLYASAWLAIILYYWRWPLPCLHDLGGEIALWSLNCPFIVTLNI(SEQ ID NO:259)

Non-limiting examples of consensus GPR polypeptides for domain V across several or many, such as 1-500, or any range or value therein, G-protein receptors are as follows:

TM5-(1) CDVFVFVDIMLCTASIFNLCAISVG(SEQ ID NO:260)

TM5-(2) YAIFVLYDIPILCTASIFNLCAISVG(SEQ ID NO:261)

TM5-(3) DYAIFVFVDIFiLMTASIFNLMAISVG(SEQ ID NO:262)

TM5-(4) DYAIFVFVDIMLHTTASTIFNLMATITVG (SEQ ID NO: 263)

TM5-(5) CDVAVVYSSDIMLFYVCTASIFSSNLCAISSVG(SEQ ID NO:264)

TM5-(6) FLFCSLGSFYIPIAVILVDIMLCTASIFNLCAISVG(SEQ ID NO:265)

TM5-(7) YAIFVLYDFLFCSLGSFYIPIAVILIMLCTASIFNLCAISVG (SEQ ID NO: 266)

TM5-(8) DYAIFVFVDIMLMTASIFLFCSLGSFYIPIAVILISVG(SEQ ID NO:267)

TM5-(9) DYAIFVFVDIMLHTTASTIFNLMAFLFCSLGSFYIPIAVILTITVG (SEQ iD NO: 268)

TM5-(10) CDVAVVYSSDIMLFYVCTASIFSSNLFLFCSLGSFYCAISSVG(SEQ ID NO:269)

TM5-(11) CDVFVFVDIMLCTASIFNWYILSSIGSFFAPCLILLVYLLCISVG (SEQ ID NO: 270)

TM5-(12) YAIFVLYDIPILCTASIFNLCAIWYILSSIGSFFAPCLILLVYLSVG (SEQ ID NO: 271)

TM5-(13) DYAIFVFVDIWYILSSIGSFFAPCLILLVYLASIFNLMAISVG(SEQ ID NO:272)

TM5-(14) DYAIWYILSSIGSFFAPCLILLVYLIMLHTTASTIFNLMATITVG (SEQ ID NO: 273 )

TM5-(15) CDVAVVYSSDIMLFYVCWYILSSIGSFFAPCLILLVYLSSNLCAISSVG(SEQ ID NO:274)

TM5-(16) CDVFVFVDIMLCTASIFWYVISSSIGSFFAPCLINHLIrYNLCAISVG(SEQ ID NO:275)

TM5-(17) YAIFVLYDIMLCTASIFNLCAIWlNSISSSIGSFFAPCLINHLVYSVG (SEQ ID NO: 276)

TM5-(18) DYAIFVFVWYVISSSIGSFFAPCLINHLVYDIMLMTASIFNLMAISVG (SEQ ID NO:277)

TM5-(19) DYAIFVFVDIMLHTTASTIFWYVISSSIGSFFAPCLINHLVYTVG (SEQ ID NO: 278)

TM5-(20) CDVAVVYSSDIMLFYVCTASIFSWYVISIGSFFAINHLVYNLCAISSVG (SEQ ID NO: 279 )

TM5-(21) CDVFVFVDIMLCTASIFNLCAITYAISSSVISFYIPVAILVTYT(SEQ ID NO:280)

TM5-(22) YAIFVLYDIMLCTATYAISSSVISFYIPVAILVTYTSIFNLCAISVG (SEQ ID NO: 281)

TM5-(23) DYAIFVFIVDIMILMTATYAISSSVISFYIPVAILYTISVG(SEQ ID NO:282)

TM5-(24) TYAISSSVISFYIPVATDYAIFVFVDIMLHTTASTIFNLMATITVG (SEQ ID NO: 283 )

TM5-(25) CDVAVVYSSDIMLFYVCTATYAISSSVISFYIPVAILVTYTSSVG(SEQ ID NO:284)

TM5-(26) CDVFVFVDFVIYSSWSFYLPFGIFfVLVYACTASIFNLCAISVG (SEQ ID NO:285)

TM5-(27) YAIFVLYDFVIYSSWSFYLPFGVTVLVYASIFNLCAISVG (SEQ ID NO :286)

TM5-(28) DYAIFVFVDFVIYSSWSFYLPFGVTVLVYATASIFNLMAISVG(SEQ ID NO:287)

TM5-(29) DYAIFVFVDFVIYSSVIZSFYLPFGVTVLVYAHTTASTIFNLMATITVG (SEQ ID NO: 288)

TM5-(30) CDVAVVYSSDFVIYSSWSFYLPFGVTVYVCTASIFSSNLCAISSVG (SEQ ID NO: 289 )

TM5-(31) CDVFVFVDIMLCTASYTIYSTCGAFYIPSVLLIILYGNLCAISVG (SEQ ID NO: 290)

TM5-(32) YAIFVLYDIMLCTASYTIYSTCGAFYIPSVLLIILYGNLCAI SVG (SEQ ID NO: 291)

TM5-(33) DYAIFVFVDIMLMTASYTIYSTCGAFYIPSVLLIILYGNLMAISVG (SEQ ID NO: 292)

TM5-(34) DYAIFVFVDIMLHTTASYTIYSTCGAFYIPSVLLIILYGMATITVG (SEQ ID NO: 293)

TM5-(35) CDVAVVYSSDIMSYTIYSTCGAFYIPSVLLIILYGIFSSNLCAISSVG (SEQ ID NO: 294)

TMS- (36) CDVFVFFVLIGSFVAVDIMLCTASIFNLCAISVG (SEQ ID NO:295 )

TM5-(37) YAIFVLYFVLIGSFVADIMLCTASIFNLCAISVG (SEQ ID NO: 296)

TM5-(38) DYAIFVFVFVLIGSFVADIMLMTASIFNLMAISVG (SEQ ID NO: 297)

TMS- (39) DYAI FVFVFVLIGSFVADIMLHTTASTIFNLMATITVG (SEQ ID NO: 298)

TM5-(40) CDVAVVYSSFVLIGSFVADIMLFYVCTASIFSSNLCAISSVG (SEQ ID NO: 299)

TM5-(41) CDVFVFVDIMLCFFIPTLIMVITYFNLCAISVG (SEQ ID NO: 300)

TM5-(42) YAIFVLYDIMLCFFIPTLIMVITYFFNLCAISVG (SEQ ID NO: 301)

TM5-(43) DYAIFVFVDIMLMFFIPTLIMVITYFNLMAISVG (SEQ ID NO: 302)

TM5-(44) DYAIFVFVDIMLHTFFIPTLIMVITYFNIIMATITVG (SEQ ID NO: 303)

TM5-(45) CDVAVVYSSDIMLFYVCFFIPTLIMVITYFSSNLCAISSVG (SEQ ID NO: 304)

TM5-(46) CDVVYGLVDGLVTFYLPLLIMCITYYDIMLCTASIFNLCAI SVG (SEQ ID NO: 305)

TM5-(47) YAIVYGLVDGLVTFYLPLLIMCITYYDIMLCTASIFNLCAISVG (SEQ ID NO: 306)

TM5-(48) DYAIVYGLVDGLVTFYLPLLIMCITYYDIMLMTASIFNLMAISVG (SEQ ID NO: 307)

TM5-(49) DYAIVYGLVDGLVTFYLPLLIMCISSDIMLHTTASTIFNLMATITVG (SEQ ID NO: 308)

TM5-(50) CDVVYDGLVTFYLPLLIMCITYYDIMLFYVCTASIFSSNLCAISSVG (SEQ ID NO: 309)

TM5-(51) CDVFVFVDIMLLVIFLGLVIVIPFVLIIVSYASIFNLCAISVG (SEQ ID NO: 310)

TM5-(52) YAIFVLYDIMLLVIFLGLVIVIPFVLIIVSYAIFNLCAISVG (SEQ ID NO: 311)

TM5-(53) DYAIFVFVDIMLMLVIFLGLVIVIPFVLIIVSYAIFNLMAISVG (SEQ ID NO: 312)

TM5-(54) DYAIFVFVDIMLHTLVIFLGLVIVIPFVLIIVSYAIFNLMATITVG (SEQ ID NO: 313)

TM5-(55) CDVAVVYSSDIMLFLVIFLGLVIVIPFVLIIVSYAIFSSNLCAISSVG (SEQ ID NO: 314)

TM5-(56) CDVFVFVDIMLCTALMIYILGGLIIIIPFLLIVMSYVSIFNLCAISVG (SEQ ID NO: 315)

TM5-(57) YAIFVLYDIMLCTALMIYILGGLIIIIPFLLIVMSYVSIFNLCAISVG (SEQ ID NO:316)

TM5-(58) DYAIFVFVDIMLMTASIFNLMIYILGGLIIIIPFLLIVMSYVLMAISVG (SEQ ID NO: 317)

TM5-(59) DYAI FVFVDIMLHTTASTI LMIYI LGGLI II IPFLLIVMSYVITVG (SEQ ID NO: 318)

TM5-(60) CDVAVVYSSDIMLFYVCTAYILGGLIPFLLIVMTYVS I FTNLCAI S SVG ((SEQ ID NO: 319)

TM5-(61) CDVFVFVDIMLCTASIFNLLMIHIMEVIIIVIPFVLIVISYACAISVG (SEQ ID NO: 320)

TM5-(62) YAIFVLYDIMLCTASIFNLLMIHIMEVIIIVIPFVLIVISYACAISVG (SEQ ID NO: 321)

TM5-(63) DYAIFVFVDIMLMTASIFIIHIEVIIIVIPFIIVISYAISVG (SEQ ID NO: 322)

TM5-(64) DYAIFVFVDIMLHTTASTILMIHIMEVIIIVIPFVLIVISYAITVG (SEQ ID NO: 323)

TM5-(65) CDVAVVYSSDIMLFYVCTASIFLMIHIMEVIIIVIPFVLIVISYAAISSVG (SEQ ID NO: 324)

Non-limiting examples of longer consensus GPR polypeptides for domain V across several or many, such as 1-500, or any value or range therein, G-protein receptors are as follows:

TM1-(1)

TM1NWPALSIWIIINTIGGNILVIMAVSIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNV FFVW IGYVCSSSLGINPVIIYTLF (SEQ ID NO: 325)

TM1- (2)

NWPALSIWIIINTIGGNILVIMAVTIYTTLDVMLCTATILNLLISLFVLIGTFVAFFIPLTIMVITYFLFNVFFV WIGY VCTTTLGINPVIIYTLF (SEQ ID NO: 326)

TM1-(3)

NWPALTIWIIIBITIGGNILVIMAVSIYTTLDVMLCTATILNLLITLFVLIGTFVAFFIPLTIMVITYFLFNVFF VWIGY VCSTSLGINPVIIYTLF (SEQ ID NO: 327)

TM1- (5)

NWPALTIWIIINTIGGNILVIMAVTIYTTLDVMLCTATILNLLITLFVLGTFVAFFIPLTIMVITYFLFNVFFVW IGY VCTLGINPVIIYTLF (SEQ ID NO: 328)

TM1-(6)

NWKNWSALLTTVVIILTIAGNILVIMAVSSLDVMLCTASILNLLISLFVLGSFVAFFIPLTIMVITYFLFNVFFV WIGY VCSSSLGINPVIIYTLF (SEQ ID NO: 329)

TM1- (7)

ITITVVLAVLILITVAGNVVVCIAVGSIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVF FVWIG YVCSSSLGINPVIIYTLF (SEQ ID NO: 330)

TM1-(8)

TLTLVCLACLUSLTVFGNVLVIIAVFSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVFFVWI GYVCS SSLGINPVIIYTLF (SEQ ID NO:331)

TM1-(9)

TAAIAAAITFLILFTIFGNALVIIAVLSIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNV FFVWI GYVCSSSLGINPVIIYTLF (SEQ ID NO: 332)

TM1-(10)

AISVGLVLGAFILFAIVGNILVILSVANWPALSIVVIIINTIGGNILVIMAVSIYTSLDVLMLCTASILNLLISL FVLIGS FVAFFIPLTIMVITYFLFNVFFVWIGYVCSSSLGINPVIIYTLF (SEQ ID NO: 333)

TM1- (ii)

AALAGALLALAVLATVGGNLLVIVAIASLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVFFVW IGYVC SSSLGINPVIIYTLF(SEQ ID NO: 334)

TM1-(12)

TAGDCLIMLIVLLIVAGNVLVIVAISLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVFFVWIG YVCSS SLGINPVIIYTLF (SEQ ID NO: 335)

TM1-(13)

VITIAVVTAVVSLMTIVGNVLVMISFSIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIILTIMVITYFLFNVF FVWIG YVCSSSLGINPVIIYTLF (SEQ ID NO: 336)

TM1(14)

IATVRGSLSLVTVVGNILVMLSISIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVFFVW IG YVCSSSLGINPVIIYTLF (SEQ ID NO: 337)

TM1-(15)

WFIAFLTGILALVTIIGNILVIVSFSIYTSLDVMLCTASILNLLISLFVLIGSFVAFFIPLTIMVITYFLFNVFF VWIGY VCSSSLGINPVIIYTLF (SEQ ID NO: 338)

Non-limiting examples of longer consensus GPR polypeptides for domain V across several or many, such as 1-500, or any value or range therein, G-protein receptors are as follows:

TM3-(165)

NWPALSIVVIIINTIGGNILVIMAFFAVWIGYVCSSSLGINPVIIYTLF (SEQ ID NO: 339)

TM3-(166)

NWPALSIWIIINTIGGNILVIMAFFACFVLVLTQSSIFSLLAIAIFVLIGSFVAFFIPLTIMVITYFLFNVFFVW IGYV CSSSLGINPVIIYTLF (SEQ ID NO: 340)

TM3-(167)

NWPALSIWIIINTIGGNILVIMAVMVACPVLILTQSSIIALLAIAVSFVAFFIPLTIMVITYFLFNVFFVWIGYV CSSS LGINPVIIYTLF (SEQ ID NO:341)

TM3-(168)

NWPALSIVVIIINTIGGNILVIMAVLWLALDYVASNASVLNLLLISFFFIPLTIMVITYFLFNVFFVWIGYVCSS SLGIN PVIIYTLF(SEQ ID NO: 342)

TM3-(169)

NWPALSIWIIINTIGGNILVIMAVLYVVSNASVMIGLLIISSFVAFFIPLTIMVITYFLFNVFFVWIGYVCSSSL GINPV IIYTLF (SEQ ID NO: 343)

TM3-(170)

NWPALSIWIIINTIGGNILVIMAVLWIAIDYVASNASVLNLLVISFGSFVAFFiPLTIMVITYFLFNVFFVWIGY VCSS SLGINPVIIYTLF (SEQ ID NO: 344)

TM3-(171)

NWPALSIWIIINTIGGNILVIMAVLFPFLQKSSVGITVLNLCALSGSFVAFFIPLTIMVITYFLFNVFFVWIGYV CSSS LGINPVIIYTLF (SEQ ID NO:345)

TM3-(172)

NWPALSIWIIINTIGGNILVIMAVCITYLQYLGINASSCSITAFTIIGSFVAFFIPLTIMVITYFLFNVFFVWIG YVCS SSLGINPVIIYTLF (SEQ ID NO:346)

TM3-(173)

NWPALSIWIIINTIGGNILVIMAVFHNFFPIAKLFASIYSMTAVAGSFVAFFIPLTIMVITYFLFNVFFVWGIGY VCSSS LGINPVIIYTLF(SEQ ID NO: 347)

TM3-(174)

NWPALSIWIIINTIGGNILVIMAVIASASVSFNLYASVFLLTCLSIGSFVAFFIPLTIMVITYFLFNVFFVWIGY VCSS SLGINPVIIYTLF (SEQ ID NO:348)

As another non-limiting, illustrative example of a GPR polypeptide consensus sequences across each individual or different transmembrane domains of 5-HT receptors may be made, such as for 5-HT, as the following:

5HT consensus(4) KNASALLSVIIINSIGGNVVTAVS (SEQ ID NO:89);

5HT consensus(5) YFLMSLAVTDLWSFVMPVSAL (SEQ ID NO:90);

5HT consensus(6) AITKIAITWAISGVSVPFIPVWG (SEQ ID NO:91); and

5HT consensus(7) LGIIFGTFIIIWLPFFITNLVSPI (SEQ ID NO:92);

Wherein variations and substitutions of amino acids may be made as described herein.

Alternatively, 5-HT consensus sequences may be provided as consensus peptides of the present invention as consensus peptides for individual transmembrane domains, such as 5-HT domains III, V and VII, e.g., as follows:

5-HT consensus (8): IWISLDVLFSTASSIMHLCAISL (SEQ ID NO:93)

3-HT consensus (9): GYTIYSTLVTFYIPSVIMVITYG (SEQ ID NO:94)

5-HT consensus (10): LLNFFNWIGYLNSLINPVIYTLF (SEQ ID NO:95)

This invention is also directed to an antibody which binds an epitope specific for a GPR polypeptide of the present invention and the use of such an antibody to detect the presence of, or measure the quantity or concentration of, the GPR protein in a cell, a cell or tissue extract, a biological fluid, an extract thereof, a solution, or sample, in vitro, in situ, or in vivo.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies specific for GPR polypeptide of the present invention, as well as fragments, consensus polypeptides or chemical derivatives thereof.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.

A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milsrein, Nature 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory (1988), the contents of which references are incoporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high miters of tabs in vivo or in si tu makes this the presently preferred method of production.

Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Neuberger et al. , Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533, (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Sahagan et al. , J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Publication No. PCT/US86/02269 (published 7 May 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Sun et al. , Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better etal., Science 40:1041-1043 (1988); and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory (1988)). These references are incorporated entirely herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody carl be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against a GPR polypeptide of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a immunogenic carrier such as keyhole limpet hemocyanin (KLH) or cationized bovine serum albumin and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a GPR polypeptide epitope.

The anti-IdmAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, fcr example, Fab and F(ab').sub.2, which are capable of binding antigen. Fab and F(ab').sub.2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab').sub.2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of a GPR polypeptide according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab').sub.2 fragments).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids. lipids or sugar side chains and have specific three dimentional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. Am antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect a GPR polypeptide in a sample or to detect presence of cells which express a GPR polypeptide of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.

The antibodies (of fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or imnunoelectron microscopy, for in si tu detection of a GPR polypeptide of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and providing the a labeled antibody of the present invention to such a specimen. The antibody (or fragment. ) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a orocedure, it is possible no determine not only the presence of a GPR polypeptide but also its distribution on the examined t. issue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Such assays for a GPR polypepnide of the present invention typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying a GPR polypeptide, and detecting the antibody by any of a number of techniques well-known in the art. See, e.g., Harlow and Lane, supra; Ausubel et al, sunera; and Sambrook et al,

The biological sample may be treated with a solid phase support or carrier, such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers, followed by treatment with a detectably labeled GPR polypeptide-specific antibody. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by known method steps, see, e.g., Harlow, supra; Ausubel, supra; or Sambrook, supra.

By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, polymer test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-GPR polypeptide antibody may be determined according to well known method steps. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. See, e.g., Harlow, supra.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which a GPR polypeptide-specific antibody, anti-idiotype antibody or fragment thereof, can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used detectably label. the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. See, Harlow, supra, Ausubel, supra.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactivity labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA maybe found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work et al., North Holland Publishing Company, N.Y. (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, incorporated entirely by reference herein. The radioactive isotope can be detected by such means as the use of a .gamma.-counter, a scintillation counter or by autoradiography.

It is also possible to label an anti-GPR polypeptide antibody, anti-idiotype antibody or fragment thereof, with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence. Among the most commonly used luorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, commercially available, e.g., from Molecular Probes, Inc. (Eugene, Oreg.).

The antibody can also be detectably labeled using fluorescence emitting metals such as .sup.152 EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).

The antibody also carl be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a hieluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence off a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigert, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigert form the sample by formation of a binary solid phase: antibody-antigen omplex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted arttigon, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigert bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present inventicn, the so-called "simultaneous" and "reverse" assays are used. A "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreated labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays. See, e.g., for the above-mentioned immunological techniques, Harlow, supra; Ausubel et al, supa; and Sambrook et al, supra. GPR polypeptides of the present invention can be made by chemical synthesis or by recombinant methods, wherein chemical synthesis is preferred.

Synthetic production of transmembrane proteins of the present invention

GPR polypeptides, variants and chemical derivatives thereof can be synthesized according to known method steps, including portions of known GPR transmembrane domains, consensus peptides thereof, conservative substitution derivative thereof or functional derivatives thereof.

Chemical polypeptide synthesis is a rapidly evolving area in the art, and methods of solid phase polypeptide synthesis are well-described in the following references, hereby entirely incorporated by reference: (Merrifield, B., J. Amer. Chem. Soc. 5:2149-2154 (1963); Merrifield, B., Science 232:341-347 (1986); Wade, J. D. et al., Biopolymers 25:S21-S-37 (1986); Fields, G. B., Int. J. Polypeptide Prot. Res. 35:161 (1990); MilliGen Report Nos. 2 and 2a, Millipore Corporation, Bedford, Mass., 1987) Ausubel et al, supra, and Sambrook et al, supra.

In general, as is known in the art, such methods involve blocking or protecting reactive functional groues, such as free amino, carboxyl and thio groups. After poiypeptide bond formation, the protective groups are removed ior de-protected). Thus, the addition of each amino acid residue requires several reaction steps lot protecting and deprotecting. Current methods utilize solid phase synthesis, wherein the C-terminal amino acid is covalently linked to an insoluble resin particle large enough to be separated from the fluid phase by filtration. Thus, reactants are removed by washing the resin particles with appropriate solvents using an automated programmed machine. The completed polypeptide chain is cleaved from the resin by a reaction which does not affect polypeptide bonds.

In the more classical method, known as the "tBoc method," the amino group of the amino acid being added to the resin-bound C-terminal amino acid is blocked with tert-butyloxycarbonyl chloride (tBoc). This protected amino acid is reacted with the bound amino acid in the presence of the condensing agent dicyclohexylcarbodiimide, allowing its carboxyl group to form a polypeptide bond the free amino group of the bound amino acid. The amino-blocking group is then removed by acidification with trifluoroacetic acid (TFA); it subsequently decomposes into gaseous carbon dioxide and isobutylene. These steps are repeated cyclically for each additional amino acid residue. A more vigorous treatment with hydrogen fluoride (HF) or trifluoromethanesulfonyl derivatives is common at the end of the synthesis to cleave the benzyl-derived side chain protecting groups and the polypeptide-resin bond.

More recently, the preferred "Fmoc" technique has been introduced as an alternative synthetic approach, offering milder reaction conditions, simpler activation procedures and compatibility with continuous flow techniques. This method was used, e.g., to prepare the peptide sequences disclosed in the present application. Here, the .varies.-amino group is protected by the base labile 9-fluorenylmethoxycarbonyl (Fmoc) group. The benzyl side chain protecting groups are replaced by the more acid labile t-butyl derivatives. Repetitive acid treatments are replaced by deprotection with mild base solutions, e.g., 20% piperidine in dimethylformamide (DMF), and the final HF cleavage treatment is eliminated. A TFA solution is used instead to cleave side chain protecting groups and the polypeptide resin linkage simultaneously.

At least three different polypeptide-resin linkage agents can be used: substituted benzyl alcohol derivatives that can be cleaved with 95% TFA to produce a polypeptide acid, methanolic ammonia to produce a polypeptide amide, or 1% TFA to produce a protected polypeptide which can then be used in fragment condensation procedures, as described by Atherton, E. etal., J. Chem. Sec. Perkin Trans. 1:538-546 (1981) and Sheppard, R. C. et al., Int. J. Polypeptide Prot. Res. 20:451-454 (1982). Furthermore, highly reactive Fmoc amine acids are available as pentafluorophenyl esters or dihydro-oxobenzotriazine esters derivatives, saving the step of activation used in the tBoc method.

Sequences available to use as a basis for polypeptide synthesis can be based on published sequences of G-protein coupled receptors, ligands and/or effectors, wherein the transmembrane or functional domains correspond to sections of hydrophobic or other amine acids of 5 to 100 amine acids, such as 5-10, 10-15, 15-25, 20-25, 23-27, 25-30, 28-35, 20-40, 10-40, 20-30, 30-40, 40-50, 10-80, 20-60 or 25-40 amine acids in length. Recombinant production of GPR polypeptides can be accomplished according to known method steps. Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J. D. et al., Molecular Biology of the Gone, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, Calif. (1987); Darnell, J. E. et al., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B. M., Genes III, John Wiley & Sons, publishers, New York, N.Y. (1989); Old, R. W., et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, publisher, Berkeley, Calif. (1981); Ausubel et al, eds. , Current Protocols in Molecular Biology, Wiley Interscience, publisher, New York, N.Y. (1987, 1992); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. (1989), the entire contents of which references are herein incorporated by reference.

A nucleic acid sequence ellcoding a GPR polypeptide of the present invention may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Ausubel et al, supra, and are well known in the art.

A nucleic acid molecule, such as DNA is said to be capable of expressing" a polypepzide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as GPR polypeptides in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook, supra and Ausubel supra.

The present invention accordingly encompasses the expression of a GPR polypeptide, in either prokaryotic or eukaryotic cells, although eukaryotic expression is preferred.

Preferred hosts are bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. It is preferred that the mammalian cell or tissue is of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used.

Further, by use of, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins may be accomplished. The fusion proteins so produced may be processed in vivo or purified and processed in vitro, allowing synthesis of a GPR polypeptide of the present invention with a specified amino terminus sequence. Moreover, problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression may be avoided. Sabin etal., Bio/Technol. 7(7): 705-709 (1989); Miller et al., Bio/Technol. 7(7): 698-704 (1989).

Any of a series of yeast gene eggression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized to obtain GPR polypeptides of the present invention. Known glycolytic genes can also provide very efficient transcriptional control signals. for example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

Production of GPR polypeptides or functional derivatives thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express transmembrane polypeptide by methods known to those of skill. See Ausubel etal, eds. Current Protocols in Molecular Biology, Wiley Interscience, .sctn..sctn.16.8-16.11 (1987, 1992).

In a preferred embodiment, the introduced nucleotide sequence will be incorporated into a plasmid or vital vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. See, e.g., Ausubel et al, supra, .sctn..sctn.1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11. Pactors of importance in selecting a particular plasmid or vital vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[Preferred prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are, for example, disclosed by Maniatis, T., et al. (Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al, eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, N.Y. (1987, 1992)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, N.Y. (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall, K. J., et al., J. Bacteriol. 169:4177-4183 (1987)) , and streptomyces bacteriophages such as .phi.C31 (Chater, K. F. , et al. , In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978); and Ausubel et al, supra).

The expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitamion, chromatography, affinity chromatography, electrophoresis, or the like. Por example, the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation. Alternatively, thetransmembrane polypeptide or functional derivative thereof may be isolated by the use of anti-transmembrane polypeptide antibodies. Such antibodies may be obtained by well-known methods, some of which are mentioned below. These antibodies may be immobilized on cellulose, agarose, hollow fibers, or cellulose filters by covalent chemical derivatives by methods well known to those skilled in the art.

As discussed herein, GPR poiypeptides of the present invention may be further modified for purposes of drug design, such is for example to reduce immunogenicity, to prevent solubility and/or enhance delivery, or to prevent clearance or degradation.

Appropriate modification of the primary amino acid sequence of GPR polypeptides of the present invention, obtained by mutagenesis or utilizing fragments of other related forms of G-protein transmembrane proteins, as described herein, will allow the creation of molecules which bind G-protein coupled receptors with higher affinity than that exhibited by naturally occurring transmembrane domains. Small polypeptides that are provided according to the present invention which polypeptides maintain G-protein coupled receptor binding inhibition activity, are expected to have two advantages over larger polypeptides. These advantages include (1) greater stability and diffusibility, and (2) less immunogenicity.

Since polypeptides according to the present invention are generally small (10-40, 20-30, 15-25, 30-45 amino acids), cell or tissue sources of G-protein coupled receptors are not required to practice the present invention, since known polypeptide syntheses steps can be used without undue experimentation to provide GPR polypeptides or sequences substantially corresponding thereto.

Preparations of GPR polypeptides for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.

By the term "protection" from infection or disease as used herein is intended "prevention," "suppression" or "treatment." "Prevention" involves administration of a GPR polypeptide, polypeptide derivative, or anti-idiotypic antibody prior to the induction of the disease.

"Suppression" involves administration of the composition prior to the clinical appearance of the disease.

"Treatment" involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term "prophylaxis" as distinct from "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis."

At least one GPR polypeptide, antibody or anti-idiotypic antibody of the present invention may be administered by any means that achieve their intended purpose, for example, to treat GPR related pathologies, such as psychotic disorders, including schizophrenia, by inhibition of binding of Dopamine D.sub.2 receptors using a GPR polypeptide corresponding to a fragment or consensus portion of a dopamine D.sub.2 transmentrane domain; in the form of a pharmaceutical composition.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. Alternatively, or concurrently, ndministration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.

A preferred mode of using a GPR pharmaceutical composition of the present invention is by intravenous or parenteral application.

A typical regimen for preventing, suppressing, or treating G-protein coupled receptor pathologies, such as dopamine receptor related schizophrenia, comprises administration of an effective amount of a GPR polypeptide, consensus sequence, or chemical derivative thereof, administered over a period of one or several days, up to and including between one week and about 24 months.

It is understood that the dosage of a GPR polypeptide of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the inventors and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

The total dose required for each treatment may be administered by multiple doses or in a single dose. a GPR polypeptide or functional a chemical derivative thereof may be administered alone or in conjunction with other therapeutics directed to GPR related pathologies, such as a the dopamine receptor related pathology as a non limiting example, or directed to other symptoms of the disease.

Effective amounts of the a GPR polypeptide or composition, which may also include a functional derivative thereof, or a GPR anti-idiotypic antibody, are from about 0.01 .mu.g to about 100 mg/kg body weight, and preferably from about 10 .mu.g to about 50 mg/kg body weight, such 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9, 1, 2, 5, 10, 20, 25, 30, 40, 45, or 50 mg/kg.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.

Pharmaceutical compositions comprising at least one GPR polypeptide of the present invention may include all compositions wherein the GPR polypeptide is contained in an amount effective to achieve its intended purposa. In addition to the GPR polypeptide, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions include suitable solutions for administration intravenously, subcutaneously, dermally, orally, mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e. the antibody) together with the excipient. Pharmaceutical compositions for oral administration include tablets and capsules. Compositions which can be administered rectally include suppositories.

The polypeptides in FIGS. 1-5 were synthesized using the following procedure and include the following characteristics.

Peptide I (SEQ ID NO:I), as shown in FIG. 1, was used as a control for hydrophobic interaction alone as the mechanism of binding and was run in parallel with the test polypeptides described below. Polypeptide II (SEQ ID NO: 2 ) , as shown in FIG. 2, represents a membrane- spanning fragment of transmembrane segment III in the dopamine D.sub.2 receptor. This particular fragment was chosen since it has been implicated in the .beta.-adrenergic receptor as having many res idues which are involved in ligand binding interaction. Polypeptide III (SEQ ID NO:3), as shown in FIG. 3, represents the consensus polypeptide which was developed as a model for the dopamine D.sub.2 system and polypeptide IV (SEQ ID NO:4), as shown in FIG. 4, is a control for length dependence to show how critical the polypeptide length is in binding studies. Polypeptide V (SEQ ID NO:5), as shown in FIG. 5, is a consensus sequence of transmembrane domains of dopamine receptors D.sub.1 and D.sub.2.

The above polypeptides I-V (SEQ ID NOS:1-5), as shown in FIGS. 1-5, respectively, were synthesized using solid phase synthesis on a Milligen 9600 polypeptide synthesizer using Fmoc amino acids (provided by Milligen/Biosearch) and PAL polystyrene resin (Milligen/Biosearch) . Coupling times were 1 hour and the polypeptides were cleaved by trifluoroacetic acid/phenol/thioanisole/ethanedithiol (82.5:5:5:5:2.5) at room temperature for 2 hours. The filtrate was collected and washed with 2 mL of trifluoroacetic acid (TFA) and 1 mL of dichloromethane (DCM). The filtrate was reduced in vacuo to 2 ml in volume and the resulting polypeptide was precipitated out by the addit ion of water. The polypeptides were then dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol [(HFIP) Eastman]; lyophilized; and stored at -20.degree. C. until purification. Polypeptides I-V (SEQ ID NOS:1-5), were purified using reverse-phase HPLC using a preparative Vydac C4 column (Vydac) at 60.degree. C. at a flow rate of 6.0 mL/min with a linear gradient of 0-100% B in a 60 min period at a UV detection wavelength of 275 mm.

Due to the highly hydrophobic nature of these polypeptides, methanol was used with 0.1% (W/V) TFA and 0.5% (W/V) HFIP as solvent A and 2-propanol with 0.1% TPA as solvent B, in order to purify these polypeptides. Further purification was performed with an analytical C4 column (Vydac) with an isocratic qradient of 40% B at a flow rate of 1 ml/min. Identity of the polypeptides was confirmed by Fast-atom bombardment mass spectrometry and electrospray mass spectrometry and amino acid analysis. Stock solutions of polypeptides were made in HFIP and stored at -20.degree. -80.degree. C.

Circular Dichroism (CD). Spectra were recorded on an Aviv model 60 DS circular dichroism spectrophotometer at room temperature with a 1 cm by 1 mm cell. The amplitude of the CD signal was calibrated using 10.1% (w/v) solution of d (+)-camphorsulfonic acid (Aldrich) and the wavelength of the CD signal was set using standard absorbance peaks of benzene vapor. Polypeptide ccncentrations were determined in a Cary 210 UV spectrophotomer wwith the absorbance measured at 280 nm. Helical content was estimated using CD signal intensity according to the method of Chen. et al Biochem. 13:3350-13359 (1974). This calculation compares the experimental ellipticity at. 222 nm ( [.theta.]222) ( [.theta.]) to a theoretical [.theta.]222. The theoretical [.theta.]222 is empirically adjusted to account for differences in polypeptide length and is based on experimental CD data from a series of proteins with known crystal structures. Since both the curve shape and magnitude are important in analysis of a CD spectrum for secondary structure contributions, we also considered qualitatively the contributions to the spectral shapes from different secondary structures using reference curves for poly (L-lysine).

FIG. 6 shows a CD spectrum of the consensus polypeptide III (SEQ ID NO: 3 ) demonstrating that the polypeptide III is only partially helical in a solvent system in which most membrane polypeptides are strongly helical.

Preparation of Small Unilamellar Visicles. Polypeptides were incorporated into DMPC vesicles at lipid:peptide ratio of 147:1 in the followinq manner: polypept ide in HFIP was mixed with dimyrystyroyl-phosphatidylcholine (synthetic) (DMPC) in dry chloroform and dried to a film with a stream of dry nitrogen at 0.degree. C. This residue was then dried further overnight under a vacuum (1.times.10.sup.-2 torr). The residue was then hydrated in 100 mL NaCl and sonicated for a 30-min period under nitrogen at 0.degree. C. The suspension was sedimented for a 30-min at 100,000 q (4.degree. C.) to remove any residual titanium particles and large unilamellar vesicles The supernatant was removed and sedimented once more at 159,000 g for a 45 min period at 4.degree. C. The supernatant in the lower portion was used immediately. This basic procedure has been shown to reliably produce small unilamellar vesicles.

Radioligand Bining Assays. A 0.50 mL volume of 1.00 nM [.sup.3 H]-spiperone (New England specific activity 21.4 Ci/mol) was added to assay tubes which contained 0.5 mL lipid/peptide supernatant, 0.5 mL Tris buffer pH 7.4 and 0.5 mL of cold drug for a final volume of 2.0 mL. Nonspecific binding was defined in the presence of 1 uM of (+) butaclamol or 1 uM spiperone. Appropriate controls for lipid vesicles containing no polypeptide were also run. Assay tubes were prepared in triplicate and the mixture was incubated for 1 h at 25.degree. C. Incubation was terminated by filtration through filters presoaked in 0.1% polyethyleneimine (w/v, Siqma) for at least 1 h prior to use.

Filters were then washed with 6.0 mL of cold 50 mM Tris-HCl buffer, pH 7.40. For detection of radioactivity, filters were placed in 2.0 mL of scintillation fluid (Scintiverse) and incubated for 24 h. The activity of the tritium was determined in a Beckman LS 7500 liquid scintillation counter. Specific binding of [.sup.3 H]-spiperone was defined as the difference in binding in the presence and absence of unlabeled (+) butaclamol.

FIG. 7 shows results of radioligand binding assays comparing polypeptide I (SEQ ID NO:1) as a control unit polypeptide iII (SEQ ID NO:3) according to the present invention. Polypeptide III (SEQ ID NO:3) is shown to unexpectedly provide receptor-like functional binding, as demonstrated by binding to the neuroleptic agent, spiperone, into a stereoselective, concentration-dependent manner.

It has also been demonstrated that as little as 0.1% of a GPR polypeptide according to the present invention is able to form a receptor-like functional binding site. Thus, a GPR polypeptide of the present invention is unexpectedly shown to act both as GPR ligands and GPR binding sites.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entire y incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.