MELANOCORTIN-4 RECEPTOR DEFICIENT CELLS, NON-HUMAN TRANSGENIC ANIMALS AND METHODS OF SELECTING COMPOUNDS WHICH REGULATE BODY WEIGHT

Abstract: Cells and non-human transgenic animals have been engineered to be deficient in the gene encoding the melcanocortin-4 receptor protein (MC-4R). These MC-4R deficient transgenic animals can be used to select for and test potential modulators of MC-4R. This data allows for methods of screening for preferential MC-4R modulators which effect body weight through modulation of both metabolic rate and food intake, as well as associated methods of treating various disorders associated with inappropriate regulation of body weight.

Description: TITLE OF THE INVENTION MELANOCORTIN-4 RECEPTOR DEFICIENT CELLS, NON-HUMAN TRANSGENIC ANIMALS AND METHODS OF SELECTING COMPOUNDS WHICH REGULATE BODY WEIGHT CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application Serial No. 60/165,074, filed November 12, 1999. STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable REFERENCE TO MICROFICHE APPENDIX Not applicable. FIELD OF THE INVENTION The present invention relates to cells and non-human transgenic animals that have been engineered to be deficient in the gene encoding the melcanocortin-4 receptor protein (MC-4R). It is shown herein that male MC-4R deficient transgenic animals of the present invention show increased fatmass and are obese, while female heterozygous MC-4R deficient transgenic animals have similar body weight to wild type mice. The MC-4R deficient transgenic animals of the present invention can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which control food intake and metabolic rate. To this end, the present invention also relates to methods of screening for MC-4R modulators which effect body weight in general but more specifically impart effects on the control of food intake and metabolic rate, in conjunction with associated methods of treating various disorders associated with inappropriate regulation of body weight. BACKGROUND OF THE INVENTION Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as -, β, or γ-melanocyte stimulating hormones (α-, β-, γ-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors. U. S. Patent No. 5,622,860 (issued April 22, 1997) and U.S. Patent No. 5,704,220 (issued December 40, 1997) to Yamada and Gantz, disclose DNA molecules which encode human MC-4R and human MC-4R, respectively (see also Gantz, et al., 1994, J. Biol. Chem. 268(11): 8246-8250). The agouti mouse represents a naturally occurring obese rodent, with a late life onset of obesity which is not corticosterone dependent. The obesity in this model results from the ectopic expression of the 141 amino acid agouti protein. Agouti is normally only expressed in the skin where it controls hair color. The protein is a paracrine antagonist of the melanocortin- 1 receptor (MC-IR), a G-protein coupled receptor of the hair follicle. MC-IR agonism, through its natural ligand, a-MSH raises cAMP and the expression of the enzyme tyrosinase. Low levels of tyrosinase, which result from agouti antagonism of MC-IR, result in reduced conversion of the hair color pigment pheomelanin to eumelanin. As a result a light (agouti) rather than black hair color results. The obese phenotype of the agouti mouse was ascribed to the expression of agouti in the brain, where it antagonizes MC-4R and MC-4R receptors. MC-Rs are expressed in a variety of tissues including the central nervous system, where the MC-4R is believed to be expressed exclusively. The generation and analysis of a melanocortin 4 receptor (MC-4R) deficient mouse (Huszar et al., 1997, Cell 88: 141-141, see also U.S. Patent No. 5,942, 779, issued August 4, 1999 to Lee et al) revealed that MC-4R knock out mice have a predisposition to obesity and hyperinsulinemia first manifested after about 8 weeks of age. Since the phenotype of MC-4R knock out mice recapitulates the obesity observed in the A vy mouse it is assumed that the effects of agouti on the MC-4R receptor mediate the obese phenotype of the A vy mouse (the effects of agouti on skin color are exerted through the MC-IR). MC-4R has also been implicated as a regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997, Nature 485: 165-168). Haynes et al. (1999, Hypertension 33: 542-547) suggest a role for the melanocortins and leptin in controlling sympathetic nervous system activity and thermogenesis. The authors showed that intra-cerebroventricular (ICV) administration of the non-selective melanocortin receptor agonist MT-II resulted in sympathoexcitation of brown adipose tissue in Sprague Dawley (SD) rats, indicative of an effect on thermogenesis. This effect could be blocked with the non-selective MC-IR, -5R agonist, -3R, -4R antagonist (SHU-9119), indicating that these effects most likely involved selective activation of melanocortin receptors. Dinulescu et al. (1998, Proc. Natl. Acad. Sci. 95: 12101-12112) show that the mahogany (mg) mutation in mice stimulates feeding and raises metabolic rate independent of the effects of mg on suppression of the action of agouti. It is desirable to discover new drugs for the treatment of body weight disorders which selectively modulate a melanocortin receptor within the host. It is also desirable to identify additional receptor targets which are involved in regulating body weight. It would be especially desirable to identify one or more melanocortin receptor targets through which regulation of body weight is mediated by both metabolic rate and food intake. The present invention addresses and meets these needs by disclosing MC-4R-deficient animal cells, related non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are also MC-4R-deficient. The present invention also addresses and meets these needs by disclosing methods of screening for compounds which effect body weight comprising the screening and selection of compounds which modulate the MC-4R, specifically by mediating both metabolic rate and food intake. SUMMARY OF THE INVENTION The present invention relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-4R deficient (MC-4R null) due to a disruption in the gene encoding MC-4R. More specifically, this portion of the invention relates to a transgenic animal, preferably a transgenic mouse, wherein homozygous or heterozygous male MC-4R deficient transgenic animals show increased fatmass and are obese, while heterozygous female MC-4R deficient transgenic animals have similar body weight to wild type mice. Transgenic female mice homozygous for alteration of the MC-4R gene become obese. The MC-4R deficient transgenic animals of the present invention can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which control both food intake and metabolic rate. The present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates heterozygous for a functional MC-4R gene native to that animal which show the male/female obesity phenotypic difference described herein. Again, the preferable transgenic animal is a mouse. The present invention also relates to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are either homozygous or heterozygous for deletion of the MC-4R gene in combination with a homozygous or heterozygous deletion at separate alleles which encode at least one additional melanocortin receptor, especially a melanocortin receptor shown to be involved in body weight regulation, such as MC-4R, wherein such transgenic animals are derived from MC-4R transgenic animals, preferably mice, which show the male/female obesity phenotype described herein. The transgenic cells and animals of the present invention are useful in the study of the effect of modulators on the activity of the MC-4R gene and/or protein or the expression of the MC-4R gene and/or protein as concerning the regulation of body weight, including but not limited to disorders such as obesity, diabetes, anorexia and cachexia, as well as diseases and disorders such as cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy. The present invention also relates to methods of selecting for modulators which regulate body weight by selecting such modulators which show an ability to effect both metabolic rate and food intake when interacting with the MC-4R. To this end, various in vivo (e.g., transgenic mice) and in vitro (e.g., recombinant cell- or membrane-based assays measuring the modulation of the MC-4R) methodology is available to specifically select for preferred modulators of MC-4R, that is, modulators of MC-4R which effect both metabolic rate and food intake. As used herein, the term "functional" is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment. Therefore, a gene which is not functional (i.e., "non-functional", "disrupted", "altered", or the like) will encode a protein which does not function as a wild type, native or non-altered protein, or encodes no protein at all. Such a non-functional gene, such as a non-functional MC-4R gene, may be the product of a homologous recombination event as described herein, where a non-functional gene is targeted specifically to the region of the target chromosome which contains a functional form of the gene, resulting in a "knock-out" of the wild type or native gene. As used herein, a "modulator" is a compound that causes a change in the expression or activity of MC-4R, or causes a change in the effect of the interaction of MC-4R with its ligand(s), or other protein(s). As used herein in reference to transgenic animals of this invention, we refer to "transgenes" and "genes". As used herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal. A gene is a nucleotide sequence that encodes a protein. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art. As used herein, the term "animal" is used herein to include all mammals, except that when referring to transgenic animals, the use of this term excludes humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra- chromosomally replicating DNA. Unless otherwise noted or understood from the context of the description of an animal, the term "transgenic animal" as used herein refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals. The genetic information is typically provided in the form of a transgene carried by the transgenic animal. As used herein, a "targeted gene" or "Knockout" (KO) is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles, especially endogenous alleles which encode MC-4R. As used herein, "MC-IR" refers to the melanocortin- 1 receptor. As used herein, "MC-3R" refers to the melanocortin-3 receptor. As used herein, "MC-4R" refers to the melanocortin-4 receptor. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence which encodes the human MC-4R (SEQ ID NO:l). Figure 2 shows the amino acid sequence of the human MC-4R (SEQ ID NO:2). Figure 3 shows a schematic description of the strategy utilized in construction of the targeting gene vector, pAJ7. Figure 4 shows the strategy utilizing the targeting gene vector, pAJ7, for homologous recombination with mouse genomic sequences encoding MC-4R. Figure 5A-B show the mean body weights of MC-4R knockout mice and control littermates. (A): Growth curve of male mice: knockout (closed diamonds), n=4; heterozygous (closed triangles), n=12; wild type (open circles), n=8. (B): Growth curve of female mice: knockout, n=10; heterozygous, n=16; wild type, n=4. Figure 6A-D show a lack of MC-4R mRNA expression in the brain of the MC-4R knock out mice. In situ hybridization was performed on 14 μm coronal brain sections from wild type control (A and B) and MC-4R knock out (C and D) mice. 33 P-labeled oligonucleotide probes were used for detecting the mRNA for MR-4R in the absence (A and C) and presence (B and D) of 100 fold molar excess of non- labeled oligonucleotide probes. Arrows indicate specific hybridization for MC-4R mRNA in the hippocampus of the wild type mouse brain. Figure 7 shows the metabolic rate measurements in wild type (+/+) and MC-4R knock out (-/-) mice. Data are the average metabolic rate (cal/hr) from 30 minutes to 4 hours post dosing, mean + SE of 8 animals/group. MT-II was administered at 20 mg/kg, ip. * P< 0.01 vs vehicle control group. Figure 8 shows twenty four hour food intake following MT-II (10 mg/kg, i.p.) administration to wild type (n=8; open histogram bar) and MC-4R knockout mice (n=8; black bar). Saline vehicle was injected to the mice for two days prior to MT-II treatment. The average daily food intake for the two days was used as vehicle baseline (open bar). **P<0.01, * P<0.05 (paired-comparisons T test). Figure 9 shows an aequorin bioluminescence assay in which the rat melanocortin 4 receptor (MC-4R), upon stimulation by the melanocortin agonist peptide αNDP-MSH (1 μM), can functionally couple to activation of phospholipase C (mobilization of intracellular calcium reported by aequorin bioluminescence) when expressed in Xenopus oocytes in the presence of the Gαl5 subunit. Each tracing represents the bioluminescence response (in counts/sec) over time (in seconds) for 1-2 individual oocytes. Figure 10 shows an experiment similar to that described for Figure 9, using the G αq i 5 subunit. In this case, the human galanin receptor 1 (hGALRl), which is coupled in its native state to inhibition of adenylate cyclase, can now when co-expressed with the G αq j 5 subunit, show functional activation in response to agonist treatment (1 μM human galanin). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-4R deficient (MC-4R null) due to a disruption in the gene encoding MC-4R. The invention also relates to a transgenic animal, preferably a transgenic mouse, wherein the male mice homozygous or heterozygous for the altered MC-4R gene show increased fatmass and are obese, while females homozygous for the altered MC-4R gene become obese while females heterozygous for the altered MC-4R gene have body weights similar to wild type female mice. In conjunction with the finding described herein that modulation of the MC-4R effects both metabolic rate and food intake, MC-4R deficient transgenic animals of the present invention can be used in methodology to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which effect both food intake and metabolic rate. As such, the MC-4R transgenic animals, especially transgenic mice, may be used in methodology to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R involved the regulation of body weight and muscle mass as defined by lean body mass, modulators which may effect such disorders as obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, and other states that are characterized as resulting from GH deficiency and cancer cachexia. Therefore, the transgenic animals of the present invention may be utilized to determine the effect of certain modulators on the activity and/or expression of the MC-4R gene or receptor protein, and aspects of disorders involving regulation of body weight. The present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates heterozygous for a functional MC-4R gene native to that animal which show the male/female obesity phenotypic difference described herein. Again, the preferable transgenic animal is a mouse and will be useful as a component or complement to the various assays described herein. The generation of MC-4R deficient transgenic non-human animals, including mice, aids in defining the in vivo function(s) of MC-4R, especially as related to the interaction of the MC-4R in the regulation of body weight. Additionally, MC-4R null animals can be used as a strain for the insertion of human MC-4R genes, and provides an animal model useful in the design and assessment of various approaches to modulating MC-4R activity and expression. Such modified transgenic non-human animals can also be used as a source of cells for cell culture. These cells can be used for corresponding in vitro studies of MC-4R expression, activity and the modulation thereof. An aspect of this invention is a method to obtain an animal in which the cells lack a functional gene MC-4R native to the animal. The method includes providing a gene for an altered form of the MC-4R gene native to the animal in the form of a transgene and targeting the transgene into a chromosome of the animal at the place of the native MC-4R gene or at another chromosomal location. The transgene can be introduced into the embryonic stem cells by a variety of methods known in the art, including electroporation, microinjection, and lipofection. Cells carrying the transgene can then be injected into blastocysts which are then implanted into pseudopregnant animals. In alternate embodiments, the transgene-targeted embryonic stem cells can be co-incubated with fertilized eggs or morulae followed by implantation into females. After gestation, the animals obtained are chimeric founder transgenic animals. The founder animals can be used in further embodiments to cross with wild-type animals to produce FI animals heterozygous for the altered MC-4R gene. In further embodiments, these heterozygous animals can be interbred to obtain the viable transgenic embryos whose somatic and germ cells are homozygous for the altered MC-4R gene and thereby lack a functional MC-4R gene. In other embodiments, the heterozygous animals can be used to produce cells lines. In preferred embodiments, the animals are mice. A further aspect of the present invention is a transgenic non-human animal which expresses a non-native MC-4R on a native MC-4R null background. In particular embodiments, the null background is generated by producing an animal with an altered native MC-4R gene that is non-functional, i.e. a knockout. The animal can be heterozygous (i.e., having a different allelic representation of a gene on each of a pair of chromosomes of a diploid genome), homozygous (i.e., having the same representation of a gene on each of a pair of chromosomes of a diploid genome) for the altered MC-4R gene, hemizygous (i.e., having a gene represented on only one of a pair of chromosomes of a diploid genome), or homozygous for the non-native MC-4R gene. In preferred embodiments, the animal is a mouse. In particular embodiments the non-native MC-4R gene can be a wild-type or mutant allele, preferably a wild-type or mutant human allele. In further embodiments the non-native MC-4R gene is operably linked to a promoter. As used herein, operably linked is used to denote a functional connection between two elements whose orientation relevant to one another can vary. In this particular case, it is understood in the art that a promoter can be operably linked to the coding sequence of a gene to direct the expression of the coding sequence while placed at various distances from the coding sequence in a genetic construct. Further embodiments are cell lines and cells derived from animals of this aspect of the invention. An aspect of this invention are transgenic animals having a transgene including a non-native MC-4R gene on a native MC-4R null background. The method includes providing transgenic animals of this invention whose cells are heterozygous for a native gene encoding a functional MC-4R protein and an altered native MC-4R gene. These animals are crossed with transgenic animals of this invention that are hemizygous for a transgene including a non-native MC-4R gene to obtain animals that are both heterozygous for an altered native MC-4R gene and hemizygous for a non-native MC-4R gene. The latter animals are interbred to obtain animals that are homozygous or hemizygous for the non-native MC-4R and are homozygous for the altered native MC-4R gene. In particular embodiments, cell lines are produced and cells isolated from any of the animals produced in the steps of the method. The transgenic animals and cells of this invention are useful in the determination of the in vivo function of a non-native MC-4R in regulation of body weight. The animals are also useful in determining the ability for various forms of wild-type and mutant alleles of a non-native MC-4R to rescue the native MC-4R null deficiency. The animals are also useful for identifying and studying the ability of a variety of compounds to act as modulators of the expression or activity of a non-native MC-4R in vivo, or by providing cells for culture, for in vitro studies. The genetic information received by the animal can cause the native gene to become non-functional to produce a "knockout" animal. Alternatively, the genetic information received by the animal can be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the information can be altered or it can be expressed differently than the native gene. The non-human transgenic animals of the present invention include non-human mammalian species, including but not limited to transgenic mice, transgenic rats, transgenic guinea pigs, transgenic rabbits, transgenic goats, transgenic non-human primates, such as chimpanzees, rhesus monkeys and green african monkeys, and transgenic cattle. Transgenic mice are preferred and exemplified herein. The present invention especially relates to analysis of the complex function(s) of MC-4R as related to obesity and diabetes by generating knock-out transgenic mice and studying how various potential modulators interact within these manipulated animals. As described herein in more detail, the native wild type gene is selectively inactivated in totipotent ES cells (such as those described herein) and used to generate the transgenic mice of the present invention. Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. The present invention relates to diploid animal cells, non-human transgenic embryos, non-human transgenic animals and non- human transgenic littermates which are heterozygous or homozygous for a disrupted MC- 4R gene resulting in deficient production of the MC-4R protein. The cells, embryos and non-human transgenic animals contain two chromosome alleles for MC-4R wherein at least one of the MC-4R alleles is mutated such that less than wild-type levels of MC-4R activity is produced. The diploid mouse cell, embryo or non-human transgenic mice homozygous for a disrupted MC-4R gene may show at least from about 50% to about 100% reduction in MC-4R activity compared to a wild type diploid cell. The diploid mouse cell, embryo or non-human transgenic mice heterozygous for a disrupted MC-4R gene may show at least from about 10% to about 100% reduction in MC-4R activity compared to a wild type diploid cell. It is within the purview of the artisan of ordinary skill to use known molecular biology techniques to measure the level of transcription, expression and/or functional MR-4R activity in mouse cell homozygous, heterozygous or hemizygous for a mutated MC-4R gene. Therefore, the present invention especially relates to analysis of the complex function(s) of MC-4R as related to obesity by generating homozygous, heterozygous or hemizygous transgenic mice and studying how various potential modulators interact within these manipulated animals. In a preferred embodiment, the assay is performed by providing an animal of the present invention, exposing the animal to the compound, and measuring the effect of said compound on body weight and other related biochemical and physiological responses. The measurement can be compared to these measurements in a genetically similar or identical animal that is not exposed to the compound. One way to facilitate such measurements would be to feed both MC-4R knock-out and wild-type mice a high fat diet to promote obesity (diet induced obesity [DIO]). After becoming obese, effects of potential MC-4R agonists may be measured for reduction of body fat in wild-type mice as opposed to no effect in MC-4R knock-out mice. Similar protocols may be useful in studying the effects of MC-4R modulators in other related disorders, such as diabetes. It will therefore be within the purview of the artisan to utilize the non-human transgenic animals of the present invention to study any number of complex events associated with modulation of the MC-4R. As additional examples, but in no way presented as limitations, the potential role of MC-4R in sexual dysfunction may be studied, in light of the fact that MC-4R is heavily expressed in lamina X of the lumbar and sacral spinal cord a key center for signaling to the penis. A MC-4R gene that naturally occurs in the animal is referred to as the native gene, and if it is not mutant, it can also be referred to as wild-type. An altered MC-4R gene should not fully encode the same MC-4R as native to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether. In cases where it is useful to express a non-native MC-4R gene in a transgenic animal in the absence of a native MC-4R gene we prefer that the altered MC-4R gene induce a null knockout phenotype in the animal. However a more modestly modified MC-4R gene can also be useful and is within the scope of the present invention. The MC-4R mutation may be a targeted deletion mutation, a targeted substitution mutation and/or a targeted insertion mutation. However, the preferred mutation is a deletion mutation, and especially preferred is a deletion mutation which results in a deletion of most if not all of the MC-4R gene. Transgenic animals are generated which have an altered, or preferably, completely deleted MC-4R gene. MC-4R gene deletions, gene modifications and or gene insertions can render the native gene nonfunctional, producing a "knockout" transgenic animal, or can lead to a MC-4R with altered expression or activity. As noted above, a non-human transgenic animal without an activated MC-4R gene can be used to evaluate the role of MC-4R in obesity and other associated disorders. The MC-4R protein is a G-protein coupled receptor comprising a ligand-binding extracellular domain, 7 transmembrane domains and an intracellular domain which couples to activation of adenyl cyclase. Melanocortin receptors belong to the rhodopsin sub-family of GPCR's. However, several features in the MC-4R are shared with all other receptors and are absent in most other GPCR's, including the EN motif in TM1, the lack of Cys in the loop between TM2 and TM3 or between TM4 and TM5, the MxxxxxxxY motif in TM5, and the DPxxY motif in TM7. Since all melanocortin receptors lack Cys residues in the extracellular loops that are present in other members of the rhodopsin sub-family, interhelical disulfide bond (e.g., between the Cys residues near the top of TM3 and TM5) may play the same function as interloop disulfide bond in most other GPCR's. Such known characteristics are useful in targeting specific host MC-4R mutations. A preferred deletion mutation may contain a deletion of anywhere from 1 nucleotide to deletion of the entire gene, including the open reading frame and associated cis-actmg regulatory sequences associated with wild type MC-4R. A smaller deletion within the open reading frame is preferably not divisible by three, so as to result in a frameshift mutation resulting in a protein which most likely is non-functional. It is preferred that any such smaller deletion not divisible by three be targeted toward the 5' region of the open reading frame to increase the possibility of generating a non-functional truncated protein product. However, as noted above, it is preferable that the deletion mutation encompass most if not all of the MC-4R gene so as to insure prevention of expression of a functional MC-4R protein. Therefore, the transgenic animals which are homozygous, heterozygous or hemizygous for a deficient MC-4R gene are useful for identifying compounds which modulate wild type MC-4R activity or expression in vivo and studying aspects of the regulation of body weight which may be imparted through activation or antagonism of the MC-4R receptor. The generation of MC-4R deficient transgenic non-human animals, including mice, aids in defining the in vivo function(s) of MC-4R. In addition, transgenic animals can be used as a strain for the insertion of human MC-4R genes and provides an animal model useful in the design and assessment of various approaches to modulating MC-4R activity and expression. An altered MC-4R gene should not fully encode the same MC-4R as native to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether. However a more modestly modified MC-4R gene can also be useful and is within the scope of the present invention. The modified cells, embryos and/or non-human transgenic animal of the present invention can also be used as a source of cells for cell culture. These cells can be used for corresponding in vitro studies of MC-4R expression, activity and the modulation thereof. The non-human transgenic animals disclosed herein are useful for drug antagonist or agonist studies, for animal models of human diseases, and for testing of treatment of disorders or diseases associated with MC-4R. Transgenic animals lacking native MC-4R are useful in characterizing the in vivo function(s) of MC-4R. A transgenic animal carrying a non- native MC-4R in the absence of native MC-4R is useful for the establishment of a non- human model for diseases involving MC-4R, such as obesity, for studies of non-native MC-4R, to study modulators of the non-native gene and to distinguish between the activities of the non-native MC-4R in in vivo and in vitro systems. In view of the teachings within this specification, it is within the purview of the artisan of ordinary skill to utilize antisense RNA transgenes to partially or totally knock out expression of the mouse MC-4R protein. The antisense transgene used herein would encode a polynucleotide which is at least partially complementary to all or a part of the host MC-4R gene and which will hybridize to a target sequence encoded by the host MC-4R gene, most specifically a mRNA transcript expressed from the host MC-4R gene. Any such oligonucleotide sequence should be at least about 15 to 30 nucleotides in length and preferably more than about 30 nucleotides, wherein this sequence in substantially complementary to the target host gene. The antisense transgene need not be a total complement, but instead should contain adequate sequence identity such that the expressed antisense RNA transgene will effective hybridize with the expressed mRNA from the host target gene so as to efficiently inhibit concomitant protein expression. These antisense polynucleotides may be produced by subcloning the sequence of interest into an appropriate gene expression vector and transferring this vector to pluripotent embryonic stem cells which may be used as described herein to generate another form of an MC-4R deficient non-human transgenic animal. A type of target cell for transgene introduction is also the embryonic stem cell (ES). ES cells can be obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, 1981, Nature 292: 154-156; Bradley et al., 1984, Nature 309: 255-258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83: 9065-9069; and Robertson et al., 1986, Nature 322: 445-448). Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science 240: 1468-1474). The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described in 1987 (Thomas et al., Cell 51:503-512, (1987)) and is reviewed elsewhere (Frohman et al., Cell 56:145-147 (1989); Capecchi, Trends in Genet. 5:70-76 (1989); Baribault et al., Mol. Biol. Med. 6:481-492, (1989); Wagner, EMBO J. 9:3025-3032 (1990); Bradley et al., Bio/Technology 10:534-539 (1992)). See also, U.S. Patent No. 5,464,764, issued to Cappecchi and Thomas on November 7, 1995; U.S. Patent No. 5,789,215, issued to Berns et al on August 4, 1998, both of which are hereby incorporated by reference). Therefore, techniques are available in the art to generate the MC-4R deficient animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates of the present invention. The methods for evaluating the targeted recombination events as well as the resulting knockout mice are also readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE), in situ hybridization and Western blots to detect DNA, RNA and protein. Therefore, the MC-4R deficient animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates of the present invention may be generated by any techniques known in the art, as sampled in the previous paragraph. The generation of a MC-4R knockout mouse with the phenotypic characteristics described herein has not been reported. The mice heterozygous for the MC-4R deletion as described by Huzar et al. (1997, Cell 88: 141-141, see also U.S. Patent No. 5,942, 779, issued August 4, 1999 to Lee et al.) showed a weight gain intermediate to that seen in wild-type and homozygous mutants for both female and male mice. On the contrary, the mean body weights of MC-4R knockout females described herein was statistically significantly different from that of wild type mice and the mean body weights were indistinguishable between wild type mice and heterozygous female MC-4R deficient mice (see Figure 5B). MC-4R knock out mice were generated and evaluated for the effect of the loss of the MC-4R receptor on metabolic rate and the potential role of other melanocortin receptors on the control of food intake. It is disclosed herein that the MC-4R knock out mice have a reduced metabolic rate when compared to their wild type littermates. In addition, following administration of MT-II to MC-4R knock out or wild type mice, conclusive evidence is disclosed to show that the MC-4R mediates the effects of this peptide on metabolic rate and food intake. A mouse 129 SJ v lambda genomic library was screened with a 1-Kb rat MC-4R cDNA clone as a probe. One of four clones was mapped in detail by restriction enzyme digestion and the sequences flanking the MC-4R mouse coding regions were chosen for further manipulation. A -1.6 Kb fragment comprising a PGK-neo cassette replaced the majority of the MC-4R coding region. The final targeting vector targeting vector was constructed in pSP72 (Promega) consisting of a 5' 3.4 Kb Hindlll to Ncol fragment, the 1.6 Kb PGK-neo and a 3' 3.5 kb Hindlll to Sail fragment. The linearize vector was electroporated into the AB2.2 embryonic stem cells and G418-resistant clones were selected. Targeted ES clones were selected and microinjected into C57B1/6J blastocysts to generate chimeric mice. Two male chimeras showed germline transmission of the targeted allele to their offspring. FI heterozygotes were interbred to produce homozygous knockout, heterozygous, and wild type F2 progeny. These F2 littermate mice were used to measure the body weight starting at 5 weeks of age. F2 homozygous knockout mice and wild type littermate mice we also used to produce F3 hybrids. In situ hybridization studies of MC-4R transgenic mice showed a lack of MC4-R expression in brain tissue. Expression of MC-4R was seen in wild type brains. Using indirect calorimetry it is shown that MC-4R knock out mice exhibit a 16% reduced metabolic rate, when compared to wild type controls. The non-selective MC-R agonist MT-II is shown to increase metabolic rate in wild type mice while these effects are not observed in MC-4R knock out mice. Taken together this data supports the notion that the MC-4R is involved in controlling metabolic rate, possibly through modulation of sympathetic nervous system output. Using MT-II, is it also shown that the MC-4R knock out mice are resistant to the effects of MT-II on food intake. It is disclosed herein that the MC-4R is involved in both the control of metabolic rate and food intake, making it an ideally suitable target for consideration in the treatment of obesity. The MC-4R knock out mice described herein fully recapitulate the effects on body weight first observed and published by Huszar et al. (1997, Cell 88: 141-141, see also U.S. Patent No. 5,942, 779, issued August 4, 1999 to Lee et al.). A similar gene dosage sensitivity in male heterozygous MC-4R knock out mice to that of the Huzar et a\.(id.) knock out mice is disclosed herein. However, a difference between the knock mice described here and those generated by Huszar et al., (id.) exists since the MC-4R knock out mice generated herein do not contain the same gene dosage sensitivity in female heterozygous MC-4R deficient mice. In this colony female heterozygous MC-4R knockout and wild type littermate mice have similar body weights. The targeting vector described in Huzar et al. contains approximately 2.4Kb of additional 3' genomic sequences, a difference in the vectors which might result in altered expression of a closely linked gene differentially affecting body weight gain in heterozygous MC-4R deficient female and male mice. The MC-4R deficient mice have an overall reduced metabolic rate (-16 %) when corrected for body mass and increased respiratory coefficient indicative of reduced fat metabolism, when compared to age matched litter-mate controls. Intraperitoneal administration of the non-selective MC-R agonist melanotan II (MT-II; a cyclic peptide) increases metabolic rate in wild type mice, while MC-4R deficient mice are insensitive to the effects of MT-II on metabolic rate. MC-4R deficient mice are also insensitive to the effects of MT-II on reducing food intake. Therefore, MC-4R mediates control of both metabolic rate and food intake in mice. The date disclosed herein show a role for the MC-4R receptor in the physiology of body weight control has not been excluded. We now show that MT-II (with MC-3R agonist activity) fails to increase metabolic rate in MC-4R knockout mice. A role for the MC-3R in the control of metabolic rate in wild type mice is therefore unlikely. The data herein showing that the MC-4R deficient mice have both a deficiency in food intake and metabolic rate allows for further definition of the neuronal pathways through which metabolic rate and appetite are controlled. Therefore, the present invention is shown to provide a model system consisting of transgenic animals, cells and assays that are useful in the study of aspects of the etiology of obesity as related to modulation of the MC-4R. The various assays are also useful for screening and selecting for compounds that have an effect on body weight regulation, the further study of these compounds and the possible administration of selected compounds to humans in order to regulate disorders which include but are not limited to obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, cancer cachexia, and other states that are characterized as resulting from GH deficiency. The finding that the MC-4R is involved in the regulation of both metabolic rate and food intake will allow testing of selected MC-4R agonists for direct measurements of their efficiency to modulate (decrease) body fat, thus assessing their therapeutic potential for the treatment of obesity. MC-4R knockout mice can be used to test melanocortin receptor subtype-specific compounds. The present invention also relates to cell- and membrane-based methods of identifying selective agonists and/or antagonists of mammalian MC-4R which affect the regulation of body weight through disorders including but not limited to obesity, diabetes, anorexia and cachexia. Therefore, an object of the present invention provides for MC-4R-based assays to select for modulators of this receptor protein which affect regulation of body weight through the various known disorders associated with regulation of body weight. The MC-4R modulators may be used to treat these body weight disorders, such as utilizing a MC-4R agonist to treat obesity or a MC-4R antagonist to treat anorexia. These assays are preferably cell-based assays whereby a DNA molecule encoding MC-4R is transfected or transformed into a host cell and this recombinant host cell is allowed to grow for a time sufficient to express MC-4R prior to use in various assays described herein. Alternatively, any "non-recombinant" cell line which has been genetically modified to overexpress MC-4R may also be utilized to screen and/or select for modulators of MC-4R useful in the treatment of body weight disorders. In addition, substantially purified membrane fractions from (1) a host cell transfected with a DNA expression vector coding for MC-4R or (2) a cell line genetically manipulated to overexpress MC-4R may be utilized to screen and/or select for modulators useful in the treatment of body weight disorders. To this end, it is a further object to provide for membrane preparations from these recombinant or genetically modified host cells for use in assays to screen and/or select for modulators of MC-4R activity associated with the regulation of body weight. Therefore, the present invention relates to methods of treating body weight disorders through administration of modulators which directly affect the MC-4R, modulators identified initially through these cell- or membrane- based screens and/or through assays utilizing the transgenic animals of the present invention. Any polynucleotide sequence which encodes a functional MC-4R may be utilized in the recombinant cell and membrane-based assays of the present invention. A preferred polynucleotide for use in constructing an appropriate DNA expression vector is a DNA molecule which comprises the open reading frame for human MC-4R as set forth in SEQ ID NO:l and disclosed in U.S. Patent No. 5,622,860, issued to Yamada and Gantz on April 22, 1997 and U.S. Patent No. 5,703,220, issued to Yamada and Gantz on December 30, 1997), as disclosed in Figure 1, and as follows: ATGGTGAACT CCACCCACCG TGGGATGCAC ACTTCTCTGC ACCTCTGGAA CCGCAGCAGT TACAGACTGC ACAGCAATGC CAGTGAGTCC CTTGGAAAAG GCTACTCTGA TGGAGGGTGC TACGAGCAAC TTTTTGTCTC TCCTGAGGTG TTTGTGACTC TGGGTGTCAT CAGCTTGTTG GAGAATATCT TAGTGATTGT GGCAATAGCC AAGAACAAGA ATCTGCATTC ACCCATGTAC TTTTTCATCT GCAGCTTGGC TGTGGCTGAT ATGCTGGTGA GCGTTTCAAA TGGATCAGAA ACCATTATCA TCACCCTATT AAACAGTACA GATACGGATG CACAGAGTTT CACAGTGAAT ATTGATAATG TCATTGACTC GGTGATCTGT AGCTCCTTGC TTGCATCCAT TTGCAGCCTG CTTTCAATTG CAGTGGACAG GTACTTTACT ATCTTCTATG CTCTCCAGTA CCATAACATT ATGACAGTTA AGCGGGTTGG GATCATCATA AGTTGTATCT GGGCAGCTTG CACGGTTTCA GGCATTTTGT TCATCATTTA CTCAGATAGT AGTGCTGTCA TCATCTGCCT CATCACCATG TTCTTCACCA TGCTGGCTCT CATGGCTTCT CTCTATGTCC ACATGTTCCT GATGGCCAGG CTTCACATTA AGAGGATTGC TGTCCTCCCC GGCACTGGTG CCATCCGCCA AGGTGCCAAT ATGAAGGGAG CGATTACCTT GACCATCCTG ATTGGCGTCT TTGTTGTCTG CTGGGCCCCA TTCTTCCTCC ACTTAATATT CTACATCTCT TGTCCTCAGA ATCCATATTG TGTGTGCTTC ATGTCTCACT TTAACTTGTA TCTCATACTG ATCATGTGTA ATTCAATCAT CGATCCTCTG ATTTATGCAC TCCGGAGTCA AGAACTGAGG AAAACCTTCA AAGAGATCAT CTGTTGCTAT CCCCTGGGAG GCCTTTGTGA CTTGTCTAGC AGATATTAA (SEQ ID NO:1), which encodes the entire open reading frame of the MC-4R protein, as disclosed in Figure 2 and set forth as SEQ ID NO:2, as follows: MV STHRGMH TSLHLWNRSS YRLHSNASES LGKGYSDGGC YEQ FVSPEV FVTLGVISLL ENI VIVAIA KNKNLHSPMY FFICS AVAD MLVSVSNGSE TIIITLLNST DTDAQSFTVN IDNVIDSVIC SSLLASICSL SIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAACTVS GILFIIYSDS SAVIICLIT FFTMLALMAS LYVHMFL AR LHIKRIAVLP GTGAIRQGAN MKGAITLTIL IGVFWCWAP FFLH IFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPL IYALRSQELR KTFKEIICCY PLGGLCDLSS RY (SEQ ID NO:2). The DNA molecule set forth as SEQ ID NO:l or a biologically equivalent polynucleotide may be inserted into an appropriate vector and linked with other DNA molecules, i.e, DNA molecules to which the MC-4R are not naturally linked, to form "recombinant DNA molecules" expressing the receptor. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode a MC-4R. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular use. A variety of mammalian expression vectors may be used to express recombinant MC-4R in mammalian cells. As noted above, expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria- yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Commercially available mammalian expression vectors which may be suitable for recombinant MC-4R expression, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565). Also, a variety of bacterial expression vectors may be used to express recombinant MC-4R in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant MC-4R expression include, but are not limited to pCR2.1 (Invitrogen), pETl la (Novagen), lambda gtl l (Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungal cell expression vectors may be used to express recombinant MC-4R in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant MC-4R expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen). Also, a variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of MC-4R include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen). Expression of MC-4R DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred. To determine the MC-4R cDNA sequence(s) that yields optimal levels of MC-4R, cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for MC-4R as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a MC-4R cDNA. The expression levels and activity of MC-4R can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the MC-4R cDNA cassette yielding optimal expression in transient assays, this MC-4R cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells. The host cells engineered to contain and/or express DNA sequences encoding the MC-4R can be cultured under suitable conditions to produce MC-4R or a biologically equivalent form. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Therefore, an expression vector containing DNA encoding a MC-4R-like protein may be used for expression of MC-4R in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines. For instance, one insect expression system utilizes Spodoptera frugiperda (Sf21) insect cells (Invitrogen) in tandem with a baculovirus expression vector (pAcG2T, Pharmingen). Also, mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209). The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce MC-4R protein. Identification of MC-4R expressing cells may be done by several means, including but not limited to immunological reactivity with anti-MC-4R antibodies, labeled ligand binding and the presence of host cell-associated MC-4R activity. In one embodiment of the present invention, assays described herein can be carried out with cells that have been genetically modified to overexpress host MC-4R, preferably resulting in at least a 5-fold increase over expression in a chosen "wild-type" host cell. Such improvements of overexpression can be brought about by any means presently known in the art, including but not limited to introducing a promoter by homologous recombination while leaving the coding region intact, or by simply selecting for cells that for whatever biological reason express a higher level of the MC-4R. In another and preferred embodiment of the present invention, assays described herein can be carried out with cells that have been transiently or stably transfected or transformed with an expression vector which directs expression of MC-4R. The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. Transformation is meant to encompass a genetic change to the target cell resulting from an incorporation of DNA. Transfection is meant to include any method known in the art for introducing MC-4R into the test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing MC-4R, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce MC-4R protein. Identification of MC-4R expressing cells may be done by several means, including but not limited to immunological reactivity with anti- MC-4R antibodies, labeled ligand binding and the presence of host cell-associated MC-4R activity. The specificity of binding of compounds showing affinity for MC-4R is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that bind to MC-4R or that inhibit the binding of a known, radiolabeled ligand of MC-4R to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for MC-4R which may be useful in the treatment of body weight disorders. Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays. Compounds identified by the above method are likely to be agonists or antagonists of MC-4R and may be peptides, proteins, or non-proteinaceous organic molecules, all of which may be useful in the treatment of body weight disorders. The present invention is directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a MC-4R protein as well as compounds which effect the function of the MC-4R protein and hence, body weight disorders. Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of MC-4R. For example, Cascieri et al. (1992, Molec. Pharmacol. 41:1096-1099) describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and thus are potential agonists or antagonists of neurokinin receptors. The method involves transfecting COS cells with expression vectors containing rat neurokinin receptors, allowing the transfected cells to grow for a time sufficient to allow the neurokinin receptors to be expressed, harvesting the transfected cells and resuspending the cells in assay buffer containing a known radioactively labeled agonist of the neurokinin receptors either in the presence or the absence of the substance, and then measuring the binding of the radioactively labeled known agonist of the neurokinin receptor to the neurokinin receptor. If the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of the neurokinin receptor. Where binding of the substance such as an agonist or antagonist to MC-4R is measured, such binding can be measured by employing a labeled substance or agonist. The substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically. Therefore, the specificity of binding of compounds having affinity for MC-4R is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that bind to MC-4R or that inhibit the binding of a known, radiolabeled ligand of MC-4R to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for MC-4R. Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays. Compounds identified by the above method are likely to be agonists or antagonists of MC-4R and may be peptides, proteins, or non- proteinaceous organic molecules which may be useful for human administration to treat various maladies , including but in no way limited to obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, cancer cachexia, and other states that are characterized as resulting from GH deficiency. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding MC-4R, or by acting as an agonist or antagonist of the MC-4R receptor protein. These compounds that modulate the expression of DNA or RNA encoding MC-4R or the biological function thereof may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Kits containing MC-4R, antibodies to MC-4R, or modified MC-4R may be prepared by known methods for such uses. To this end, the present invention relates in part to methods of identifying a substance which modulates MC-4R receptor activity, which involves: (a) combining a test substance in the presence and absence of a MC-4R receptor protein, including but not limited to the MC-4R proteins comprising the amino acid sequence as set forth in SEQ ID NO:2; and (b) measuring and comparing the effect of the test substance in the presence and absence of the MC-4R receptor protein. In addition, several specific embodiments are disclosed herein to show the diverse type of screening or selection assay which the skilled artisan may utilize in tandem with an expression vector directing the expression of the MC-4R receptor protein. Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of MC-4R. Therefore, these embodiments are presented as examples and not as limitations. To this end, the present invention includes assays by which MC-4R modulators (such as agonists, inverse agonists and antagonists) may be identified. Accordingly, the present invention includes a method for determining whether a substance is a potential agonist or antagonist of MC-4R useful in the treatment of body weight disorders, comprising: (a) transfecting or transforming cells with an expression vector that directs expression of MC-4R in the cells, resulting in test cells; (b) allowing the test cells to grow for a time sufficient to allow MC-4R to be expressed; (c) exposing the cells to a labeled ligand of MC-4R in the presence and in the absence of the substance; and, (d) measuring the binding of the labeled ligand to MC-4R; where if the amount of binding of the labeled ligand is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of MC-4R. The conditions under which step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C. The test cells may be harvested and resuspended in the presence of the substance and the labeled ligand. In a modification of the above-described method, step (c) is modified in that the cells are not harvested and resuspended but rather the radioactively labeled known agonist and the substance are contacted with the cells while the cells are attached to a substratum, e.g., tissue culture plates. The present invention also includes a method for determining whether a substance is capable of binding to MC-4R or a mutant MC-4R that is no longer functional but nonetheless may be involved in ligand binding, i.e., whether the substance is a potential agonist, inverse agonist or an antagonist of MC-4R and hence useful in the treatment of body weight disorders, where the method comprises: (a) transfecting or transforming cells with an expression vector that directs the expression of MC-4R in the cells, resulting in test cells; (b) exposing the test cells to the substance; (c) measuring the amount of binding of the substance to MC-4R; (d) comparing the amount of binding of the substance to MC-4R in the test cells with the amount of binding of the substance to control cells that have not been transfected with MC-4R; wherein if the amount of binding of the substance is greater in the test cells as compared to the control cells, the substance is capable of binding to MC-4R. Determining whether the substance is actually an agonist or antagonist can then be accomplished by the use of functional assays such as, e.g., the assay involving the use of promiscuous G-proteins described below. The conditions under which step (b) of the method is practiced are conditions that are typically used in the art for the study of protem-ligand interactions: e.g , physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C. The test cells are harvested and resuspended in the presence of the substance. It is shown herein (see Example Section 4) that a MC-4R-ιnduced functional response may be measured in the Xenopus oocyte system, or within any useful eukaryotic cell line. In conducting the assay, either the subunit itself or a nucleic acid encoding the subunit (the nucleic acid molecule may be a DNA expression vector or possible a cRNA transcπpt generated from an equivalent DNA expression vector, preferably in vitro) or both protein and nucleic acιd(s) may be added, and the addition events need not occur together. Next, a nucleic acid or pool of nucleic acids, wherein at least one nucleic acid encoding a MC-4R is introduced into the cell To this end, another embodiment of the present invention is the use of promiscuous G-protems (such as G αl5 , G α ι 6 , and G qι5 ) in assays which rely on phospholipase C activation to measure receptor-mediated changes in mtracellular calcium and which are amenable to high throughput screening. These and other related promiscuous G-proteins permit the use of the phospholipase C signaling pathway by G-protem coupled receptors that normally signal via reduction in mtracellular cAMP via G_. Measurement in the reduction m cAMP has drawbacks because of a small dynamic range in the assay and low throughput. Assays which rely on phospholipase C activation, as noted above, instead measure changes in calcium via assays utilizing detector molecules (e.g., such as aequoπn bioluminescence [see Example Section 4] or FLIPR), which makes this type of assay amenable to high throughput screening, such as but not limited to detection via a lummometer or a charge-coupled device (CCD) camera system (see Milligan et al, 1996, TiPS 17:235-237 for a review). Of course, virtually any convenient eukaryotic cell may be used in the assays developed herein These would include oocytes (preferred ones are from Xenopus sp.) but cell lines may be used as well as Examples of preferred cell lines are mammalian cell lines, including COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 and VERO. One important component of such an assay utilizing a promiscuous G-protem is the above-mentioned detector molecule. Preferably, the detector molecule is responsive to an mtracellular event which is part of the biochemical cascade initiated by GHS-GHSR binding. One class of preferred detector molecules can respond to changes in calcium concentrations. A preferred detector molecule which responds to calcium concentrations is aequoπn (a jellyfish photoprotem; see Inouye et al., 1885, Proc. Natl. Acad. Sci. U.S.A. 82: 3154-3158, hereby incorporated by reference), which emits a characteristic fluoresence in the presence of Ca 2+ , which acts on the substrate coelenterazine. Other detector molecules include calcium chelators with fluorescence capabilities, such as FURA-2 and indo-1. In addition, several modifications to aequorin have disclosed and may also be utilized to practice this portion of the invention (for example, see U.S. Patent Nos. 6,027,881; 5,625,048; and 5,804,387, which are also incorporated by reference). The detector molecule itself may be introduced into the cell, or nucleotides which encode the detector molecule may be introduced into the cell, under conditions which will allow the expression of the detector molecule. Generally, it is preferred to introduce nucleotides, such as DNA or cRNA which encode the detector molecule into the cell, under conditions wherein the cell will express the detector molecule. Heterotrimeric G proteins, consisting of α, β, γ subunits, serve to relay information from cell surface receptors to intracellular effectors, such as phospholipase C and adenylate cyclase. The G-protein alpha subunit (α-subunit) is an essential component of the intracellular signal transduction pathway activated by receptor-ligand interaction. In the process of ligand-induced GPCR activation, the Gα subunit of a trimeric Gαβγ complex will exchange its bound GDP for GTP and dissociate from the βγ heterodimer. The dissociated Gα-protein serves as the active signal transducer, often in concert with the βγ complex, thus starting the activation of the intracellular signal transduction pathway. G-alpha subunits are classified into sub-families based on sequence identity and the main type of effectors are coupled: G s activate adenylate cyclase, Gi /0/t , inhibit adenylate cyclase, G q /π 5 activate β-class phospholipase C polypeptides (PI-PLC), and G 12/ ι 3 , effector unknown (see Milligan and Rees, TiPS 20: 118-124, for a review of Gα subunit classification and activity. The artisan will realize upon review of this specification that any known Gα subunit may be tested for use in the MC-4R-based assays of this portion of the invention. Therefore, as shown in Example Section 4, the present invention relates in part to methods of identifying MC-4R agonists or antagonists for use in treating body weight disorders (i.e., body weight regulation) which includes but is by no means limited to the use of promiscuous G-proteins (such as G α ι 5 , G α ι 6 , and G q ι 5 ) in assays which rely on phospholipase C activation to receptor-activated changes in intracellular calcium levels. These changes in intracellular calcium levels may be detected by use of a "detector molecule", which includes but is in no way limited to the exemplified detector molecule, aequorin, such that detection of intracellular calcium as reported by aequorin bioluminescence will be amenable to high throughput screening, as shown in Example Section 4, comprising such steps as follows: I. (a) transfecting or transforming cells (such as a Xenopus oocyte or any convenient eukaryotic cell known in the art) with a first expression vector (or equivalent cRNA molecule) which directs expression of MC-4R and introducing a promiscuous G-protein subunit into the test cells or transfecting or transforming the test cells with a second expression vector (or equivalent cRNA molecule) which directs expression of the G-protein subunit; (b) introducing a detector molecule into the test cells or transfecting or transforming the test cells with an expression vector (or equivalent cRNA molecule) which directs expression of a detector molecule; resulting in test cells (d) contacting the test cells with a compound suspected of being a modulator of MC-4R; and, (e) determining whether the compound modulates MC-4R activity by monitoring the detector molecule, where an increase in detection level of the detector molecule in the cells as compared to the level of detection in the absence of the suspected agonist indicates that the substance is an agonist of MC-4R. II. (a) transfecting or transforming cells (such as a Xenopus oocyte or any convenient eukaryotic cell known in the art) with a first expression vector (or equivalent cRNA molecule) which directs expression of MC-4R and introducing a promiscuous G-protein subunit into the test cells or transfecting or transforming the test cells with a second expression vector (or equivalent cRNA molecule) which directs expression of the G-protein subunit; (b) introducing a detector molecule into the test cells or transfecting or transforming the test cells with an expression vector (or equivalent cRNA molecule) which directs expression of a detector molecule; resulting in test cells; (c) contacting the test cells with a compound that is an agonist of MC-4R; (d) subsequently, concurrently or prior to step (c), exposing the test cells to a substance that is a suspected antagonist of MC-4R; and, (e) determining whether the compound modulates MC-4R activity by monitoring the detector molecule, where a decrease in the level of detection in the test cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of MC-4R. III. The above methods wherein the first and second expression vectors of step (a) are replaced with a single expression vector which expresses a chimeric MC-4R protein fused at its C-terminus to a respective promiscuous G-protein. Of course, compounds selected through such assays may then be administered to non-human animals, such as the transgenic mice described herein, to further characterize potential modulators of MC-4R activity, and hence, the biological events characterized by modulation of this receptor, as described herein. Chen et al. (1995, Analytical Biochemistry 226: 349-354) describe a colorimetric assay which utilizes a recombinant cell transfected with an expression vector encoding a G-protein coupled receptor with a second expression vector containing a promoter with a cAMP responsive element fused to the LacZ gene. Activity of the overexpressed G-protein coupled receptor is measured as the expression and OD measurement of β-Gal. Therefore, another aspect of this portion of the invention includes a non-radioactive method for determining whether a substance is a potential agonist or antagonist of MC-4R that comprises: (a) transfecting or transforming cells with an expression vector encoding MC-4R, resulting in test cells; (b) transfecting or transforming the test cells of step (a) with an expression vector which comprises a cAMP-inducible promoter fused to a colorimetric gene such a LacZ; (c) allowing the transfected cells to grow for a time sufficient to allow MC-4R to be expressed; (d) harvesting the transfected cells and resuspending the cells in the presence of a known agonist of MC-4R and/or in both the presence and absence of the test compound; (e) measuring the binding of the known agonist and test compound to overexpressed MC-4R by a colorimetric assay which measures expression off the cAMP- inducible promoter and comparing expression levels in the presence of the known agonist as well as in the presence and absence of the unknown substance so as to determine whether the unknown substance acts as either a potential agonist or antagonist of MC-4R. Again, the expression vector may be a DNA expression vector or a cRNA transcript generated from an equivalent DNA expression vector, preferably in vitro. Additional methods of identifying MC-4R agonists or antagonists for use in treating body weight disorders include but are by no means limited to the following: I. (a) transfecting or transforming cells with a first expression vector which directs expression of MC-4R and a second expression vector which directs the expression of a promiscuous G-protein, resulting in test cells; (b) exposing the test cells to a substance that is a suspected agonist of MC-4R; (c) measuring the level of inositol phosphates in the cells; where an increase in the level of inositol phosphates in the cells as compared to the level of inositol phosphates in the cells in the absence of the suspected agonist indicates that the substance is an agonist of MC-4R. II. (a) transfecting or transforming cells with a first expression vector which directs expression of MC-4R and a second expression vector which directs the expression of a promiscuous G-protein, resulting in test cells; (b) exposing the test cells to a substance that is an agonist of MC-4R; (c) subsequently or concurrently to step (b), exposing the test cells to a substance that is a suspected antagonist of MC-4R; (d) measuring the level of inositol phosphates in the cells; where a decrease in the level of inositol phosphates in the cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of MC-4R. III. The method of II wherein the first and second expression vectors of step (a) are replaced with a single expression vector which expresses a chimeric MC-4R protein fused at its C-terminus to a promiscuous G-protein. Methods described herein can be modified in that, rather than exposing the test cells to the substance, membranes can be prepared from the test cells and those membranes can be exposed to the substance. Such a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268. Accordingly, another embodiment of the present invention includes a method for determining whether a substance binds and/or is a potential agonist or antagonist of MC-4R wherein membrane preparations from the test cells are utilized in place of the test cells. Such methods comprise the following and may utilized the physiological conditions as noted above: (a) transfecting or transforming cells with an expression vector that directs the expression of MC-4R in the cells, resulting in test cells; (b) preparing membranes containing MC-4R from the test cells and exposing the membranes to a ligand of MC-4R under conditions such that the ligand binds to the MC-4R in the membranes; (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance; (d) measuring the amount of binding of the ligand to the MC-4R in the membranes in the presence and the absence of the substance; (e) comparing the amount of binding of the ligand to MC-4R in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to MC-4R in the membranes in the presence of the substance indicates that the substance is capable of binding to MC-4R. The present invention also relates to a method for determining whether a substance is capable of binding to MC-4R comprising: (a) transfecting or transforming cells with an expression vector that directs the expression of MC-4R in the cells, resulting in test cells; (b) preparing membranes containing MC-4R from the test cells and exposing the membranes from the test cells to the substance; (c) measuring the amount of binding of the substance to the MC-4R in the membranes from the test cells; (d) comparing the amount of binding of the substance to MC-4R in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with MC-4R, where if the amount of binding of the substance to MC-4R in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to MC-4R. A preferred embodiment of the present invention is determining various ligand binding affinities using 125 I-labeled NDP-α-MSH as the labeled ligand in the presence of varying concentration of unlabeled ligands. The activation of the second messenger pathway may be determined by measuring the intracellular cAMP elicited by agonist at various concentration. It will be within the scope of the invention to submit screened compounds which show an in vitro modulation effect on MC-4R to in vivo analysis, preferably by administering the compound of interest to either a transgenic and/or wild-type animal as described herein to measure in vivo effects of the compound on the MC-4R receptor and to further measure biological and physiological effects of compound administration on the non-human animal. These in vivo studies may be done either alone or in combination with a known MC-4R ligand, such as but not limited to α-MSH, the agouti protein or the agouti like protein. For example, the MC-4R KO and wild-type mice can be used for in vivo testing of candidate compounds for their effects on several different parameters such as food intake, body weight, body composition, glucose, insulin, leptin and cholesterol levels, sexual function, memory, learning, nerve regeneration, and pain. In order to facilitate such measurement relating to body weight and diabetes both knockout and wild- type mice can be made DIO (diet-induced obesity) first before subject to compounds testing. The comparison of the effects on wild type, knock and heterozygote mice is an essential component of the evaluation of the selectivity of said compounds. It is also an essential part of the present invention to measure sensitivity to other melanocortin or other pathways that may have been up or down regulated and the measure changes in sensitivity of compounds that modulate these pathways. To this end, testing of compounds that affect MC-4R, or other melanocortin receptors, NPY receptors, galanin receptors, MCH receptors, Orexin receptors, Insulin receptors, receptors belonging to the bombesin family of receptors (BRS-3, neuromedin receptors, gastrin releasing peptide receptors), motilin receptors, neuromedin U receptors, adrenergic receptors, leptin receptors, modulators of STATs and SOCs transcription factors, phoshpodiesterase enzymes and others are within the scope of uses for the non-human transgenic animals of the present invention. Pharmaceutically useful compositions comprising modulators of MC-4R may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, modified MC-4R, or either MC-4R agonists or antagonists- Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences. Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times. The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug. The following examples are presented by the way of illustration and, because various other embodiments will be apparent to those in the art, the following is not to be construed as a limitation on the scope of the invention. EXAMPLE 1 Construction of MC-4R Targeting Vector And Associated Oligonucleotide Primers Construction ofMC-4R targeting vector and gene targeting in ES cells - To isolate genomic DNA containing the murine MC-4R gene, a mousel29 SjV lambda genomic library (Lambda FIX II Library, Stratagene, La Jolla,CA) was screened using a 1-Kilobasepair (Kb) rat MC-4R cDNA clone as a probe. One of four clones was mapped in detail by restriction enzyme digestion. The clone consisted of the 29-kilobase pair (Kb) lambda vector and a 15 Kb genomic insert encoding the 1 Kb MC-4R coding sequence, between 10 Kb of 5' and 4 Kb of 3' flanking sequences. An approximately 1.5 Kb fragment extending from an Ncol site located approximately 20 basepair (bp) downstream of the MC-4R translation initiation codon to the Hindlll site situated approximately 0.5 Kb downstream of the ATG stop codon of the MC-4R coding sequence was replaced with a PGK-neo cassette (neomycin phosphotransferase gene under the control of the phosphoglycerokinase promoter [pPGKneobpA, obtained from Dr. Alan Bradely; also see e.g., Tybulewicz et al., 1991, Cell 65:1153-1163). The targeting vector was constructed in pSP72 (Promega) consisting of a 5' 3.4 Kb Hindlll to Ncol fragment, the 1.6 Kb PGK-neo fragment and a 3' 3.5 kb Hindlll to Sail fragment. A complete schematic diagram for constructing pAJ7 is shown in Figure 3 while the strategy for recombination with mouse genomic sequences is shown in Figure 4. The gene targeting vector pAJ7 was linearized at a unique Seal site and electroporated into the AB2.2 embryonic stem cells (Lexicon Genetics) under standard condition using a Gene Pulser (Bio-Rad). Selection of the G418-resistant clones was performed as previously described (Von Koch et al., 1997). PCR Analysis of Knockout Mice - To facilitate the identification of a large number of knockout and wild type mice, 3 oligonucleotides were designed to distinguish the knockout allele from the wild-type allele by PCR. The synthetic oligonucleotides 5'-CTAACCATAAGAAATCAGCAGCCCG-3'(SEQ ID NO:3) and 5'-AGGGAAGTATACATG CCATGGTGGT-3' (SEQ ID NO:4) result in 500 bp PCR product by wild type allele. Oligonucleotides 5'-CTAACCATAAGAAATCAG CAGCCCG-3' (SEQ ID NO:5) and 5'-TACCGGTGGATGTGGAATGTGTGC-3' (SEQ ID NO:6) result in 650 bp product derived from the mutant allele. EXAMPLE 2 Generation of MC-4R Knockout Mice Animal care and maintenance - All animal protocols used in these studies were approved by Merck Institutional Animal care and Use Committee. The mice were group housed in microisolator cages in a specific pathogen-free facility with an air shower entrance. The mice were maintained on a Teklad 7012 chow diet and ad libitum water. Generation ofMC-4R Knockout Mice - Targeted ES clones were identified by Southern blotting analysis, using a 700-bp Ncol -Hindlll fragment located 5' outside of the targeting vector as a probe. Of the 600 clones selected, 3 showed a 7 Kb targeted Apal restriction enzyme fragment in addition to the expected 5 Kb wild-type fragment (the Apal site in the MC-4R coding region was eliminated during the homologous recombination event). These positive clones were microinjected into C57B1/6J blastocysts to generate chimeric mice. Two male chimeras showed germline transmission of the targeted allele to their offspring. FI heterozygotes were interbred to produce homozygous knockout, heterozygous, and wild type F2 progeny. These F2 littermate mice were used to measure the body weight starting at 5-week of age. F2 homozygous knockout mice and wild type littermate mice were also used to produce F3 hybrids. For metabolic rate studies, F3 hybrids were used. Since the knock out mice become obese with increasing age, we hypothesized that the homozygous mice might have reduced fertility and therefore would not be suitable for efficient mass-production of knockout mice. However, the litter size of homozygous breeding pairs appeared normal when compared to that of heterozygous breeding pairs. The knockout males of different ages (2 to 4.5 months old) were also evaluated for reproductive performance by mating with young SW females in estrous. Results indicated a normal plug rate (production of vaginal plugs overnight) and litter size for males between 2 and 4.5 months old. EXAMPLE 3 Analysis of MC-4R Knock Out Mice In Situ Hybridization - MC-4R knock out and age/sex matched wild type control mice were killed by decapitation, and brains were quickly removed and frozen in -40°C isopentane, and stored at -80°C until use. Coronal brain sections (14 μm) were cut at -17°C with a cryostat microtome, and thaw -mounted onto baked microslides. Following fixation in ice-cold 4% phosphate-buffered paraformaldehyde, the tissue sections were stored in 95% ethanol at 4°C until use. The hybridization probes consist of an equal molar mixture of three non-overlapping, antisense oligonucleotides against the coding region of MC-4R. Their nucleotide sequences are 5'-ATCCTCTTAATGTGAAGCCTCGCCATCAGGAACATGT GGACATAGAGAGA -3' (SEQ ID NO:7), 5'-GAAAGCAGGCTGCAAATGGA TGCGAGCAAGGAGCT ACAGATCAC -3' (SEQ ID NO: 8), and 5'-AACAGATGATCTCTTTGAAGGTTTTCCTCAGTTCTTGACTCCG -3' (SEQ ID NO:9). The oligonucleotide probes were terminally labeled with [α 33 PjdATP and terminal transferase, and hybridization and washing conditions were as described in Guan et al. (1998, Molecular Brain Res. 59: 273-279). Indirect calorimetry - Metabolic rate was measured in male MC-4R knock out and their wild type syngeneic control mice. Metabolic rate was measured by indirect calorimetry using a 16-chamber open-circuit Oxymax system (Columbus Instruments, Columbus, Ohio). Mice (approximately 10 weeks of age) were maintained at 21 to 24°C in a 12 hour light dark cycle, (light period 7:00 am to 7:00 pm). Food (autoclaved pelleted chow: Harlan Teklad 7012, Madison, WI), and water were available ad libitum except where noted below. Animals were individually housed in specially built plexiglas cages (20 cm x 10.5 cm x 12 cm) through which room air was passed at a flow rate of 0.52 liter/min. Exhaust air from each chamber was sampled at 10 minute intervals for a period of one minute. Sample air was sequentially passed through O 2 and CO 2 analyzers (Columbus Instruments, Columbus, Ohio) for determination of O and CO 2 content. Metabolic rate (cal/hr) was calculated from the following equation; 3.815 + 1.232 x RER x vO where RER is the respiratory exchange ratio (volume of CO produced (ml/kg body weight/ hour) per volume of O 2 consumed (ml/kg body weight/ hour) and vO 2 is the volume of O consumed per hour. Baseline metabolic rate was measured over a 4 hour period commencing 30 minutes following food removal. RER was measured over the 2 hour period prior to food removal. For baseline measurements of metabolic rate, food was removed at 12:00 pm and returned at 5 pm that evening. The effects of the nonselective MC-R agonist MT-II (Peninsula Laboratories, San Carlos, CA; 20 mg/kg, intraperitoneally administered) or an equivalent volume of vehicle (saline) on metabolic rate were similarly determined over a 4 hour period commencing 30 minutes following food removal and dosing. Each animal received both treatments with at least a 5 day recovery period between treatments. The MC-4R knock out mice described above were evaluated for body weight gain in the MC-4R colony. Twenty-four male littermates (4 knockout [diamond symbols], 12 heterozygous [squares] and 8 wild type [triangles] mice) and thirty female littermates (10 knockout, 16 heterozygous and 4 wild type mice) were monitored every week from 5-weeks of age (Figure 5A-B). The mean body weight of knockout males was statistically significantly different (p<0.05) from that of wild type male mice by 6-weeks of age. After 8 weeks, the mean body weight of heterozygous males was also statistically significantly different (p<0.05) from that of wild type males. By 9 weeks of age, both homozygous male and female mice were approximately 50% heavier than their wild type controls (Figure 5A). Homozygous knockout males and females gained more weight than heterozygous mice, while these in turn gained more weight than their wild type littermates. This result suggested a gene dosage effect of MC-4R on body weight regulation and we observed a similar phenotype with MC-4R male knock out, heterozygous and wild type mice. However, although the mean body weights of MC-4R knockout females was statistically significantly different from that of wild type mice the mean body weights were indistinguishable between wild type mice and heterozygous female MC-4R deficient mice (Figure 5B). The lack of MC4-R expression in brains from MC-4R knock out mice was confirmed by in situ hybridization. In wild type brains, intense hybridization signals were detected in several regions, including the hippocampus (Figure 6A). This hybridization signal was specific for the MC-4R since it could be blocked by the addition of 100 fold molar excess of the non-labeled probe (Figure 6B). In contrast, specific hybridization signals for the MC-4R could not be observed in MC-4R knock out mouse brains (Figures 6C and 6D) providing conclusive evidence for the notion that the MC-4R receptor was functionally inactivated. The mean body weight of the MC-4R knock out mice (39.6 + 1.7 gm, mean + sem, n=8) used for determination of metabolic rate was significantly (P<0.01) greater than that of the age-matched wild type littermate control mice (25.5 + 0.7 gm, n=8). Correspondingly, the average resting metabolic rate (4 hour average of baseline values from approximately 1:30 pm to 4:30 pm) of the MC-4R knock out mice (408 + 14 cal/hr, n=8) was significantly greater (P<0.01) than that of wild type mice (313 + 6 cal/hr, n=8). However, representing the data on a per gram body weight basis, the metabolic rate of MC-4R knock out mice was well below that of wild type littermates (a roughly 16 % reduction, when compared to wild type mice). Furthermore the RER of MC-4R knock out mice, measured prior to removal of food but during the light period, was significantly (p<.01) greater (approximately 8%) than that measured in wild type controls. These data are shown in Figure 7. Administration of MT-II to MC-4R wild type animals evoked a significant (P<0.01) increase in metabolic rate of approximately 19% over the 4 hour period following dosing (397 + 15 cal/hr, n=8) when compared to vehicle administration (333 + 12 cal/hr, n=8). MT-II administration to MC-4R knock out mice had no effect on metabolic rate (422 + 25 cal/hr, n=8) over the 4 hour period following dosing when compared to vehicle administration (425 + 14 cal/hr, n=8). Effects of MT-II on RER could not be determined as RER was declining following removal of food in all groups. The effects of MT-II on food intake were evaluated and were fully mediated through its action at the MC-4R. Wild type and MC-4R knock out mice were evaluated for food intake over a 24 hour period following a 10 mg/kg intraperitoneal administration of MT-II. While a roughly 20% reduction of food intake resulted in wild type mice, a reduction of only 3% was observed in the MC-4R knock out mice, indicating that the effects of MC-4R to reduce food intake in wild type mice are largely mediated through the MC-4R (Figure 8). EXAMPLE 4 Use of Promiscuous Gα Protein in a Aequorin Bioluminescence Assay (ABA) to Measure MC-4R Activity Oocyte Preparation and Selection - Xenopus laevis oocytes were isolated and injected using standard methods previously described by Arena, et. al., 1991, Mol. Pharmacol. 40: 368-374, which is hereby incorporated by reference. Adult female Xenopus laevis frogs (purchased from Xenopus One, Ann Arbor, MI) were anesthetized with 0.17% tricaine methanesulfonate and the ovaries were surgically removed and placed in a 60 mm culture dish (Falcon) containing OR-2 medium without calcium (82.5 mM NaCl, 2 mM KC1, 2.5 mM sodium pyruvate, 1 mM MgCl 2 , 100 u/ml penicillin, 1 mg/ml streptomycin, 5 mM HEPES, pH=7.5; ND-96 from Specialty Media, NJ). Ovarian lobes were broken open, rinsed several times, and oocytes were released from their sacs by collagenase A digestion (Boehringer- Mannheim; 0.2% for 2-3 hours at 18°C) in calcium-free OR-2. When approximately 50% of the follicular layers were removed, Stage V and VI oocytes were selected and placed in ND-86 with calcium (86 mM NaCl, 2 mM KC1, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 2.5 mM sodium pyruvate, 0.5 mM theopylline, 0.1 mM gentamycin, 5 mM HEPES [pH=7.5]). For each round of injection, typically 3-5 frogs were pre-tested for their ability to express a control G-protein linked receptor (human gonadotropin- releasing hormone receptor) and show a robust phospholipase C intracellular signaling pathway (incubation with 1% chicken serum which promotes calcium mobilization by activation of phospholipase C). Based on these results, 1-2 frogs were chosen for library pool injection (50 nl of cRNA at a concentration of 25 ng (complex pools) to 0.5 ng (pure clone) per oocyte usually 24 to 48 hours following oocyte isolation. Plasmid DNA Preparation and cRNA Transcription - Plasmid DNA (plasmids included rat MC4R, rat GALRl, Gα 15 and G αθt s) was purified from pellets of bacteria (using the Wizard Miniprep kit according to the manufacturer's instructions (Promega Biotech, Madison, WI). The nucleotide sequence encoding respective proteins utilized herein are known in art, for example, see Alvaro, et al., 1996, Mol. Pharmacol. 50 (3): 583-591 (rat MC-4R); Inouye et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3154-3158 (aequroin); Strathmann and Simon, 1990, Proc. Natl. Acad. Sci. U.S.A. 87 (23): 9113-9117 (G aq); Coward et al., 1998, Proc Natl Acad Sci U S A. 95(l):352-357 (Gqi5). In preparation for cRNA synthesis, 4 μg of DNA was digested with Not I, and the subsequent linearized DNA was made protein and RNase-free by proteinase K treatment (10 μg for 1 hour at 37°C), followed by two phenol, two chloroform/isoamyl alcohol extractions, and two ethanol precipitations. The DNA was resuspended in approximately 15 μl of RNase-free water and stored at -70°C until needed. cRNA was synthesized using a kit from Promega Biotech with modifications. Each 50 μl reaction contained: 5 μl of linearized plasmid (approximatelyl mg), 40 mM Tris-HCl (pH=7.5), 6 mM MgCl 2 , 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 0.05 mg/ml bovine serum albumin, 2 units/ml RNasin, 800 μM each of ATP, CTP and UTP, 200 μM GTP, 800 μM m7G(5')ppp(5')G, 80 units of T7 RNA polymerase, and approximately 20,000 cpm of 32 P-CTP as a trace for quantitation of synthesized RNA by TCA precipitation. The reaction was incubated for 3 hrs. at 30°C; 20 units of RNase-free DNase was added, and the incubation was allowed to proceed for an additional 15 min. at 37°C. cRNA was purified by two phenol, chloroform/isoamyl alcohol extractions, two ethanol precipitations, and resuspended at a concentration of 500 ng/ml in RNase-free water immediately before use. Aequorin Bioluminescence Assay (ABA) and Clone Identification - The ABA requires injection of G protein-coupled receptor cRNA (1 ng/egg) with aequorin cRNA (2 ng/egg) supplemented with the G-protein alpha subunits G α ι 5 (2 ng/egg) or G αq i 5 (2 ng/egg). To facilitate stabilization of synthetic transcripts from aequorin plasmid, the expression vector pCDNA-3 was modified (termed pcDNA-3v2) by insertion (in the Apa I restriction enzyme site of the polylinker) of a cassette to append a poly (A) tract on all cRNA's which initiate from the T7 RNA polymerase promoter. This cassette includes (5' to 3'): a Bgl II site, pA (20) and a Sfi I site which can be used for plasmid linearization. Polymerase chain reaction (PCR) was utilized to generate a DNA fragment corresponding to the open reading frame (ORE) of the aequorin cDNA with an optimized Kosak translational initiation sequence (Inouye et. al, 1985, Proc. Natl. Acad. Sci. USA 82:3154-3158). This DNA was ligated into pCDNA-3v2 linearized with EcoR I and Kpn I in the EcoR I/Kpn I site of pCDNA-3v2. G α ι ι cDNA was excised as a Cla I/Not I fragment from the pCMV-5 vector (Woon, C. et. al., 1989 J. Biol. Chem. 264: 5687-5693), made blunt with Klenow DNA polymerase and inserted into the EcoR V site of pcDNA-3v2. cRNA was injected into oocytes using the motorized "Nanoject" injector (Drummond Sci. Co., Broomall, PA.) in a volume of 50 nl. Injection needles were pulled in a single step using a Flaming/Brown micropipette puller, Model P-87 (Sutter Instrument Co) and the tips were broken using 53X magnification such that an acute angle was generated with the outside diameter of the needle being <3 μm. Following injection, oocytes were incubated in ND-96 medium, with gentle orbital shaking at 18°C in the dark. Oocytes were incubated for 24 to 48 hours (depending on the experiment and the time required for expression of the heterologous RNA) before "charging" the expressed aequorin with the essential chromophore coelenterazine. Oocytes were "charged" with coelenterazine by transferring them into 35 mm dishes containing 3 ml charging medium and incubating for 2-3 hours with gentle orbital shaking in the dark at 18°C. The charging medium contained 10 μM coelenterazine (Molecular Probes, Inc., Eugene, OR.) and 30 μM reduced glutathione in OR-2 media (no calcium). Oocytes were then returned to ND-86 with calcium medium described above and incubation continued in the dark with orbital shaking until bioluminescence measurements were initiated. Measurement of GHSR expression in oocytes was performed using a Berthold Luminometer LB953 (Wallac Inc., Gaithersburg, MD) connected to a PC running the Autolumat-PC Control software (Wallac Inc., Gaithersburg, MD). Oocytes (singly or in pairs) were transferred to plastic tubes (75 x 12 mm, Sarstedt) containing 2.9 ml Ca ++ -free ND-96 medium. Each cRNA pool was tested using a minimum of 3 tubes containing oocytes. Bioluminescence measurements were triggered by the injection of 0.1 ml of 30 μM galanin or αNDP-MSH (1 μM final concentration) and recordings were followed for 2 min. to observe kinetic responses consistent with an _P3-mediated response. Background and Summary of Results - Melanocortins interact with 5 known G-protein-coupled receptors (GPC-R) which in turn activate the intracellular effector adenylate cyclase to generate elevated concentrations of the cyclic nucleotide cAMP (Cone, et al. 1996, Recent Progress Hormone Res., 51, 287-317). This example shows an alternative assay to measure the activation of melanocortin receptors that is more robust, of a higher throughput, and reduced cost than assays which directly measure cAMP. In addition, this assay would have the added feature of finding ligands to receptors in which the signaling properties are unknown (orphan receptors) that may represent novel melanocortin receptors. The assay described herein relies on the use of G protein alpha subunits which have the unique property of bypassing the normal route of receptor activation and subsequent coupling to intracellular effectors. In the search for GPC-R agonists and antagonists, it is generally easier to develop assays which rely on the activation of phospholipase C, rather than the modulation of the activity of adenylate cyclase (Coward et al., 1999, Anal. Biochem 270:242-248 ). Thus, a goal would be to switch the native intracellular coupling pathway from adenylate cyclase to phospholipase C. Previous studies have shown that promiscuous Ga subunits (Gal5 and Gal6) can functionally couple a variety of GPC-R' s to activation of the phospholipase C signal transduction pathway (Offermans and Simon, 1995, J. Biol. Chem. 270, 15175-15180; Burg et al., 1995, FEBS Lett. 377: 426-428; Milligan et al., 1996, Trends Pharmacol. Sci., 17: 235- 237). Similarly, chimeric Gα subunits in which C-terminal amino acids (final 3 to 10 residues) have been replaced, permit the activation of phospholipase C (Conklin et al., 1993, Nature 363, 274-276; Conklin, et al., 1996, Mol. Pharmacol. 50: 885-90; Milligan and Rees, 1999, Trend Pharmacol Sci. 20, 118-24). This is typified in the G αq i5 protein in which a G αq backbone with 3 amino acid changes at the extreme C-terminus from the G α ; subunit results in a switch in the coupling pathway from inhibition of adenylate cyclase to activation of phospholipase C. Figure 9 shows an aequorin bioluminescence assay in which the rat melanocortin 4 receptor (MC-4R), upon stimulation by the melanocortin agonist peptide αNDP-MSH (1 μM), can functionally couple to activation of phospholipase C (mobilization of intracellular calcium reported by aequorin bioluminescence) when expressed in Xenopus oocytes in the presence of the Gαl5 subunit. Each tracing represents the bioluminescence response (in counts/sec) over time (in seconds) for 1-2 individual oocytes. Figure 10 shows a similar experiment using the G α s subunit. In this case, the human galanin receptor 1 (hGALRl), which is coupled in its native state to inhibition of adenylate cyclase, can now when co- expressed with the G αq i 5 subunit, show functional activation in response to agonist treatment (1 μM human galanin).

Claims: WHAT IS CLAIMED: 1. A transgenic non-human animal whose somatic cells and germ cells contain at least one non-functional gene coding for a MC-4R protein, wherein females heterozygous for the non-functional gene have a body weight similar to a wild type animal. 2. A cell line derived from a transgenic animal of claim 1. 3. A transgenic non-human animal of claim 1 wherein said animal exhibits a disorder selected from the group consisting of an obesity syndrome, diabetes, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGFl function, and other states resulting from GH deficiency. 4. A transgenic non-human animal of claim 1 wherein said animal exhibits an obesity syndrome. 5. A transgenic non-human animal of claim 1 which is a transgenic mouse. 6. A transgenic mouse of claim 5 wherein said animal exhibits a disorder selected from the group consisting of an obesity syndrome, diabetes, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGFl function, and other states resulting from GH deficiency. 7. A transgenic mouse of claim 6 wherein said mouse exhibits an obesity syndrome. 8. A cell line derived from a transgenic mouse of claim 5. 9. A cell line derived from a transgenic mouse of claim 6. 10. A cell line derived from a transgenic mouse of claim 7. 11. A mouse of claim 5, wherein the mouse is fertile and capable of transmitting the altered MC-4R gene to its offspring. 12. A mouse of claim 6, wherein the mouse is fertile and capable of transmitting the altered MC-4R gene to its offspring. 13. A mouse of claim 7, wherein the mouse is fertile and capable of transmitting the altered MC-4R gene to its offspring. 14. A method of identifying a compound that effects metabolic rate and food intake by modulation of a melanocortin-4 receptor protein, which comprises: (a) exposing a compound with the melanocortin-4 receptor protein; determining the interaction between the compound and the melanocortin-4 receptor protein; (b) administering the compound to a wild type mouse and the transgenic mouse of claim 1 ; and, (c) determining the effect of the compound on metabolic rate and food intake. 15. A method of identifying a compound that effects metabolic rate and food intake by modulation of a melanocortin-4 receptor protein, which comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of MC-4R in the cells; (b) exposing the test cells to the compound; (c) measuring the amount of binding of the compound to MC-4R; (d) comparing the amount of binding of the compound to MC-4Rin the test cells with the amount of binding of the compound to control cells that have not been transfected with MC-4R, and, (e) administering the compound to a wild type mouse and the transgenic mouse of claim 5 and determining the effect of the compound on metabolic rate and food intake. 16. A method of identifying a compound that effects metabolic rate and food intake by modulation of a MC-4R, which comprises: (a) transfecting or transforming cells with an expression vector which directs expression of MC-4R in the cells, resulting in test cells; (b) preparing membranes containing MC-4R from the test cells and exposing the membranes to a the compound; (c) measuring the amount of binding of the compound to the MC-4R in the membranes in the presence and the absence of the compound; (d) comparing the amount of binding of the compound to MC-4R in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to MC-4R in the membranes in the presence of the substance indicates that the substance is capable of binding to MC-4R; and, (e) administering the compound to a wild type mouse and the transgenic mouse of claim 5 and determining the effect of the compound on metabolic rate and food intake. 17. A method of identifying a compound that effects metabolic rate and food intake by modulation of a MC-4R, which comprises: (a) transfecting or transforming test cells with an expression vector or cRNA molecule which directs expression of MC-4R; (b) introducing a Gαprotein subunit into the test cells or transfecting or transforming the test cells with an expression vector or cRNA molecule which directs expression of the Gα-protein subunit; (c) introducing a detector molecule into the test cells or transfecting or transforming the test cells with an expression vector which directs expression of a detector molecule; (d) contacting the test cells with a compound suspected of being a modulator of MC-4R; and, (e) determining whether the compound modulates MC-4R activity by monitoring the detector molecule. 18. A method of claim 17 wherein the compound is administered to a wild type mouse and the transgenic mouse which shows a homozygous, hemizygous or heterozygous deficiency in MC-4R, and determining the effect of the compound on metabolic rate and food intake. 19. A method according to Claim 18 wherein the Gα protein subunit is selected from the group consisting of a G α π subunit, a G α ι 5 subunit, a G α ι 6 subunit, and a G αq j 5 subunit. 20. A method according to claim 17 wherein the detector molecule is aequorin, or a biologically active form thereof. 21. A method of claim 18 wherein the Gα protein subunit is a G « ι 5 subunit. 22. A method according to claim 21 wherein the detector molecule is aequorin, or a biologically active form thereof. 23. A method of claim 17 wherein the test cells are Xenopus oocytes. 24. A method according to claim 23 wherein the Gα protein subunit is selected from the group consisting of a G α n subunit, G α ι 5 subunit, G α ι 6 subunit, and a G αq i 5 subunit. 25. A method of claim 24 wherein the Gα protein subunit is a G α ι 5 subunit. 26. A method according to claim 25 wherein the detector molecule is aequorin, or a biologically active form thereof.

Inventors: CHEN AIRU S, CHEN HOWARD Y, FEIGHNER SCOTT D, FORREST MICHAEL J, HRENIUK DONNA, MACINTYRE DUNCAN E, METZGER JOSEPH M, PALYHA OKSANA C, VAN DER PLOEG LEONARDUS H T, CHEN, AIRU, S., CHEN, HOWARD, Y., FEIGHNER, SCOTT, D., FORREST, MICHAEL, J., HRENIUK, DONNA, MACINTYRE, DUNCAN, E., METZGER, JOSEPH, M.., PALYHA, OKSANA, C., VAN DER PLOEG, LEONARDUS, H., T.
Applicants: CHEN AIRU S, CHEN HOWARD Y, FEIGHNER SCOTT D, FORREST MICHAEL J, HRENIUK DONNA, MACINTYRE DUNCAN E, MERCK & CO INC, METZGER JOSEPH M, PALYHA OKSANA C, PLOEG LEONARDUS H T V D, CHEN, AIRU, S., CHEN, HOWARD, Y., FEIGHNER, SCOTT, D., FORREST, MICHAEL, J., HRENIUK, DONNA, MACINTYRE, DUNCAN, E., MERCK & CO., INC., METZGER, JOSEPH, M.., PALYHA, OKSANA, C., VAN DER PLOEG, LEONARDUS, H., T.
Application year: 2000
Main classification: A01K67
IPC codes: A01K67, C07K14, C12N5, C12N15, C12Q1