WO2001068706A1

WO2001068706A1 - Melanin concentrating hormone receptor chimeric and fusion proteins

Info

Publication number: WO2001068706A1
Application number: PCT/US2001/008071
Authority: WO
Inventors: Donald J. Marsh
Original assignee: Merck & Co., Inc.
Priority date: 2000-03-15
Filing date: 2001-03-14
Publication date: 2001-09-20
Also published as: EP1265926A1; CA2402147A1; JP2003532388A

Abstract

The present invention features melanin concentrating hormone receptor (MCH-R) chimeric and fusion proteins. MCH-R chimeric proteins comprise an MCH-R polypeptide region made up of at least two or more polypeptide regions characteristic of MCH-R found in different species. MCH-R fusion proteins comprise an MCH-R polypeptide region and a fluorescent protein region.

Description

TITLE OF THE INVENTION

MELANIN CONCENTRATING HORMONE RECEPTOR CHIMERIC AND

FUSION PROTE1-NS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to provisional application U.S. Serial No. 60/189,698, filed March 15, 2000, hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION The references cited herein are not admitted to be prior art to the claimed invention.

Neuropeptides present in the hypothalamus play a major role in mediating the control of body weight. (Flier et al, 1998. Cell, 92, 437-440.) Melanin- concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide synthesized as part of a larger pre-prohormone precursor in the hypothalamus which also encodes neuropeptides NEI and NGE. (Nahon et al, 1990. Mol. Endocrinol. 4, 632-637.) MCH was first identified in salmon pituitary, and in fish MCH affects melanin aggregation thus affecting skin pigmentation. L trout and in eels MCH has also been shown to be involved in stress induced or CRF-stimulated ACTH release. (Kawauchi et al, 1983. Nature 305, 321-323.)

In humans two genes encoding MCH have been identified that are expressed in the brain. (Breton et al, 1993. Mol Brain Res. 18, 297-310.) In mammals MCH has been localized primarily to neuronal cell bodies of the hypothalamus which are implicated in the control of food intake, including perikarya of the lateral hypothalamus and zona inertia. (Knigge et al, 1996. Peptides 17, 1063- 1073.)

Pharmacological and genetic evidence suggest that the primary mode of MCH action is to promote feeding (orexigenic). MCH mRNA is up regulated in fasted mice and rats and in the ob/ob mouse. (Qu et al, 1996. Nature 380, 243-247.) Injection of MCH centrally (ICN) stimulates food intake and MCH antagonizes the hypophagic effects seen with -melanocyte stimulating hormone (odVISH). (Qu et al, 1996. Nature 380, 243-247.) MCH-deficient mice are lean, hypophagic, and have increased metabolic rate. (Shimada et al, 1998. Nature 396, 670-673.)

MCH action is not limited to modulation of food intake as effects on the hypothalamic-pituitary-axis have been reported. (Νahon 1994. Critical Rev. in Neurobiol 8, 221-262.) MCH may be involved in the body response to stress as MCH can modulate the stress-induced release of CRF from the hypothalamus and ACTH from the pituitary. In addition, MCH neuronal systems may be involved in reproductive or maternal function.

Several references describe a receptor that is indicated to bind MCH. (Chambers et al, 1999. Nature 400, 261-265; Saito et al, 1999. Nature 400, 265-269; Bachner et al, 1999. FEBS Letters 457:522-524; Shimomura et al, 1999. Biochemical and Biophysical Research Communications 261, 622-626; and Lembo et al, 1999. Nat. Cell Biol. 1, 267-271.)

SUMMARY OF THE INVENTION

The present invention features melanin concentrating hormone receptor

(MCH-R) chimeric and fusion proteins. MCH-R chimeric proteins comprise an

MCH-R polypeptide region made up of at least two or more polypeptide regions characteristic of MCH-R found in different species. MCH-R fusion proteins comprise an MCH-R polypeptide region and a fluorescent protein region.

An MCH-R polypeptide region provides a functional G-protein coupled receptor region able to bind MCH and transduce an intracellular signal.

Examples of MCH-R polypeptide regions include naturally occurring MCH-R, chimeric MCH-R containing two or more regions from naturally occurring MCH-R, and functional derivatives thereof.

Reference to the terms "characteristic" and "derivatives thereof describe a relationship to a reference sequence. In both cases, there is at least about

75% sequence similarity to the reference sequence. Thus, a first aspect of the present invention describes a fusion protein comprising (a) an MCH-R polypeptide region and (b) a fluorescent polypeptide region. The fluorescent polypeptide region is joined directly, or though a polypeptide linker, to the carboxy side of the MCH-R polypeptide region.

Another aspect of the present invention describes an MCH-R chimeric protein. The protein comprises: (a) an MCH-R binding region characteristic of a human MCH-R, (b) a transmembrane domain characteristic of a non-human MCH-R, and (c) an intracellular domain characteristic of a non-human MCH-R.

Another aspect of the present invention describes a nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein. Such nucleic acid comprises either a contiguous nucleotide sequence that codes for the protein or a sequence that is processed by a host cell to produce a contiguous nucleotide sequence encoding for the protein. Processing of a nucleic acid sequence to produce a contiguous nucleotide sequence encoding for a protein can occur by the splicing together of exons resulting in intron removal. Another aspect of the present invention describes an expression vector comprising a nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.

Another aspect of the present invention describes a recombinant cell comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein. The nucleic acid may be part of the host genome or may exist independently of the host genome.

Another aspect of the present invention describes a non-human transgenic animal comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein. Another aspect of the present invention describes a method for assaying for MCH-R active compounds by measuring the effect of a test preparation on one or more MCH-R activities. The method is performed using either an MCH-R fusion protein or an MCH-R chimeric protein described herein.

Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates aequorin assay results comparing a mouse MCH-R fusion with a human wild type MCH-R and a CMV-EGFP control.

Figure 2 illustrates a cAMP flashplate assay of CHO cell clones stably expressing mMCH-lR-EGFP. Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH at the indicated concentrations in the presence of 10 μM forskolin. Cells were then lysed and assayed for bound [¹²⁵I]cAMP. Mouse MCH-IR-EGFP clones exhibited EC50 values (0.1111, 0.1255, 0.1291, or 0.2304 nM) indistinguishable from that of a CHO cell clone expressing the wild-type human short isoform of MCH-IR ( 0.1282 nM). Figure 3 illustrates a cAMP flashplate assay of CHO cell clones stably expressing human short/mouse species chimeric MCH-IR-EGFP. Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH at the indicated concentrations in the presence of 10 μM forskolin. Cells were then lysed and assayed for bound [¹²⁵I]cAMP. Human short/mouse species chimeric MCH-IR-EGFP clones exhibited EC50 values (0.0366, 0.0462, 0.2117, or 0.2499 nM) indistinguishable from that of a CHO cell clone expressing the wild-type human short isoform of MCH-IR (0.1137 nM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features MCH-R chimeric and fusion proteins. Such proteins have a variety of different uses including being used as a research tool to study MCH-R function and dynamics, and being used to screen for MCH-R agonists and antagonists. The MCH-R provides a target to achieve different beneficial effects in a patient. Preferably, MCH-R activity is modulated to achieve one or more of the following: weight loss, weight gain, treat cancer (e.g., colon or breast), reduce pain, treat diabetes, reduce stress, or teat sexual dysfunction.

Modulation of MCH-R activity can be achieved by evoking a response at the MCH receptor or by altering a response evoked by an MCH receptor agonist or antagonist. Compounds modulating MCH-R receptor activity include agonists, antagonists, and allosteric modulators. Generally, MCH-R antagonists and allosteric modulators negatively affecting activity will be used to achieve weight loss, treat cancer (e.g., colon or breast), reduce pain, reduce stress, or teat sexual dysfunction; and MCH-R agonists and allosteric modulators positively affecting activity will be used to produce a weight gain.

Preferably, MCH-R activity is modulated to achieve a weight loss or to treat diabetes in a patient. Diabetes mellitus can be treated by modulating MCH-R activity to achieve, for example, one or both of the following: enhancing glucose tolerance or decreasing insulin resistance.

Excessive body weight is a contributing factor to different diseases, including hypertension, diabetes, dyslipidemias, cardiovascular disease, gall stones, osteoarthritis, and certain forms of cancers. Bringing about a weight loss can be used, for example, to reduce the likelihood of such diseases and as part of a treatment for such diseases. Weight reduction can be achieved by modulating MCH-R activity to obtain, for example, one or more of the following effects: reducing appetite, increasing metabolic rate, reducing fat intake, or reducing carbohydrate craving.

Increasing body weight is particularly useful for a patient having a disease or disorder, or under going a treatment, accompanied by weight loss. Examples of diseases or disorders accompanied by weight loss include anorexia, AIDS, wasting, cachexia, and frail elderly. Examples of treatments accompanied by weight loss include chemotherapy and radiation therapy.

MCH-R Chimeric Proteins MCH-R chimeric proteins contain an MCH-R polypeptide region made up by at least two or more polypeptide regions characteristic of MCH-R found in different species. The different polypeptide regions that are present provide for an N~ terminal extracellular domain; a transmembrane domain made up of transmembrane regions, extracellular loop regions, and intracellular loop regions; and an intracellular carboxy terminus domain. Examples of MCH-R amino acid sequences include the following: SEQ. ID. NO. 1 (human MCH1R long form), SEQ. ID. NO. 2 (human MCH1R short form), and SEQ. ID. NO. 3 (mouse MCH1R).

Preferably, the MCH-R chimeric protein comprises an MCH-R binding region characteristic of a human MCH-R along with transmembrane and intracellular domains characteristic of a non-human MCH-R. There are substantial amino acid differences between the N-terminus of the MCH-R present in humans and that present in other species such as mice. Such differences could result in, for example, the mouse MCH-R having different intrinsic properties and responsiveness to agonists and/or antagonists than the human MCH-R. The presence of a human MCH-R binding region provides for a "humanized" MCH-R chimeric receptor.

The transmembrane and intracellular domains characteristic of a non- human MCH-R can be used in conjunction with a non-human host to provide a more naturally occurring environment for these regions. For example, an MCH-R chimeric having mouse transmembrane and intracellular domains are preferably used in murine cells lines or in transgenic mice.

MCH-R chimeric proteins may contain regions other than extracellular, transmembrane, and intracellular domains that do not substantially decrease the activity of the protein. Preferably, additional regions do not cause a decrease of more than about 25% of MCH-R activity as measured using one or more of the assays described in the examples provided below. Examples of additional regions that may be present include fluorescent protein regions and linker regions.

In an embodiment of the present invention, the MCH-R chimeric protein comprises: (a) an MCH binding region characteristic of a first species and (b) a transmembrane and intracellular domain region characteristic of a second species joined directly, or though a linker, to the carboxy side of the MCH binding region. Preferably, the protein comprises, consists, or consists essentially of an MCH-R polypeptide having a sequence similarity of at least about 75%, at least 85%, or at least 95% with either SEQ. ID. NO. 4 (human short form/mouse species chimeric MCHIR) or SEQ. ID. NO. 5 (human long form/mouse species chimeric). Even more preferably, the protein comprises, consists essentially of, or consists of, SEQ. ID. NO. 4 or SEQ. ID. NO. 5.

Sequence similarity for polypeptides can be determined by BLAST. (Altschul et al, 1997. Nucleic Acids Res. 25, 3389-3402, hereby incorporated by reference herein.) In an embodiment of the present invention, sequence similarity is determined using tBLASTn search program with the following parameters: MATRIX:BLOSUM62, PER RESIDUE GAP COST: 11, and Lambda ratio: 1.

Differences in naturally occurring amino acids are due to different R groups. An R group effects different properties of the amino acid such as physical size, charge, and hydrophobicity. Amino acids can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine); basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid). Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing a change in polypeptide functioning. Changes outside of different amino acids groups can also be made.

Preferably, such changes are made taking into account the position of the amino acid to be substituted in the polypeptide. For example, arginine can substitute more freely for nonpolor amino acids in the interior of a polypeptide then glutamate because of its long aliphatic side chain. (See, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix lC.) MCH-R Fusion Proteins

MCH-R fusion proteins contain an MCH-R polypeptide region and a fluorescent protein region either directly joined together or joined together through a linker. These regions provide MCH-R activity and a marker for evaluating MCH-R dynamics.

An MCH-R polypeptide region provides functional MCH-R activity and includes naturally occurring MCH-R, chimeric MCH-R, and derivatives thereof.

Preferred derivatives thereof have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to a naturally occurring MCH-R or a chimeric

MCH-R described herein.

A fluorescent protein region contains a chromophore that fluoresces.

Preferably, the fluorescent protein region is the green fluorescent protein of the jellyfish Aequorea victoria or a derivative thereof. Preferred derivatives have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to the Aequorea victoria green fluorescent protein (GFP). The Aequorea victoria green fluorescent protein and examples of derivatives thereof are described by Cormack et al, 1996. Gene 17, 33-38; Yang et al, 1996. Nucleic Acids Research 24, 4592-4593;

Tsien et al, U.S. Patent No. 5,625,048; Tsien et al, U.S. Patent No. 5,777,079; and Cormack et al, U.S. Patent No. 5,804,387 (each of which are hereby incorporated by reference herein).

In different embodiments the MCH-R polypeptide region comprises, consists essentially of, or consists of, a sequence selected from the group consisting of: SEQ. LD. NO. 1, SEQ. D. NO. 2, SEQ. LD. NO. 3, SEQ. LD. NO. 4, and SEQ. ID. NO. 5; and the fluorescent polypeptide region comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of SEQ. ID.

NO. 6 (GFP), SEQ. ID. NO. 7 (EGFP), SEQ. ID. NO. 8 (Emerald), SEQ. ID. NO. 9

(Topaz), and SEQ. ID. NO. 10 (Wlb). EGFP, Emerald, Topaz, and Wlb are derivatives of GFP. The optionally present linker is a polypeptide region that is preferably from 1 to about 100 amino acids in length. In different embodiments the linker is up to 75, 50 or 25 amino acids in length.

Preferably, the MCH-R fusion protein comprises, consists essentially of, or consists of, the MCH-R polypeptide region and the fluorescent polypeptide region. More preferably, the protein comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 11 (mouse MCHlR-linker-EGFP), SEQ. ID. NO. 12 (mouse MCHIR/EGFP direct fusion), SEQ. D. NO. 13 (human short form/mouse species chimeric MCHlR-linker- EGFP), or SEQ. ID. NO. 14 (human long form/mouse species chimeric MCH1R- linker-EGFP).

MCH-R Chimeric and Fusion Proteins Nucleic Acid and Expression

MCH-R chimeric and fusion proteins can be produced using techniques well known in the art. Preferably, such proteins are produced by recombinant expression inside a host cell by way of an expression vector or by way of nucleic acid integrated into the host genome. Examples of nucleic acid sequences encoding for MCH-R polypeptide regions, fluorescent protein regions, MCH-R chimeric proteins, and MCH-R fusion proteins are provided for by SEQ. D. NOs. 15-

29 (see Example 1, infra). Starting with a particular amino acid sequence and the known degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost all amino acids are encoded for by different combinations of nucleotide triplets or codons. The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990).

Amino acids are encoded for by codons as follows:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteine: codons UGC, UGU

D=Asp= Aspartic acid: codons GAC, GAU E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, UUU

G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His=Histidine: codons CAC, CAU

I=Le=Isoleucine: codons AUA, AUC, AUU K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

M=Met=Methionine: codon AUG

N=Asn=Asparagine: codons AAC, AAU

P=Pro=Proline: codons CCA, CCC, CCG, CCU Q=Gln=Glutamine: codons CAA, CAG R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU T=Thr=Threonine: codons ACA, ACC, ACG, ACU V=Val=Valine: codons GUA, GUC, GUG, GUU W=Trp=Tryρtophan: codon UGG Y=Tyr=Tyrosine: codons UAC, UAU

Examples of techniques for introducing nucleic acid into a cell and expressing the nucleic acid to produce protein are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989.

An expression vector contains recombinant nucleic acid encoding for a polypeptide along with regulatory elements for proper transcription and processing. The recombinant nucleic acid contains two or more nucleic acid regions not naturally associated with each other. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing recombinant nucleic acid in a particular host. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses.

Generally, the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal providing for processing in eukaryotic cells. Preferably, an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.

Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. Mammalian expression vectors well known in the art include pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), pCI- neo (Promega) and .lambda.ZD35 (ATCC 37565). Bacterial expression vectors well known in the art include pETlla (Novagen), lambda gtll (Invitrogen), pcDNAJJ (Invitrogen), and pKK223-3 (Pharmacia). Fungal cell expression vectors well known in the art include pYES2 (Invitrogen) and Pichia expression vector (Invitrogen). Insect cell expression vectors well known in the art include Blue Bac HI (Invitrogen).

Recombinant host cells may be prokaryotic or eukaryotic. Examples of recombinant host cells include the following: bacteria such as E. coli; fungal cells such as yeast; mammalian cells such as human, bovine, porcine, monkey, hampster, and rodent; and insect cells such as Drosophila and silkworm derived cell lines. Commercially available mammalian cell lines include L cells L-M(TK.sup.-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 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) and MRC-5 (ATCC CCL 171).

To enhance expression in a particular host it may be useful to modify the sequence to take into account codon usage of the host. Codon usage of different organisms are well known in the art. (See, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix lC.)

Expression vectors may be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation. Nucleic acid encoding for a polypeptide can be expressed in a cell without the use of an expression vector employing, for example, synthetic mRNA or native mRNA. Additionally, mRNA can be translated in various cell-free systems such as wheat germ extracts and reticulocyte extracts, as well as in cell based systems, such as frog oocytes. Introduction of mRNA into cell based systems can be achieved, for example, by microinjection.

Techniques for producing transgenic animals are well known in the art. Examples of such techniques are provided for by Teratocarcinomas and embryonic stem cells: a practical approach. Ed. By E. J. Robertson, JJRL Press Limited, Oxford, England (1987); and Gene Targeting: a practical approach. Ed. By A. L. Joyner, Oxford University Press Inc. New York, NY (1993).

G-Protein Coupled Receptor Assays

MCH-R is G-protein coupled receptor. Techniques for measuring different G-protein activities, such as Gi/o, Gs, and Gq are well known in the art. MCH-R activity is preferably assayed for by measuring either Gi/o or Gq. Gi/o and Gs activity can be measured using techniques such as a melonaphore assay, measuring cAMP production, measuring inhibition of cAMP accumulation, and measuring binding of 35S-GTP. cAMP can be measured using different techniques such as radioimmunoassay and indirectly by cAMP responsive gene reporter proteins.

Gq activity can be measured using techniques such as those measuring intracellular Ca . Examples of techniques well known in the art that can be employed to measure Ca include the use of dyes such as Fura-2 and the use of Ca - bioluminescent sensitive reporter proteins such as aequorin. An example of a cell line employing aequorin to measure G-protein activity is HEK293/aeql7. (Button et al, 1993. Cell Calcium 14, 663-671, and Feighner et al, 1999. Science 284, 2184- 2188, both of which are hereby incorporated by reference herein.)

Functional assays can be performed using individual compounds or preparations containing different compounds. A preparation containing different compounds where one or more compounds affect MCH-R chimeric or fusion protein activity can be divided into smaller groups of compounds to identify the compound(s) affecting MCH-R chimeric or fusion protein activity. In an embodiment of the present invention a test preparation containing at least 10 compounds is used in a functional assay. Functional assays can be performed using recombinantly produced

MCH-R chimeric or fusion protein present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the MCH-R chimeric or fusion protein expressed from recombinant nucleic acid and an appropriate membrane for the polypeptide; and the use of a purified MCH-R chimeric or fusion protein produced by recombinant means that is introduced into a different environment suitable for measuring G-protein activity.

Fluorescent Protein Assays

Fluorescent protein joined to an MCH receptor can be employed to study different aspects of receptor dynamics including receptor sequestration, receptor densitization, and receptor localization. The fluorescent protein can be used in in vitro or in vivo systems.

In vitro applications of fluorescent proteins can be performed using techniques well known in the art. Examples of such techniques are provided by Barak et al, 1997. Mol Pharm. 5, 177-184; Tarasova et al, 1997. J. Biol. Chem. 272, 14817-14824; Lin et al, 1998. Mol. Cell. Endo. 146, 27-37; Tarasova et al, 1998. J. Biol Chem. 273, 15883-15886; Kallal et al, 1998. J. Biol. Chem. 273, 322-328; Groake et al, 1999. J. Biol. Chem. 274, 23263-23269; Doherty et al, 1999. Biochem. J. 341, 415-422; Brock et al, 1999. Proc. Natl Acad. Sci. USA 96, 10123- 10128; Cornea et al, 1999. Endocrinology 140, 4272-4280; and Lembo et al, 1999. Nat. Cell Biol. 1, 267-271 (these references are not admitted to be prior art to the claimed invention).

In vivo applications of fluorescent proteins can be performed using techniques well known in the art. Examples of such techniques are provided by Mombaerts et al, 1996. Cell 87, 675-686; Rodriquez et al, 1999. Cell 97, 199-208; Spergel et al, 1999. J. Neurosci. 1, 2037-2050; and Zuo et al, 1999. Proc. Natl. Acad. Sci. USA 96, 14100-14105 (these references are not admitted to be prior art to the claimed invention).

EXAMPLES

Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.

Example 1:

Amino acid and nucleic acid sequence information for SEQ. ID. NOs. 1-29 are provided below. SEQ. ID. NOs. 1-29 include examples of polypeptide and encoding nucleic acid sequences for MCH-R polypeptide regions, fluorescent polypeptide regions, fusion proteins and chimeric proteins. some cases the encoding nucleic acid is shown with additional nucleic acid upsteam or downstream from an open reading frame.

SEQ. ID. NO. 1: Human long form MCHIR

MSVGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR TGSISYlϊ tfPSWGTICIXGπGNSTVjEAVVK^

LLFLLGMPFMMQLMGNGVWFIFGETMCTLITAMDANSQFTSTYILTAMAIDR YLATVHPISSTK KPSVATLVICLLWALSFLSRRPVWLYAP IPFPGGAVGCGI IΛLPNPDTΌLYWFTLYQFFLAFALPFVVΠ^,AAYVPJLQRMTSSVAPASQRS]_RLR TKRVTRTNIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANS CLNPFVYΓVLCETFRKRLVLSVKPAAQGQLRAVSNAQTADEERTESKGT

SEQ. ID. NO. 2: Human short form MCHIR MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISYΓNΠMPSVFGTICLLGΠG

FGETMCTLITAMDANSQFTSTYILTAMAIDRYLATVHPISSTKFRKPSVATLVI CLLWALSHSL PVWLYARLIPFPGGAVGCGΠILPNPDTDLYWFTLYQFFLAFA LPFVVΓΓAAYVRILQRMTSSVAPASQRSIRLRTKRVTRTAIAICLVFFVCWAPY YVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLSV KPAAQGQLRAVSNAQTADEERTESKGT

SEQ. ID. NO. 3: Mouse MCHIR

MDLQASLLSTGPNASNISDGQDNFrLAGPPPRTRSVSYJ-NiTMPSVFGTICLLGI VGNSTVlTAVVia SKLFiWCSNVPDlEjXNLSVVDLLFLLGMPF mQLMGNG WHFGETMCTLITAMDANSQFTSTYJI.TAMAIDRYLATVHPISSTKFRKPSMAT LVICIiWALSHSITPVWLYARLIPFPGGAVGCGIPJ PNPDTDLYWFTLYQFFLA FALPFVVITAAYVKJLQRMTSSVAPASQRSIRLRTKRVTRTAJNICLVFFVCWA PYYVLQLTQLSISRPTLTFVYLYΝAAISLGYAΝSCLΝPFVYTVLCETFRKRLVLS VKPAAQGQLRTVSΝAQTADEERTESKGT

SEQ. ID. NO. 4: Human short form/mouse species chimeric MCHIR

MDLEASIXPTGPNASNTSDGPDNLTSAGSPPRTGSISYINΠMPSVFGTICLLGΠG NSTVIFAVVKKSKLFJNVCNNVPDLPIINLSVVDLLFLLGMPFMMQLMGNGVWH FGETMCTLLTAMDANSQFTSTYILTAMARORYLATVHPISSTKFRKPSMATLVI CIXWALSHS PVWLYARLJTFPGGAVGCGIRLPNPDTDLYWFTLYQFFLAFA LPFVVJTAAYVKL QRMTSSVAPASQRS LRTKRVTRTAINICLVFFVCWAPY YVLQLTQLSISRPTLTFVYLYΝAAISLGYAΝSCLΝPFVYΓVLCETFRKRLVLSV KPAAQGQLRTVSΝAQTADEERTESKGT

SEQ. ID. NO.5: Human long form/mouse species chimeric MCHIR

MSVGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR TGSISYINJTMPSWGTICI GHGNSTVIFAVVKKSKLHWCNNWDIFJXN^ Ll-J^LLGMPFNπHQLMGNGVWHFGETMCTLrTAMDANSQFTSTYJXTAMAro YLNTVHPISSTKFT KPSMNTLVICLLWALSπSITPVWLYARLIPFPGGAVGCGI RLPΝPDTDLYWFTLYQΪΨLAFALPFVVITAAYVKILQRMTSSVAPASQRSIRLR TKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYΝAAISLGYAΝS CLΝPFVYTVLCETFRKRLVLSVKPAAQGQLRTVSΝAQTADEERTESKGT

SEQ. ID. NO. 6: GFP

MSKGEELFTGVWILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL PVPWPTLVTTFSYGVQCFSRYPDFIMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKG1OFKEDGNILGHKLEYNYNSHNVYIMADKQ KNGlKVNFK]TFLNmDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKD PNEKRDHMVLLEFVTAAGITHGMDELYK

SEQ. ID. NO. 7: EGFP

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDF KQHDFFKSAMPEGYVQERTJJFFKDDG NYKTRAEVKIΕGDTLVNRffiLKGroFKEDGNILGHKLEYNYNSHNVYIMADK QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK DPNEKRDHMVLLEFVTAAGITLGMDELYK

SEQ. ID. NO. 8: Emerald

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro He Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe lie Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gin Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg Thr lie Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Le Glu Leu Lys Gly lie Asp Phe Lys Glu Asp Gly Asn lie Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Lys Val Tyr Le Thr Ala Asp Lys Gin Lys Asn Gly Le Lys Val Asn Phe Lys Thr Arg His Asn He Glu Asp Gly Ser Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro lie Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly He Thr Leu Gly Met Asp Glu Leu Tyr Lys SEQ. ID. NO. 9: Topaz

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Le Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe He Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gin Cys Phe Ala Arg Tyr Pro Asp His Met Arg Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg Thr Le Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg He Glu Leu Lys Gly Ee Asp Phe Lys Glu Asp Gly Asn He Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr lie Met Ala Asp Lys Gin Lys Asn Gly He Lys Val Asn Phe Lys Ue Arg His Asn He Glu Asp Gly Ser Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro De Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gin Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Le Thr Leu Gly Met Asp Glu Leu Tyr Lys

SEQ. ID. NO. 10: W1B

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Le Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe He Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg Thr Le Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg lie Glu Leu Lys Gly lie Asp Phe Lys Glu Asp Gly Asn Le Leu Gly His Lys Leu Glu Tyr Asn Tyr Ee Ser His Asn Val Tyr Ue Thr Ala Asp Lys Gin Lys Asn Gly Le Lys Ala His Phe Lys He Arg His Asn He Glu Asp Gly Ser Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro He Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly He Thr Leu Gly Met Asp Glu Leu Tyr Lys

SEQ. ID. NO. 11: Mouse MCHlR-linker-EGFP MDLQASLLSTGPNASMSDGQDNFTLAGPPPRTRSVSYINΠMPSVFGTICLLGI VGNSTVJTAVVKKSKLHWCSNWD LNLSVVDLLFLLGMPFNIIHQLMGNGV WHFGETMCTLITAMDANSQFΓSTYILTAMAJΌRYLATVHPISSTKFRKPSMAT LVICLLWALSMSITPVWLYAI^IPFPGGAVGCG]TLPNPDTDLYWFTLYQFFLA FALPFVVTT'AAYVKILQPJVITSSVAPASQRSII^RTK-RVTRTAIAICLVFFVCWA PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYTVLCETFRKRLVLS VKPAAQGQLRTVSNAQTADEERTESKGTVDGTAGPGSINTMVSKGEELFTGV VPILVELDGDVΝGHKΓSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGΝYKTRAEVKFE

GDTLVΝPJELKGIDFKEDGΝILGHKI^YΝYΝSHΝVYJ ADKQKΝGIKVΝFKR HΝFFIDGSVQLADHYQQΝTPIGDGPVLLPDΝHYLSTQSALSKDPΝEKRDHMVL LEFVTAAGΓTLGMDELYK

SEQ. ID. NO. 12: Mouse MCHIR/EGFP direct fusion

MDLQASI STGPΝASMSDGQDΝFTLAGPPPRTRSVSYJMIMPSWGTICLLGI VGΝSTVlFAVVTαMKIirWCSΝV

WHFGETMCTLlTAMDAΝSQFTSTYILTAMAroRYLATVHPISSTKFRKPSMAT LVICII.WALSHSITPVWLYAPJ-,IPFPGGAVGCG]TLPΝPDTDLYWFTLYQFFLA FALPFVVITAAYV]5ILQRMTSSVAPASQRSJTLRTKRVTRTAIAICLVFFVCWA PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIΛT.CETFRKRLVLS VKPAAQGQLRTVSNAQTADEERTESKGTMVSKGEELFTGVVPILVELDGDVN GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIT^KDDGNYKTRAEVKFEGDTLVNRIELKGI DFKEDGNILGHKLEYNYNSHNVYMADKQK-NGKVNFKmHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEK-RDHMVLLEFVTAAGITLG MDELYK

SEQ. ID. NO. 13: Human short form/mouse species chimeric MCHlR-linker- EGFP

MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISYINILMPSVFGTICLLGΠG NSTVIFAVVKKSKLFJWCNNWDIFJINLSVVDLLFLLGMPFMIHQLMGNGVWH

FGETMCTLITAMDANSQFTSTYILTAMAIDRYLATVHPISSTKFRKPSMATLVI CLLWALSHSJ PVWLYAP IPFPGGAVGCGMLPNPDTDLYWFTLYQFFLAFA LPFVVITAAYVKL QRMTSSVAPASQRSIRLRTKRVTRTAIAICLVFFVCWAPY YVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYΓVLCETFRKRLVLSV KPAAQGQLRTVSNAQTADEERTESKGTVDGTAGPGSIATMVSKGEELFTGVV P1-LVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT

YGVQCFSRYPDFΪMKQHDFFKSAMPEGYVQERTJPFKDDGNYKTRAEVKFEG DTLVNRffiLKGroFKEDGNILGHKI^YNY

NffiDGSVQLADHYQQNTPIGDGPVI PDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK SEQ. ID. NO. 14: Human long form/mouse species chimeric MCHlR-linker- EGFP

MSVGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR TGSISYj-NDMPS GTICLLGHGNSTVff^

LLFIXGMPFMFFLQLMGNGVWHFGETMCTLITAMDANSQFTSTYILTAMAIDR YLATVFJPISSTKFRKPSMATLVICLLWALSHSITPNVVLYARLIPFPGGAVGCGI P PNPDTDLYWFTLYQI^FLAFALPFVVITAAYVKILQRMTSSVAPASQRSIRLR TKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANS CLNPFVYTVLCETFRKRLVLSVKPAAQGQLRTVSNAQTADEERTESKGTVDGT AGPGSL^TMVSKGEELFTGVWILVELDGDVNGHKFSVSGEGEGDATYGKLT LKHCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQE RTIJ^FKDDGNYKTRAJIVKFEGDTLVNRFFILKGIDFKEDGNILGFIKLEYNYNSH NVYIMADKQKNGKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGΓΓLGMDELYK

SEQ. ID. NO. 15: Human long form MCHIR cDNA

ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT CATCATCAACCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTT CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC ATCTCTTCCACGAAGTTCCGGAAGCCCTCTGTGGCCACCCTGGTGATCTGC CTCCTGTGGGCCCTCTCCTTCATCAGCATCACCCCTGTGTGGCTGTATGCC AGACTCATCCCCTTCCCAGGAGGTGCAGTGGGCTGCGGCATACGCCTGCC CAACCCAGACACTGACCTCTACTGGTTCACCCTGTACCAGTTTTTCCTGGC CTTTGCCCTGCCTTTTGTGGTCATCACAGCCGCATACGTGAGGATCCTGCA GCGCATGACGTCCTCAGTGGCCCCCGCCTCCCAGCGCAGCATCCGGCTGC GGACAAAGAGGGTGACCCGCACAGCCATCGCCATCTGTCTGGTCTTCTTT GTGTGCTGGGCACCCTACTATGTGCTACAGCTGACCCAGTTGTCCATCAGC CGCCCGACCCTCACCTTTGTCTACTTATACAATGCGGCCATCAGCTTGGGC TATGCCAACAGCTGCCTCAACCCCTTTGTGTACATCGTGCTCTGTGAGACG TTCCGCAAACGCTTGGTCCTGTCGGTGAAGCCTGCAGCCCAGGGGCAGCT TCGCGCTGTCAGCAACGCTCAGACGGCTGACGAGGAGAGGACAGAAAGC AAAGGCACCTGA

SEQ. ID. NO. 16: Human short form MCHIR cDNA

ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT GCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCGGTCGTGAAG AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC TCATCNCGGCCATGGNTGCCNNTNGTCNGTTCNCCNGCNCCTNCATCCTG ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC NCGNNGTTCCGGAAGCCCTCTGTGGCCACCCTGGTGATCTGCCTCCTGTGG GCCCTCTCCTTCATCAGCATCACCCCTGTGTGGCTGTATGCCAGACTCATC CCCTTCCCAGGAGGTGCAGTGGGCTGCGGCATACGCCTGCCCAACCCAGA CACTGACCTCTACTGGTTCACCCTGTACCAGTTTTTCCTGGCCTTTGCCCTG CCTTTTGTGGTCATCACAGCCGCATACGTGAGGATCCTGCAGCGCATGAC GTCCTCAGTGGCCCCCGCCTCCCAGCGCAGCATCCGGCTGCGGACAAAGA GGGTGACCCGCACAGCCATCGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG CACCCTACTATGTGCTACAGCTGACCCAGTTGTCCATCAGCCGCCCGACCC TCACCTTTGTCTACTTATACAATGCGGCCATCAGCTTGGGCTATGCCAACA GCTGCCTCAACCCCTTTGTGTACATCGTGCTCTGTGAGACGTTCCGCAAAC GCTTGGTCCTGTCGGTGAAGCCTGCAGCCCAGGGGCAGCTTCGCGCTGTC AGCAACGCTCAGACGGCTGACGAGGAGAGGACAGAAAGCAAAGGCACCT GA SEQ. ID. NO. 17: Mouse MCHIR cDNA

Nucleic acid sequence start and stop codons are highlighted:

GGCGGTAGAGGAAGACCCTTTTCTGGACTGCGGGGCTCAAGCTCCGGACA AGGCGGTGGAGGGCGCTGGAGGCTGCCGCAGCCTGCGTGGGTGGACGGG CGCTCCACTCCAGGGAGCAGGCGACCTGCACCGGCTGCATGGATCTGCAA GCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACATCTCCGATGGCCA GGATAATTTCACATTGGCGGGGCCACCTCCTCGCACAAGGAGTGTCTCCT NCNTCNACNTCNTCNTGCCTTCNGTGTTTGGTNCCNTCTGTCTCCTGGGCN TTGTGGGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTG CACTGGTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTG GATCTGCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGT AATGGTGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCAT GGACGCCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCA TTGACCGCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGA AGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCA TTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGG GTGCTGTGGGCTGTGGCNTCCGCCTNCCNNNCCCNGNTNCTGNTCTTTNCT GGTTCNCTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCAT CACTGCTGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCC CAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACA GCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTG CTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTAC CTGTACAATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCC CTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTC GGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGA CAGCTGACGAGGAGAGGACAGAAAGCAAAGGCACCTGACAATCCCCCCC GGTCACCTCCAAGTCAGGTCACCGCATCAAACCATGGGGAGAGATACTGA GATAAACCCGGGGCTACCCTGGGAGGATGCAGAAGCTGGAGGCTGGGGG CTTGTAGCAAACCACATTCCACGGGGCCCACAAATTGCTAGGGAGGCTTG CAGCCTGGTTTGGGGGGGAAGCCTCAGACTGCAGGGATCCCCTTGACAGA ATAGAAGCGGAGCAAGAAGGAAAGGGTGGTTTGACTGGTTCTCGGGGTCT GTATCTGTTGGCTCGCATATATCTTTCTCTCAAGGGAAGAAGGCGGAGGT GCCTAGCTGGGTTCCTTTAAAACTAGGCAGGGCTAGGATCTGAGCAGCTA GGGCTCTACTGTGAGACTGGGCAAGCCGAGCGTTCCCTCCCATCTCTCATT GGTGTTGATAGAAGGCAGTCTTTCTCCCAAGCTGGTGGATCTCCTGAAGC ACGCTGCCTGGGCTCCAGCATCCTGTGCGGATTTCACGTTCTCTTTAGGGG ATGCATGTTGACACTGGGGTGTGGGCTCTGAGCCCACAGGAGTTTAAAAA ACCAAAAGAGCTCAGAGTGTCGAGAGAGACCCAATCACCGAGAATGACA AGGCAACCTGGGGTGGATGTGGATCTTGAAACTAATAAAAAGGGGTTTTC ACAGTGACAGCGACATTCTCTTCATAGGGCACAGCTGTCAGTCTATGGCT GATCCAGAGCGAGCATCCATGAATTCTGCATGTGCAGGGGTCACTCTAAT ACCTGATATGTTGGCATCATCTTTGTGCTTGAGCCTTCCNCTCCCAAATGG GAATGAAATAAAGGCAAATTCCCNCCCCCCCCAAAAAAGGGGNAAAAAA AAAAAAAAAAAAAAAAAAAA

SEQ. ID. NO. 18: Mouse MCHIR genomic DNA

Nucleic acid sequence start and stop codons, as well as intron borders, are highlighted:

GGCGGTAGAGGAAGACCCTTTTCTGGACTGCGGGGCTCAAGCTCCGGACA AGGCGGTGGAGGGCGCTGGAGGCTGCCGCAGCCTGCGTGGGTGGACGGG CGCTCCACTCCAGGGAGCAGGCGACCTGCACCGGCTGCATGGATCTGCAA GCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACATCTCCGATGGCCA GGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTCCTCCCTCCTCCG GGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAGCCAAACTGCCTG GGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCAGTCGGCTTTGCC TGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGACAGAAGGGGCA GGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAACAACAAGGGTTG GTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGCTGATGAGTCCCA AATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGGCTGGAGAAGCAA GCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGTGGGAGCCAGCAG AAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTCTGGCCTCGCACTG GGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGCAAAGGCTGCTGG GAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTTTCTGGTGAATTA AGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATTCTAGCTCTGAAG GAGAAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGTGGAGAAATTCCC AAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCTATTGGGAAAGCC CAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACAGAAAACCGGGAG AGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACCCATGGTGACAGTC ACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTTGGCCATCTCTCTC TGAAAAGATGAGGCTGCAAATACACACACACACACACACACACACACAC ACACACACACACACACACACACACACACACACACAAATGTCCTTCAAGCC TTTTTGACAAGGTTTTCTGGTGGATCCCGGGGATATGAAGTTGTTCTCAGC AGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAAATGGATGAAGCG AAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTAGAAAATCCTATAG GAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACAGAACTAACAATAG CCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAGACGTAGTGACCCC AAGCTGCAAGGGAAAGCATGAGGGGCCAGCAGGAAGGCCGACATGGCAG GTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTGCTCTTTCTATCCTC AGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATCAACATCATCATGC CTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTGGGAAACTCCACAG TCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTGGTGCAGCAACGTC CCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCTGCTTTTCCTGCTGG GCATGCCTTTCATGATCCACCAGCTCATGGGTAATGGTGTCTGGCACTTTG GGGAAACCATGTGCACCCTCATCACAGCCATGGACGCCAACAGTCAGTTC ACCAGCACCTACATCCTGACTGCTATGGCCATTGACCGCTACTTGGCCACC GTCCNTCCCNTCTCCTCCNCCNNGTTCCGGNNGCCCTCCNTGGCCNCCCTG GTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGG CTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATC CGCCTACCAAACCCAGATACTGATCTTTACTGGTTCACTCTGTATCAGTTT TTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAA ATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCAT CCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGG TCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGT CCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCA GCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCT GTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAG GGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTGACGAGGAGAGGA CAGAAAGCAAAGGCACCTGACAATCCCCCCCGGTCACCTCCAAGTCAGGT CACCGCATCAAACCATGGGGAGAGATACTGAGATAAACCCGGGGCTACC CTGGGAGGATGCAGAAGCTGGAGGCTGGGGGCTTGTAGCAAACCACATTC CACGGGGCCCACAAATTGCTAGGGAGGCTTGCAGCCTGGTTTGGGGGGGA AGCCTCAGACTGCAGGGATCCCCTTGACAGAATAGAAGCGGAGCAAGAA GGAAAGGGTGGTTTGACTGGTTCTCGGGGTCTGTATCTGTTGGCTCGCATA TATCTTTCTCTCAAGGGAAGAAGGCGGAGGTGCCTAGCTGGGTTCCTTTA AAACTAGGCAGGGCTAGGATCTGAGCAGCTAGGGCTCTACTGTGAGACTG GGCAAGCCGAGCGTTCCCTCCCATCTCTCATTGGTGTTGATAGAAGGCAG TCTTTCTCCCAAGCTGGTGGATCTCCTGAAGCACGCTGCCTGGGCTCCAGC ATCCTGTGCGGATTTCACGTTCTCTTTAGGGGATGCATGTTGACACTGGGG TGTGGGCTCTGAGCCCACAGGAGTTTAAAAAACCAAAAGAGCTCAGAGTG TCGAGAGAGACCCAATCACCGAGAATGACAAGGCAACCTGGGGTGGATG TGGATCTTGAAACTAATAAAAAGGGGTTTTCACAGTGACAGCGACATTCT CTTCATAGGGCACAGCTGTCAGTCTATGGCTGATCCAGAGCGAGCATCCA TGAATTCTGCATGTGCAGGGGTCACTCTAATACCTGATATGTTGGCATCAT CTTTGTGCTTGAGCCTTCCNCTCCCAAATGGGAATGAAATAAAGGCAAAT TCCCNCCCCCCCCAAAAAAGGGGNAAAAAAAAAAAAAAAAAAAAAAAA AA

SEQ. ID. NO. 19: Human short form/mouse species chimeric MCHIR

ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT GCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCGGTCGTGAAG AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC TCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACCTACATCCTG ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC ACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTG GGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTAT CCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCAAACCCAG ATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCTTCGCCCT TCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGCGCATGAC GTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGA GGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG CGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACC CTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTATGCCAAC AGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAA CGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGT CAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAAGGCACC TGA SEQ. ID. NO. 20: Human long form/mouse species chimeric MCHIR

ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT CATCATCAACCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTT CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC ATCTCTTCCACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGC CTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCC AGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCA

TCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGC GCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGG ACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTG TGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGC CCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTAT GCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTT CGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCG CACGGTCAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAA GGCACCTGA

SEQ. ID. NO. 21: Aequorea victoria Green Fluorescent Protein (GFP) cDNA

Nucleic acid sequence start and stop codons are highlighted:

TACACACGAATAAAAGATAACAAAGATGAGTAAAGGAGAAGAACTTTTC NCTGGNGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCAC AAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACT TACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAAC ACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCNGNT CNTNTGNNNCNGCNTGNCTTTTTCNNGNGTGCCNTGCCCGNNGGTTNTGT NCAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTG CTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAA GGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATA CAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATG GAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTT CAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT CCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGA TCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTG CTGGGATTACACATGGCATGGATGAACTATACAAATAAATGTCCAGACTT CCAATTGACACTAAAGTGTCCGAACAATTACTAAAATCTCAGGGTTCCTG GTTAAATTCAGGCTGAGATATTATTTATATATTTATAGATTCATTAAAATT GTATGAATAATTTATTGATGTTATTGATAGAGGTTATTTTCTTATTAAACA GGCTACTTGGAGTGTATTCTTAATTCTATATTAATTACAATTTGATTTGACT TGCTCAAA

SEQ. ID. NO. 22: EGFP + Linker

Nucleic acid sequence start and stop codons are highlighted and a 12 amino acid linker sequence is denoted in lower case: gtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGAGCTGTT CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACNNCTNCNNCNGCCNCAACGTCTATATCATGGCCGACAAGCAG AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG GCNGCGTGCNGCTCGCCGNCCNCTNCCNGCNGNNCNCCCCCNTCGGCGNC GGCCCCGTGCTGCTGCCCGNCNNCCNCTNCCTGNGCNCCCAGTCCGCCCT GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGC GGCCGC

SEQ. ID. NO. 23: Emerald ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGNGCTGGACGGCGACGTNNNCGGCCNCNNGTTCNGCGTGTCCGGCGNG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCT ACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCNNGTCCGCCNTGCCCGNAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA AGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC AAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCG CGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAA

SEQ. ID. NO. 24: Topaz

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCT ACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGCGCCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG CATGGACGAGCTGTACAAGTAA

SEQ. ID. NO. 25: W1B ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT GGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGTACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTC AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG CATGGACGAGCTGTACAAGTAA

SEQ. ID. NO. 26: Mouse MCHlR-linker-EGFP

Nucleic acid sequence start codon and start and stop codons for mouse MCHIR and EGFP, respectively, as well as intron borders, are highlighted and a 12 amino acid linker sequence is denoted in lower case: ATGGATCTGCAAGCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACAT CTCCGATGGCCAGGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTC CTCCCTCCTCCGGGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAG CCAAACTGCCTGGGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCA GTCGGCTTTGCCTGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGA CAGAAGGGGCAGGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAAC AACAAGGGTTGGTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGC TGATGAGTCCCAAATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGG CTGGAGAAGCAAGCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGT GGGAGCCAGCAGAAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTC TGGCCTCGCACTGGGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGC AAAGGCTGCTGGGAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTT TCTGGTGAATTAAGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATT CTNGCTCTGNNGGNGNAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGT GGAGAAATTCCCAAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCT ATTGGGAAAGCCCAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACA GAAAACCGGGAGAGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACC CATGGTGACAGTCACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTT GGCCATCTCTCTCTGAAAAGATGAGGCTGCAAATACACACACACACACAC ACACACACACACACACACACACACACACACACACACACACACACACAAA TGTCCTTCAAGCCTTTTTGACAAGGTTTTCTGGTGGATCCCGGGGATATGA AGTTGTTCTCAGCAGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAA ATGGATGAAGCGAAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTA GAAAATCCTATAGGAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACA GAACTAACAATAGCCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAG ACGTAGTGACCCC AAGCTGC AAGGGAAAGC ATGAGGGGCC AGC AGGAAG GCCGACATGGCAGGTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTG CTCTTTCTNTCCTCAGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATC AACATCATCATGCCTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTG GGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTG GTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCT GCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGTAATGG TGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCATGGACG CCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCATTGACC GCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGAAGCCCT CCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCA TCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTG TGGGCTGTGGCATCCGCCTACCAAACCCAGATACTGATCTTTACTGGTTCA CTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGC TGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCT CTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATT GCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAG CTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTAC AATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTG TACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAA GCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTG ACGAGGAGAGGACAGAAAGCAAAGGCACCgtcgacggtaccgcgggcccgggatccatcg ccaccATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCC TGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT CTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCG AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCC ACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAA CTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC ACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAA GCGCGATC AC ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC ACTC TCGGCATGGACGAGCTGTACAAGTAA

SEQ. ID. NO. 27: Mouse MCH1R EGFP direct fusion

Nucleic acid sequence start codon and start and stop codons for mouse MCHIR and EGFP, respectively, as well as intron borders, are highlighted:

ATGGATCTGCAAGCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACAT CTCCGATGGCCAGGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTC CTCCCTCCTCCGGGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAG CCAAACTGCCTGGGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCA GTCGGCTTTGCCTGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGA CAGAAGGGGCAGGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAAC AACAAGGGTTGGTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGC TGATGAGTCCCAAATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGG CTGGAGAAGCAAGCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGT GGGAGCCAGCAGAAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTC TGGCCTCGCACTGGGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGC AAAGGCTGCTGGGAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTT TCTGGTGAATTAAGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATT CTAGCTCTGAAGGAGAAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGT GGAGAAATTCCCAAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCT ATTGGGAAAGCCCAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACA GAAAACCGGGAGAGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACC CATGGTGACAGTCACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTT GGCCATCTCTCTCTGAAAAGATGAGGCTGCAAATACACACACACACACAC ACACACACACACACACACACACACACACACACACACACACACACACAAA TGTCCTTCAAGCCTTTTTGACAAGGTTTTCTGGTGGATCCCGGGGATATGA AGTTGTTCTCAGCAGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAA ATGGATGAAGCGAAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTA GAAAATCCTATAGGAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACA GAACTAACAATAGCCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAG ACGTAGTGACCCCAAGCTGCAAGGGAAAGCATGAGGGGCCAGCAGGAAG GCCGACATGGCAGGTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTG CTCTTTCTATCCTCAGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATC AACATCATCATGCCTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTG GGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTG GTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCT GCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGTAATGG TGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCATGGACG CCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCATTGACC GCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGAAGCCCT CCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCA TCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTG TGGGCTGTGGCATCCGCCTACCAAACCCAGATACTGATCTTTACTGGTTCA CTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGC TGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCT CTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATT GCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAG CTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTAC AATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTG TACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAA GCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTG ACGAGGAGAGGACAGAAAGCAAAGGCACCATGGTGAGCAAGGGCGAGG AGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAG CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAAC TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGG GCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACAT CGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCC ATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGC TGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC AAGTAA

SEQ. ID. NO. 28: Human short form mouse species chimeric MCHlR-linker- EGFP

Nucleic acid sequence start codon and start and stop codons for mouse MCHIR and EGFP, respectively, are highlighted and a 12 amino acid linker sequence is denoted in lower case: ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT GCCTCCTGGGCATCATCGGGNNCTCCNCGGTCNTCTTCGCGGTCGTGNNG AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC TCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACCTACATCCTG ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC ACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTG GGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTAT CCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCAAACCCAG ATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCTTCGCCCT TCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGCGCATGAC GTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGA GGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG CGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACC CTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTATGCCAAC AGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAA CGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGT CNGCNNTGCTCNGNCNGCTGNCGNGGNGNGGNCNGNNNGCNNNGGCNCC gtcgacggtaccgcgggcccgggatccatcgccaccATGGTGNGCAAGGGCGAGGAGCTGTT CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG AAGA ACGGC ATCA AGGTGAACTTC AAGATCCGCC AC AAC ATCGAGGACG GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

SEQ. ID. NO. 29: Human long form/mouse species chimeric MCHlR-linker-

EGFP

Nucleic acid sequence start codon and start and stop codons for mouse MCHIR and

EGFP, respectively, are highlighted and a 12 amino acid linker sequence is denoted in lower case:

ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT CNTCNTCNNCCTCTCGGTNGTNGNTCTCCTCTTTCTCCTGGGCATGCCCTT CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC ATCTCTTCCACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGC CTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCC AGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCA AACCCAGATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCT TCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGC GCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGG ACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTG TGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGC CCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTAT GCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTT CGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCG C ACGGTC AGC AATGCTC AGAC AGCTGACGAGGAGAGGAC AGAAAGC AAA GGCACCgtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGA GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGC ATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGG AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG TAA

Example 2: Generation of Chimeric and Fusion Proteins

DNA vectors encoding fusion proteins between a MCH-R receptor (MCHIR) and several different superbright variants of Green Fluorescent Protein (GFP) were generated. GFP variants were fused either via a 12 amino acid linker: TCGACGGTACCGCGGGCCCGGGATCCATCGCCACC (SEQ. ID. NO. 30), amino acid sequence: VDGTAGPGSIAT (SEQ. ID. NO. 31) (linker fusions) or directly to the C-terminus of MCHIR (direct fusions).

Mouse MCHlR-linker-GFP Variant Fusion Constructs Initially, mouse MCHIR was fused in frame via the linker to Enhanced Green Fluorescent Protein (EGFP). MCHIR was PCR-amplified (95°C for 5 minutes; 95°C for 30 seconds, 60°C for 45 seconds, 68°C for 3.5 minutes, for 15 cycles; 68°C for 7 minutes) from a full-length mouse MCHIR genomic DNA lambda clone utilizing a high fidelity polymerase mix (Expand High Fidelity PCR System from Boehringer Mannheim) and PCR primers [MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO. 32), MCHIR (Sal I) 3': GCGTCGACGGTGCCTTTGCTTTCTGTCC (SEQ. ED. NO. 33)] that generated Eco RI and Sal I enzymatic restriction sites at the N- and C-terminus, respectively. The MCHIR N-terminal PCR primer was also designed to introduce a Kozak consensus sequence for translation which contained an Nco I site (5'-ACCATGG-3'), and the MCHIR C-terminal PCR primer was also designed to eliminate the endogenous stop codon present in the mouse MCHIR gene. The resulting PCR product was phenol/chloroform extracted, restriction digested with Eco RI and Sal I, gel purified, and subcloned in frame into the multicloning site of Clontech's pEGFP- N3 vector between Eco RI and Sal I sites. Several resulting clones for this construct were sequenced to identify a clone with an entirely correct nucleotide sequence. This clone was named mMCHlR-1-EGFP for mouse MCHlR-linker-EGFP. An approximately 760 bp Sal I to Not I fragment of mMCHlR-1-EGFP was excised, gel purified, and subcloned into the multicloning site of pBluescript (SK+) (Stratagene) between Sail and Not I sites. An approximately 710 bp Nco I to Bsr Gl fragment of EGFP was excised from the resulting pBluescript-EGFP vector and replaced with the corresponding Nco I to Bsr Gl fragment of either Emerald, Topaz, or W1B (other superbright GFP variants), which were excised from vectors pRSET-Emerald, pRSET-Topaz, and pRSET-WlB, respectively. pRSET-Emerald, pRSET-Topaz, and pRSET-WlB were obtained from Aurora Biosciences Co. Sal I to Not I fragments containing either Emerald, Topaz, or W1B were excised from the resulting pBluescript-Emerald, pBluescript-Topaz, and pBluescript-WlB vectors, respectively. Appropriate fragments were gel purified and subcloned into mMCHlR- 1-EGFP digested with Sal I and Not I, replacing the Sal I to Not I EGFP fragment with the corresponding Sal I to Not I fragment from either Emerald, Topaz, or W1B. Several clones for each construct were sequenced to confirm the presence of the appropriate GFP variant. The resulting vectors were named mMCHlR-1-Emerald, mMCHlR-1-Topaz, and mMCHlR-1-WlB for mouse MCHlR-linker-Emerald, mouse MCHlR-linker-Topaz, and mouse MCHlR-linker-WlB, respectively.

Mouse MCHIR/GFP Variant Direct Fusion Constructs A two step PCR strategy was employed to generate the direct fusion constructs. First, mouse MCHIR, EGFP, and Emerald were PCR-amplified from a full-length mouse MCHIR genomic DNA lambda clone, Clontech's pEGFP-N3 vector, and Aurora's pRSET-Emerald vector, respectively. Mouse MCHIR was PCR-amplified according to the previously mentioned conditions utilizing the same N-terminal PCR primer [MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCA AGCCTCG (SEQ. ID. NO. 32)], but in this case a different C-terminal PCR primer was employed. The C-terminal PCR primer [MCHIR (EGFP/Emerald) 3': CCTTGCTCACCATGGTGCCTTTGCTTTCTGTCC (SEQ. LD. NO. 34)] eliminated the endogenous stop codon of mouse MCHIR as before and introduced a region of nucleotide sequence complementary to the nucleotide sequence of the N-terminus of EGFP.

EGFP and Emerald were PCR-amplified (95°C for 5 minutes; 95°C for 30 seconds, 60°C for 45 seconds, 68°C for 1.5 minutes, for 15 cycles; 68°C for 7 minutes) separately with a high fidelity polymerase mix (Advantage HF-2 from Clontech) from their respective templates utilizing a common N-terminal PCR primer [EGFP/Emerald (MCHIR) 5': CAGAAAGCAAAGGCACCATGGTGAGCAA GGGCGAGGAGC (SEQ. ID. NO. 35)] that generated a region of nucleotide sequence complementary to the C-terminus of mouse MCHIR and C-terminal PCR primers [EGFP 3': GGCGGATCCTCTAGAGTCGCGGCC (SEQ. ID. NO. 36), or Emerald (EGFP) 3': GCTCTAGAGTCGCGGCCGCTTACTTGTACAGCTCGTCC (SEQ. ED. NO. 37)] that generated a Not I site at the C-terminus. The resulting PCR products were electrophoresed on an agarose gel and the appropriate fragments were gel purified.

In a second PCR step, PCR reactions were set up between the previously generated mouse MCHIR and EGFP, or mouse MCHIR and Emerald PCR products. Following an initial 5 minute denaturation step at 95°C, two rounds of thermocycling (95°C for 30 seconds, 60°C for 45 seconds, 68°C for 4 minutes) were performed in the absence of PCR primers. This allowed the mouse MCHIR and GFP variants to anneal at their complementary regions and to be filled in by the high fidelity polymerase mix (Expand High Fidelity PCR System from Boehringer Mannheim), yielding double stranded template DNA.

Subsequently, the common N-terminal mouse MCHIR [MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO. 32)] and appropriate C-terminal PCR primers [EGFP 3': GGCGGATCCTCTAGAGTC GCGGCC (SEQ. ID. NO. 36) or Emerald (EGFP) 3': GCTCTAGAGTCGCGG CCGCTTACTTGTACAGCTCGTCC (SEQ. LD. NO. 37)] were added to the reactions and thermocycling was continued for an additional fifteen cycles followed by a final extension at 68°C for 7 minutes. The resulting PCR products were phenol/chloroform extracted, restriction digested with Eco RI and Not I, electrophoresed on an agarose gel, and appropriate fragments were gel purified. These Eco RI to Not I fragments represent direct fusions between either mouse MCHIR and EGFP, or mouse MCHIR and Emerald. Clontech's pEGFP-N3 vector was restriction digested with Eco RI and Not I liberating an approximately 780 bp Eco RI to Not I EGFP fragment. This restriction digest was electrophoresed on an agarose gel and the approximately 3.9 Kb pEGFP-N3 vector backbone was gel purified. Eco RI to Not I mouse MCHIR/EGFP or mouse

MCHIR/Emerald direct fusion fragments were subcloned into the pEGFP-N3 vector backbone between Eco RI and Not I sites. Several resulting clones for each of these two constructs were sequenced to identify clones with correct nucleotide sequence; however, no clones with entirely correct nucleotide sequences were identified. Fortunately, several clones for each of the two constructs only had nucleotide mismatches in the intron region of mouse MCHIR, and therefore, were not expected to effect the functionality of the resulting fusion proteins. These clones were named mMCHlR/EGFP and mMCHlR/Emerald for mouse MCHIR/EGFP direct fusion and mouse MCHIR/Emerald direct fusion, respectively.

Human Short and Long Form/Mouse Species Chimeric

MCHlR-linker-GFP Variant Fusion Constructs The initial mouse MCHlR-linker-GFP variant fusion constructs were modified to generate both human short form and human long form/mouse species chimeric MCHlR-linker-GFP variant fusion constructs. An approximately 1.7 kb Hind El to Bsp El fragment of the mouse MCHIR gene containing exon 1, the intron, and 127 amino acids of exon 2 was excised from the various mouse MCHlR-linker- GFP variant fusion constructs and replaced by either an approximately 470 bp Hind LTJ to Bsp El fragment from the wild-type human MCHIR short form or an approximately 670 bp Hind IJJ to Bsp El fragment from the wild-type human MCHIR long form.

Several clones for each construct were sequenced to confirm the presence of the N-terminal region of either the human MCHIR short or long forms. These clones were named hshort/mMCHlR-1-GFP variant or hlong/mMCHlR-1-GFP variant for human short form mouse species chimeric MCHlR-linker-GFP variant and human long form/mouse species chimeric MCHlR-linker-GFP variant, respectively.

Example 3: Functional Evaluation of MCHIR/GFP Variant Fusion Proteins

Both HEK293 Aequorin (National Institutes of Health) and CHO mammalian cell lines were transiently transfected with the various MCHIR/GFP variant fusion constructs, as well as the appropriate control constructs. Transfection was performed using Lipofectamine 2000 (Gibco BRL) per the manufacturer recommended protocol. Approximately 48 hours after transfection cells were harvested, stimulated with various concentrations of human MCH, and assayed for either aequorin bioluminescence (HEK293 Aequorin cells) or cAMP production (CHO cells). Aequorin bioluminescence is a representative measure of intracellular Ca²⁺ mobilization. cAMP production was measured with the Adenylyl Cyclase Activation FlashPlate Assay (NEN Life Science Products, Inc.).

Following transient transfection of the mMCHlR-linker-EGFP construct (MCH-R-1-EGFP) into HEK293 Aequorin cells, the resulting fusion protein exhibited functional activity comparable to that of the wild-type human MCHIR short form (MCH-R wt). By this functional assay, the EC50 value for mMCHlR-1-EGFP was nearly identical to that of the wild-type human short form receptor (Figure 1). Following transient transfections of the mMCHlR-1-EGFP and mMCHlR/EGFP fusion constructs into CHO cells, the resulting fusion proteins exhibited functional activity comparable to that of the wild-type human MCHIR short form. By this functional assay, the EC50 values for mMCHlR-1-EGFP and mMCHlR EGFP were comparable to that of the wild-type human receptor (Table 1). Transient transfections with the corresponding Emerald constructs yielded similar results (data not shown).

Table 1

Following transient transfections of the human short form/mouse species chimeric MCHlR-1-EGFP (HuShort/mMCHlR-1-EGFP) and human long form/mouse species chimeric MCHlR-1-EGFP (HuLong/mMCHlR-1-EGFP) constructs into HEK293 cells, the resulting fusion proteins exhibited functional activity comparable to that of the wild-type human MHCIR short and long forms, respectively. By this functional assay, the EC50 value for each fusion proteins was nearly identical to that of the corresponding wild-type human receptor (Table 2).

Table 2

Following transient transfections of the human short form/mouse species chimeric MCHlR-1-EGFP (HuShort/mMCHlR-1-EGFP) and human long form/mouse species chimeric MCHlR-1-EGFP (HuLong/mMCHlR-1-EGFP) constructs into CHO cells, the resulting fusion proteins exhibited functional activity comparable to or less than that of the wild-type human MHCIR short and long forms, respectively (Table 3). By this functional assay, the EC50 value for the human short form/mouse species chimeric MCHlR-1-EGFP fusion protein was comparable to that of the corresponding wild-type human receptor, whereas, the human long form/mouse species chimeric MCHlR-1-EGFP fusion protein had an EC50 value approximately 7.5-fold higher than that of its corresponding wild-type control.

Table 3

Transient expression of all the MCHIR/GFP variant fusion proteins that underwent functional evaluation resulted in fluorescence primarily associated with the plasma membrane in both HEK293 and CHO cells (data not shown). This pattern of fluorescence is consistent with a predominant membrane associated localization.

Example 4: Generation of Stable Cell Lines

Wild-type CHO cells were transfected using SuperFect (Qiagen) and either mouse MCH-IR-EGFP or human short/mouse species chimeric MCH-IR- EGFP. Forty-eight hours after transfection, transfected cells were subjected to positive selection for approximately ten days in media containing G418. Following selection, MCH-IR-EGFP expressing CHO cells were bulk sorted by Fluorescence Assisted Cell Sorting (FACS) for one or two rounds on the basis of fluorescence intensity to increase the population of cells expressing EGFP. Following bulk sorts, individual clones of varying fluorescence intensities were isolated by FACS and expanded.

Fluorometric Microvolume Assay Technology (FMAT) was initially employed to screen a large number of stable clones by whole cell binding with a fluorescently labeled MCH derivative (SymJz-MCH, PE Biosystems) to identify those clones with good specific binding windows. Several clones exhibiting specific binding windows greater than 3 -fold were further evaluated for MCH binding with the SPA-based Binding Assay. Cells from individual clones were dissociated in enzyme free dissociation media and cell membranes were prepared and subsequently tested for their ability to bind [¹²⁵I]Phe¹³Tyr¹⁹-MCH in the presence of human MCH. CHO cell lines expressing either mouse MCH-IR-EGFP or human short/mouse species chimeric MCH-IR-EGFP (Figure 4) displayed IC50 values with MCH that were indistinguishable from the corresponding IC50 values obtained with a CHO cell line expressing the wild-type human short isoform of MCH-IR.

The functional activity of these clones was evaluated with the cAMP Flashplate Assay (Figures 2 and 3). CHO cell lines expressing either mouse MCH- IR-EGFP (Figure 2) or human short/mouse species chimeric MCH-IR-EGFP (Figure 3) displayed EC50 values with human MCH that were indistinguishable from the EC50 value obtained with a CHO cell line expressing the wild-type human short isoform of MCH-IR.

The subcellular localization of the MCH-IR-EGFP fusion proteins were determined by confocal microscopy utilizing EGFP fluorescence as a marker for MCH-IR expression. CHO cell lines stably expressing either mouse MCH-IR-EGFP or human short/mouse species chimeric MCH-IR-EGFP displayed EGFP fluorescence primarily associated with the plasma membrane, demonstrating that these MCH-IR-EGFP fusion proteins are primarily associated with the plasma membrane.

Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising: a) a melanin concentrating hormone receptor polypeptide region comprising a sequence selected from the group consisting of: SEQ. ED. NO. 1, SEQ. ED. NO. 2, SEQ. SEQ. ED. NO. 3, SEQ. ED. NO. 4, and SEQ. ID. NO. 5; and b) a fluorescent polypeptide region joined directly, or though a linker, to the carboxy side of said melanin concentrating hormone receptor polypeptide region.

2. The protein of claim 1, wherein said fluorescent polypeptide region consists of an amino acid sequence selected from the group consisting of SEQ. ED. NO. 6, SEQ. ED. NO. 7, SEQ. ED. NO. 8, SEQ. ED. NO. 9, and SEQ. ED. NO. 10.

3. The protein of claim 2, wherein said melanin concentrating hormone polypeptide region consists of a sequence selected from the group consisting of: SEQ. ED. NO. 1, SEQ. ED. NO. 2, SEQ. ED. NO. 3, SEQ. ED. NO. 4, and SEQ. ED. NO. 5.

4. The protein of claim 3, wherein said protein consists essentially of said melanin concentrating hormone receptor polypeptide region and said fluorescent polypeptide region.

5. The protein of claim 4, wherein said protein consists of the amino acid sequence of SEQ. ED. NO. 11 or SEQ. ED. NO. 12.

6. The protein of claim 1, wherein said melanin concentrating hormone polypeptide region is a chimeric polypeptide comprising (a) an MCH binding region from a first species and (b) a transmembrane and intracellular domain region from a second species joined directly, or though a linker, to the carboxy side of said MCH binding region.

7. The protein of claim 6, wherein said fluorescent polypeptide region consists of an amino acid sequence selected from the group consisting of: SEQ. ED. NO. 6, SEQ. ED. NO. 7, SEQ. ED. NO. 8, SEQ. ED. NO. 9, and SEQ. ED. NO. 10.

8. The protein of claim 7, wherein said protein consists of the amino acid sequence of SEQ. ED. NO. 13 or SEQ. ED. NO. 14.

9. A chimeric melanin concentrating hormone protein comprising: a) a melanin concentrating hormone binding region characteristic of a human melanin concentrating hormone receptor; b) a transmembrane domain characteristic of a non-human melanin concentrating hormone receptor; and c) an intracellular domain characteristic of a non-human melanin concentrating hormone receptor.

10. The protein of claim 9, wherein said protein comprises a melanin concentrating hormone receptor polypeptide having a sequence similarity of at least 75% with either SEQ. ED. NO. 4 or SEQ. ED. NO. 5.

11. The protein of claim 10, wherein said protein comprises the sequence of SEQ. ID. NO. 4 or SEQ. ED. NO. 5.

12. The protein of claim 11, wherein said protein consists of the sequence of SEQ. ED. NO. 4 or SEQ. ED. NO. 5.

13. A nucleic acid comprising a nucleotide sequence encoding for the protein of claim 1.

14. The nucleic acid of claim 13, wherein said nucleotide sequence is a contiguous sequence.

15. The nucleic acid of claim 13, wherein said nucleotide sequence is selected from the group consisting of SEQ. ED. NO. 26, SEQ. ED. NO. 27, SEQ. ED.

NO. 28 and SEQ. ED. NO. 29.

16. A nucleic acid comprising a nucleotide sequence encoding for the protein of claim 9.

17. The nucleic acid of claim 16, wherein said nucleotide sequence is a contiguous sequence.

18. The nucleic acid of claim 16, wherein said nucleotide sequence is selected from the group consisting of SEQ. ED. NO. 19 and SEQ. ED. NO. 20.

19. An expression vector comprising the nucleic acid of claim 13.

20. An expression vector comprising the nucleic acid of claim 16.

21. A recombinant cell comprising the nucleic acid of claim 13.

22. The recombinant cell of claim 21, wherein said nucleic acid is present in an expression vector.

23. The recombinant cell of claim 21, wherein said nucleic acid is present in the genome of said cell.

24. A recombinant cell comprising the nucleic acid of claim 16.

25. The recombinant cell of claim 24, wherein said nucleic acid is present in an expression vector.

26. The recombinant cell of claim 24, wherein said nucleic acid is present in the genome of said cell.

27. A non-human transgenic animal comprising the nucleic acid of claim 13.

28. A non-human transgenic animal comprising the nucleic acid of claim 16.

29. A method for assaying for melanin concentrating hormone receptor active compounds comprising the steps of: a) contacting the cell of claim 21 with a test preparation comprising one or more test compounds; and b) measuring the effect of said test preparation on one or more melanin concentrating hormone receptor activities.

30. A method for assaying for melanin concentrating hormone receptor active compounds comprising the steps of: a) contacting the cell of claim 24 with a test preparation comprising one or more test compounds; and b) measuring the effect of said test preparation on one or more melanin concentrating hormone receptor activities.