EP1228096A1

EP1228096A1 - Dog and rabbit motilin receptor orthologs

Info

Publication number: EP1228096A1
Application number: EP00975380A
Authority: EP
Inventors: Carina Tan; Karen Mckee
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1999-10-29
Filing date: 2000-10-25
Publication date: 2002-08-07
Also published as: WO2001032710A1; CA2389329A1; EP1228096A4; JP2003512859A

Abstract

The present invention features polypeptides and nucleic acids related to the dog and rabbit motilin receptor, and uses of such polypeptides and nucleic acids. The dog motilin receptor exon 1 amino acid sequence is provided for by SEQ. ID. NO. 1, the rabbit motilin receptor amino acid sequence is provided for by SEQ. ID. NO. 2, the nucleic acid sequence encoding for exon 1 of the dog motilin receptor is provided for by SEQ. ID. NO. 3, and the nucleic acid sequence encoding for the rabbit motilin receptor is provided for by SEQ. ID. NO. 4.

Description

TITLE OF THE INVENTION

DOG AND RABBIT MOTILIN RECEPTOR ORTHOLOGS

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Serial No. 60/162,264, filed

October 29, 1999, hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The references cited herein are not admitted to be prior art to the claimed invention.

Motilin is a 22 amino acid peptide hormone affecting gastric motility. Motilin has been found to induce smooth muscle contractions in the gastrointestinal tract of different species including humans, cats, rabbits, dogs, and chickens. (Peeters and Depoortere, Digestive Diseases and Sciences 59:765-785, 1994; Van Assche, et al, European Journal of Pharmacology 337:267-274, 1997; Depoortere and Peters, American Journal of Physiology 272.G994 (1997); Kitazawa, et al, Peptides 76: 1243-1252, 1995; and Itoh, Peptides 75:593- 608, 1997.)

The effects of motilin include inducing interdigestive (phase III) antrum and duodenal contractions. (Itoh, Peptides 75:593-608, 1997; Poitras, in Gut Peptides: Biochemistry and Physiology, J. H. Walsh and G. J. Dockray, Eds. (Raven, New York, 1994), pp. 261-304; and Tonini, Pharmacol. Res. 33:217-226, 1996.) The antibiotic erythromycin induces similar effects that may be mediated by motilin receptors. (Itoh, et al, American Journal of Physiology 247:G688-G694, 1984; and Weber, et al, American Journal of Gastroenterology 5S:485-490, 1993.) Erythromycin produces side effects including vomiting, nausea, diarrhea and abdominal discomfort. (Tonini, Pharmacol. Res. 33:217-226, 1996.)

SUMMARY OF THE INVENTION

Polypeptides related to the dog or rabbit motilin receptor contain a region of at least 9 contiguous amino acids that are present in the dog" or rabbit motilin receptor. Such polypeptides may contain additional regions including regions present, or not present, in the dog or rabbit motilin receptor.

Nucleic acids related to the dog or rabbit motilin receptor contain a region of at least 18 contiguous nucleotides that is present in the dog or rabbit motilin receptor nucleic acid or the complement thereof. Such nucleic acids may contain additional regions including regions present, or not present, in the dog or rabbit motilin receptor nucleic acid.

Thus, a first aspect of the present invention describes a purified polypeptide comprising a unique amino acid region of a dog or rabbit motilin receptor. The unique region is at least 9 amino acids in length.

A "unique amino acid region" is a region of contiguous amino acids present in SEQ. ID. NOs. 1 or 2 that is not present in SEQ. ID. NOs. 5 or 6. SEQ. ED. NO. 5 is a human motilin receptor amino acid sequence and SEQ. ID. NO. 6 is an amino acid sequence for Spheroides nephelus 75E7. The unique region may contain segments of contiguous amino acids present in SEQ. ID. NOs. 5 or 6 smaller than the indicated unique region size.

A "purified polypeptide" represents at least 10% of the total protein present in a sample or preparation. In preferred embodiments, the purified polypeptide represents at least about 50%, at least about 75%, or at least about 95% of the total protein in a sample or preparation. Reference to "purified polypeptide" does not require that the polypeptide has undergone any purification and may include, for example, chemically synthesized polypeptide that has not been purified. Another aspect of the present invention describes a purified nucleic acid comprising a nucleotide sequence encoding for a unique amino acid region from the dog or rabbit motilin receptor or the complement thereof. The encoded for region is at least 9 amino acids in length.

A "purified nucleic acid" represents at least 10% of the total nucleic acid present in a sample or preparation. In preferred embodiments, the purified nucleic acid represents at least about 50%, at least about 75%, or at least about 95% of the total nucleic acid a sample or preparation. Reference to "purified nucleic acid" does not require that the nucleic acid has undergone any purification and may include, for example, chemically synthesized nucleic acid that has not been purified. Another aspect of the present invention describes a purified nucleic acid comprising a unique nucleotide sequence region of a dog or rabbit motilin receptor nucleic acid sequence. The unique nucleotide sequence region is at least 18 nucleotides in length. A "unique nucleotide sequence region" is a region that comprises at least 18 contiguous nucleotides of SEQ. ID. NOs. 3 or 4 that is not present in SEQ. ID. NOs. 7 or 8. SEQ. ID. NO. 7 is the nucleotide sequence encoding for a human motilin receptor and SEQ. ID. NO. 8 is the nucleotide sequence encoding for Spheroides nephelus 75E7. The unique region may contain segments of contiguous nucleotides present in SEQ. ID. NOs. 7 or 8 smaller than the indicated unique region size. Another aspect of the present invention describes an expression vector. The expression vector comprises a recombinant nucleotide sequence encoding for a unique amino acid region of a dog or rabbit motilin receptor.

A "recombinant nucleotide sequence" is a sequence that is present on a nucleic acid containing one or more nucleic acid regions not naturally associated with that sequence. Examples of such regions that may be present include one or more regulatory elements not naturally associated with the sequence, viral elements, and selectable markers.

Another aspect of the present invention describes a recombinant cell comprising an expression vector encoding for a unique amino acid region of a dog or rabbit motilin receptor. The expression vector contains a promoter that is functionally coupled to nucleic acid encoding for the unique region and is recognized by an RNA polymerase present in the cell.

Another aspect of the present invention describes a recombinant cell made by introducing an expression vector encoding for a unique amino acid region of a dog or rabbit motilin receptor into a cell. The expression vector can be used to insert the dog or rabbit nucleic acid into the genome of the host, or can exist as an autonomous piece of nucleic acid. Another aspect of the present invention describes a method of measuring the ability of a compound to effect motilin receptor activity. The method involves providing the compound to a recombinant cell expressing a functional motilin receptor containing a unique dog or rabbit amino acid region from a recombinant nucleic acid and measuring motilin receptor activity. Preferably, the recombinant nucleic acid is present on an expression vector. Another aspect of the present invention describes a method of producing a motilin receptor polypeptide. The method involves the step of growing a recombinant cell able to express a dog or rabbit motilin receptor polypeptide under conditions wherein the polypeptide is expressed from an expression vector. 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 a comparison of the protein sequence for the dog motilin receptor exon 1 (SEQ. ID. NO. 1), the rabbit motilin receptor (SEQ. ID. NO. 2), the human motilin receptor (SEQ. ID. NO. 5) and Spheroides nephelus 75E7 (SEQ. ED. NO. 6).

Figures 2A-2C illustrate a comparison of the DNA sequence encoding for the dog motilin receptor exon 1 (SEQ. ID. NO. 3), the rabbit motilin receptor (SEQ. ID. NO. 4), the human motilin receptor (SEQ. ED. NO. 7) and Spheroides nephelus 75E7 (SEQ. ID. NO. 8).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features polypeptides and nucleic acids related to the dog and rabbit motilin receptor. Preferred polypeptides contain an amino acid region not present in the human motilin receptor or Spheroides nephelus 75E7. Preferred nucleic acids contain a nucleotide region not present in nucleic acid encoding for the human motilin receptor or Spheroides nephelus 75E7.

The amino acid sequence and encoding DNA sequence for two alternatively spliced forms of the human motilin receptor (MTL-R1 and MTL-R2) are described by Feighner, et al, Science 254:2184-2188, 1999 (not admitted to be prior art to the claimed invention). Additionally, an amino acid sequence for genomic DNA encoding for "GPR38" is described by McKee, et al, Genomics 46:426-434, 1997. Feighner, et al, identifies GPR38 as the motilin receptor and indicates the presence of an intron.

The Spheroides nephelus gene 75E7 has a high level of homology to the human motilin receptor. The protein sequence of 75E7 is 54% identical to human motilin receptor (MTL-R1) and contains a similar exon-intron structure. (Feighner, et al, Science 254:2184-2188, 1999.)

A prefeπed use of dog and rabbit motilin receptor polypeptides and nucleic acids is in an in vitro functional assay that measures whether a compound acts differently at the dog or rabbit receptor than at the human receptor. Such an assay can be used to help evaluate whether a dog or rabbit model provides a useful test system in looking for a human therapeutic compound.

Therapeutic uses of compounds active at the motilin receptor include the treatment gastrointestinal diseases and disorders such as gastric motility disorders (intrinsic myopathies or neuropathy), gastroparesis, irritable bowel syndrome, and diarrhea. Additionally, compounds active at the motilin receptor can be used as a research tool for studying motilin receptor activity.

MOTILIN RECEPTOR RELATED POLYPEPTIDES

Polypeptides related to the dog and rabbit polypeptide contain a unique dog or rabbit amino acid region. In addition to the unique amino acid region, regions that may, or may not, be related to the dog or rabbit motilin receptor polypeptide may be present in the polypeptides. Such polypeptides have a variety of uses, such as providing a component of a functional motilin receptor; being used as an immunogen to produce antibodies binding to the dog or rabbit motilin receptor; being used as a target to identify compounds binding to the motilin receptor; and/or being used in assays to measure the ability of a compound to effect motilin receptor activity.

Unique dog and rabbit amino acid regions can readily be identified based on a comparison of the dog and rabbit motilin receptor sequences described herein, with the human motilin receptor and the Spheroides nephelus 75E7 protein sequences. Such a sequence comparison is illustrated in Figure 1. Examples of unique dog amino acid regions include the following: GPGNSSDGA (SEQ. ID. NO. 9); VCLGLFAVGV (SEQ. ID. NO. 10); ALLSSRRRA (SEQ. ID. NO. 11); APFFFLVGVEQDAGG (SEQ. ID. NO. 12); and CLCVLYGRI (SEQ. ED. NO. 13). Examples of unique rabbit amino acid regions include the following: DPAVFAAPDR (SEQ. ED. NO. 14); NGTVPLDPS (SEQ. ED. NO. 15); SPAPASPPSGPG (SEQ. ID. NO. 16); RRLLRESRAG (SEQ. ID. NO. 17); and SGVCGSRGPEQD (SEQ. ID. NO. 18).

The definition of unique amino acid region is with respect to human motilin receptor and Spheroides nephelus 75E7 protein sequences. Thus, a unique amino acid region may be present in a motilin receptor sequence from one or more species other than the human motilin receptor or Spheroides nephelus 75E7 protein sequence, or in a non-motilin receptor sequence. For example, SEQ. ED. NO. 10 is present in both the dog and rabbit motilin receptor. In different embodiments a dog or rabbit motilin receptor related polypeptide comprises or consists of a unique amino acid region at least 18, at least 27, or at least 54, bases in length. Preferably, the dog or rabbit motilin receptor related polypeptide comprises or consists of the amino acid sequence of SEQ. ID. NO. 1 or SEQ. ED. NO. 2. Polypeptides can be produced using standard techniques including those involving chemical synthesis and those involving biochemical synthesis. Techniques for chemical synthesis of polypeptides are well known in the art. (See e.g., Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990.)

Biochemical synthesis techniques for polypeptides are also well known in the art. Such techniques employ a nucleic acid template for polypeptide synthesis. The genetic code providing the sequences of nucleic acid triplets coding for particular amino acids is well known in the art. (See, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990.) 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.

Functional Motilin Receptor Derivatives

Functional motilin receptors can bind motilin and transduce an intracellular signal. Functional motilin receptors include the dog and rabbit motilin receptors, and receptors having motilin receptor activity that contain a unique dog or rabbit amino acid region as a component.

Starting with a motilin receptor obtained from a particular source, derivatives can be produced having motilin receptor activity. Such derivatives include polypeptides with amino acid substitutions, additional and deletions. Changes made to produce functional derivatives should be made outside of the motilin-binding domain and in a manner not altering the tertiary structure. The ability of a polypeptide to have motilin receptor activity can be confirmed using techniques such as those measuring G-protein activity.

The sequence comparison provided in Figure 1 illustrates amino acids that vary between the human, dog, and rabbit motilin receptors. Such variable amino acids are good targets for alterations.

Additionally, amino acids are classified into certain types based on the structure of their R-groups. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine may not cause a change in functionality of the polypeptide.

Motilin Antibodies Antibodies recognizing a dog or rabbit motilin receptor polypeptide can be produced using a polypeptide of SEQ. ED. NO. 1, SEQ. ED. NO. 2, or a fragment thereof as an immunogen. Fragments should be at least 9 amino acids in length and preferably consist of a unique amino acid region.

Antibodies to the motilin receptor have different uses such as being used to identify the presence of motilin receptor polypeptides and for isolating motilin receptor polypeptides. Examples of techniques for producing and using antibodies are described in

Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Harlow, et al,

Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and Kohler, et al,

Nature 256:495-497, 1975.

Binding Assays

Assays measuring the ability of a compound to bind the dog or motilin receptor can be performed using a polypeptide of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or a fragment thereof as a target. Fragments should be at least 9 amino acids in length and contain a site to which either an agonist, antagonist, or allosteric modulator binds. Different types of assay formats can be employed including competitive and non-competitive assays. Compounds identified as binding to a full-length receptor or a receptor fragment can be used to determine the locus of a binding site by testing out the ability of the compound to bind to smaller length fragments. For example, motilin binds to the motilin receptor and labeled motilin can be used to identify that portion of the receptor to which motilin binds. Fragments identified as containing a compound binding site can be used to test for additional compounds that bind to the binding site.

Preferred polypeptide fragments used in a binding assay consist of a unique amino acid region. However, fragments containing additional amino acid sequences can be employed, for example, to facilitate attachment to a column.

Binding assays can be performed using individual compounds or preparations containing different compounds. A preparation containing different compounds wherein one or more compounds bind to the motilin receptor can be divided into smaller groups to identify compound(s) binding to the motilin receptor. In an embodiment of the present invention a test preparation containing at least 10 compounds is used in a binding assay. Binding assays can be performed using recombinantly produced motilin receptor polypeptides present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the motilin receptor polypeptide expressed from recombinant nucleic acid; and the use of a purified motilin receptor polypeptide produced by recombinant means which is introduced into a different environment.

Functional Assays

Assays involving functional dog or rabbit motilin receptors can be employed to select for compounds active at the motilin receptor and to evaluate the ability of a compound to effect receptor activity. Motilin receptor activity can be measured using different techniques such as detecting a change in the intracellular conformation of the motilin receptor, measuring G-protein activity, or measuring the level of intracellular messengers.

Recombinantly expressed motilin receptor polypeptides can be used to facilitate determining whether a compound is active at the motilin receptor or another receptor. For example, the motilin receptor can be expressed by an expression vector in a cell line such as HEK 293, COS 7, and CHO not normally expressing the receptor, wherein the same cell line without the expression vector or with an expression vector not encoding for a motilin receptor can act as a control.

Motilin receptors appear to activate the phospholipase C signal transduction pathway. Activity of the phospholipase C signal transduction pathway can be measured using standard techniques such as those measuring intracellular Ca ⁺. Examples of techniques well known in the art that can be employed to measure Ca²⁺ include the use of dyes such as Fura-2 and the use of Ca²⁺-bioluminescent sensitive reporter proteins such as aequorin. An example of a cell line employing aequroin to measure G protein activity is HEK293/aeql7. (Button, et al, Cell Calcium 74:663-671, 1993, and Feighner, et al, Science 254:2184-2188, 1999, both of which are hereby incorporated by reference herein.)

Chimeric receptors containing a motilin binding region functionally coupled to polypeptides from other G protein can also be used to measure motilin receptor activity. Such chimeric receptors preferably contain at least one unique dog or rabbit amino acid region. A chimeric motilin receptor contains an N-terminal extracellular domain; a transmembrane domain made up of transmembrane regions, extracellular loop domains, and intracellular loop domains; and an intracellular carboxy terminus. Prefeπed chimerics contain the extracellular domain of a motilin dog or rabbit receptor.

The specificity of G protein coupling is determined by intracellular domain(s). A chimeric motilin receptor can be produced to functionally couple to a particular G protein such as a Gq protein or a Gi protein. Such signal swapping allows for the detection of motilin receptor activity by measuring Gq or Gi activity. Techniques for producing chimeric receptors and measuring G protein coupled responses are provided for in, for example, International Application Number WO 97/05252, and U.S. Patent Number 5,264,565, 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 motilin receptor activity can be divided into smaller groups of compounds to identify the compound(s) affecting motilin receptor 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 motilin receptor polypeptides present in different environments. Such environments include, for example, cell extracts, and purified cell extracts, containing the motilin receptor polypeptide expressed from recombinant nucleic acid; and the use of a purified motilin receptor polypeptide produced by recombinant means which is introduced into a different environment.

MOTILIN RECEPTOR RELATED NUCLEIC ACID Nucleic acids related to the dog and rabbit motilin receptor nucleic acid contain a unique dog or rabbit nucleotide sequence region. Such nucleic acids have a variety of uses, such as being used as a hybridization probe or PCR primer to identify the presence of dog or rabbit motilin nucleic acid; being used as a hybridization probe or PCR primer to identify nucleic acid encoding for receptors related to the motilin receptor from different sources; and/or being used for recombinant expression of dog or rabbit motilin receptor polypeptide.

Unique dog and rabbit nucleic acid regions can readily be identified based on a comparison of the dog and rabbit motilin receptor nucleic acid sequences described herein, with the human motilin receptor and the Spheroides nephelus 75E7 nucleic acid sequences.

Such a sequence comparison is illustrated in Figure 2.

Examples of unique dog nucleic acid regions include the following:

GGCCCCGGGAACAGCAGCGACGGCGCG (SEQ. ID. NO.19); GGCCGTGTGCCTGGGCCT (SEQ. ED. NO.20);

CGCGCGCTGCTGTCCCGG (SEQ. ID. NO.21);

AGGACGCGGGCGGCCCCG (SEQ. ID. NO.22); and

CCGCGAGCTGCGGAGGCG (SEQ. ID. NO.23).

Examples of unique rabbit nucleic acid regions include the following: TTCGGCCGGGCCCTTCTTCTTT (SEQ. ID. NO. 24);

GGTCTTCGCGGCCCCGGA (SEQ. ID. NO. 25);

CGGTACTGTGCCGCTGGA (SEQ. ID. NO. 26);

GCTTTTCTACCTGAGTGCGTCC (SEQ. ID. NO. 27); and

CGAGCGGGGCCCAGTGGTG (SEQ. ID. NO. 28). The guidance provided in the present application can be used to obtain the nucleic acid sequence encoding for the full-length dog motilin receptor, to obtain nucleic acids encoding for motilin receptors from additional sources, and to artificially produce a motilin receptor. Obtaining nucleic acids encoding for a motilin receptor from different sources is facilitated using sets of degenerative probes and primers and by the proper selection of hybridization conditions. Sets of degenerative probes and primers are produced taking into account the degeneracy of the genetic code. Adjusting hybridization conditions is useful for controlling probe or primer specificity to allow for hybridization to nucleic acids having similar sequences.

Techniques employed for hybridization detection and PCR cloning are well known in the art. Nucleic acid detection techniques are described, for example, in Sambrook, et al, in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor

Laboratory Press, 1989. PCR cloning techniques are described, for example, in White,

Methods in Molecular Cloning, volume 67, Humana Press, 1997.

Motilin receptor probes and primers can be used to screen nucleic acid libraries containing, for example, genomic DNA or cDNA. Such libraries are commercially available, and can be produced using techniques such as those described in Ausubel, Current

Protocols in Molecular Biology, John Wiley, 1987-1998.

Starting with a particular motilin receptor 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. 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=Ile=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=Tryptophan: codon UGG Y=Tyr=Tyrosine: codons UAC, UAU

In different embodiments dog or rabbit motilin receptor related nucleic acid comprises or consists of a unique nucleic acid region at least 27 or at least 54 bases in length.

Preferably, the dog or rabbit motilin receptor related nucleic acid comprises or consists of the nucleic acid sequence of SEQ. ID. NO. 3 or SEQ. ID. NO. 4. Nucleic acid having a desired sequence can be synthesized using chemical and biochemical techniques. Examples of chemical techniques are described in 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. Biochemical synthesis techniques involve the use of a nucleic acid template and appropriate enzymes such as DNA and/or RNA polymerases. Examples of such techniques include in vitro amplification techniques such as PCR and transcription based amplification, and in vivo nucleic acid replication. Examples of suitable techniques are provided by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Sambrook, et al, in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989, and Kacian, et al, U.S. Patent No. 5,480,784.

Motilin Receptor Probes

Detection probes for the dog or rabbit motilin receptor preferably contain a unique dog or rabbit nucleic acid region, or the complement thereof. Such probes can contain additional nucleic acid that may, or may not, be complementary to dog or rabbit motilin receptor nucleic acid. Preferably, additional nucleic acid that is present has a particular purpose such as providing for increased specificity, being a reporter sequence, or being a capture sequence. However, additional nucleic acid need not have a particular purpose.

Probes for the motilin receptor can specifically hybridize to motilin receptor target nucleic acid under appropriate hybridization conditions (i.e., distinguish target nucleic acid from one or more non-target nucleic acid molecules). A preferred non-target nucleic acid is either nucleic acid encoding for the human motilin receptor or the complement thereof. Hybridization occurs through complementary nucleotide bases present on the probe and motilin receptor nucleic acid. Hybridization conditions determine whether two molecules have sufficiently strong interactions with each other to form a stable hybrid.

Probes are composed of nucleic acids or derivatives thereof such as modified nucleic acid and peptide nucleic acid. Modified nucleic acid includes nucleic acid with one or more altered sugar groups, altered internucleotide linkages, and/or altered nucleotide purine or pyrimidine bases. References describing modified nucleic acid include WO 98/02582, U.S. Patent No. 5,859,221 and U.S. Patent No. 5,852,188, each of which are hereby incorporated by reference herein.

The degree of interaction between two molecules that hybridize together is reflected by the Tm of the produced hybrid. The higher the Tm the stronger the interactions and the more stable the hybrid. Tm is effected by numerous factors well known in the art such as the degree of complementarity, the type of complementary bases present (e.g., A-T hybridization versus G-C hybridization), the structure of the nucleic acid backbones, and solution components. E.g., Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989.

Stable hybrids are formed when the Tm of a hybrid is greater than the temperature employed under a particular set of hybridization assay condition. The degree of specificity of a probe can be varied by adjusting the hybridization stringency conditions. Detecting probe hybridization is facilitated through the use of a detectable label. Examples of detectable labels include luminescent, enzymatic, and radioactive labels.

Examples of stringency conditions are provided in Sambrook, et al, in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989. An example of high stringency conditions is as follows: Prehybridizadon of filters containing DNA is carried out for 2 hours to overnight at 65 °C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hours at 65°C in prehybridizadon mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 minutes before autoradiography. Other procedures using conditions of high stringency would include, for example, either a hybridization step carried out in 5X SSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.

Recombinant Expression

Motilin receptor related polypeptides can be expressed from recombinant nucleic acid in a suitable host or in a test tube using a translation system. Recombinantly expressed motilin receptor polypeptides are preferably used in assays to screen for compounds that bind to the motilin receptor and modulate the activity of the receptor.

Preferably, expression is achieved in a host cell using an expression vector.

An expression vector contains recombinant nucleic acid encoding for a desired polypeptide along with regulatory elements for proper transcription and processing. The regulatory elements that may be present include those naturally associated with the recombinant nucleic acid and exogenous regulatory elements not naturally associated with the recombinant nucleic acid. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing recombinant nucleic acid in a particular host. Generally, the regulatory elements that are present 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. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.

Expression vectors that can be used to provide 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 gtl l (Invitrogen), pcDNAII (Invitrogen), and pKK223-3 (Pharmacia). Fungal cell expression vectors well known in the art include pYES2 (Invitrogen), Pichia expression vector (Invitrogen). Insect cell expression vectors well known in the art include Blue Bac III (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 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-K 1 (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).

Expression vectors may be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation.

Motilin receptor nucleic acid 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.

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 : Cloning of the Rabbit Motilin Receptor A rabbit motilin receptor was identified and cloned from a λDashll genomic library (Stratagene, La Jolla, CA) by screening with a human MTLR probe (exon I & II, GPR38; McKee, et al, Genomics 46:426-434, 1977, hereby incorporated by reference herein). Hybridization was performed using reduced-stringency conditions as described below. 1.1 x 10⁶ plaque forming units (pfu) were plated on E. coli XLBlue MRA (P2) and transferred to nylon membranes (NEF-978A; NEN, Boston, MA). Duplicate membranes were incubated overnight at 30°C in prehybridizadon solution (50% formamide, 2X Denhardt's, 5X SSPE, 0.1% SDS, 100 μg/ml salmon sperm DNA) followed by overnight incubation in hybridization solution (50% formamide, 2X Denhardt's, 5X SSPE, 0.1% SDS, 10% dextran sulfate, 100 μg/ml salmon sperm DNA) with 1 x 10⁶ cpm/ml labeled probe and final wash conditions of IX SSC at 55°C. A clone was identified after two rounds of screening and sequenced with BIG DYE terminator cycle sequencing Ready Reactions (Perkin Elmer, Foster City, CA) on a 377 ABI Prism cycle sequencer (Perkin Elmer, Foster City, CA). To generate a contiguous open reading frame (ORF) for the rabbit motilin receptor, overlapping PCR was performed on exons I and II. PCR products for exons I and II were produced each containing a small portion of the other exon. The primers for exon I, SEQ. ID. NO. 29 (5' gggcccgaattcgccgccATGGGCAGCCCCTGGAACGGCAGC) and SEQ. ID. NO. 30 (5'GGCCAGAACCACCACCAGCAGGACGCGGACGGTCTG), contained an EcoRI site and a "GCC GCC" Kozac sequence. The primers for exon II, SEQ. XD. NO. 31 (5'GTCCGCGTCCTGCTGGTGGTGGTTCTGGCCTTTATAGTG) and SEQ. ID. NO. 32 (5'agtttagcggccgcCTATGCAGCCGTCTTTGTGTTAGC3'), contained a Notl site. The rabbit motilin ORF was then generated from exon I and II templates and primers SEQ. ID. NO. 29 and SEQ. ID. NO. 32. An Advantage cDNA PCR kit (Clontech, Palo Alto, CA) was used in the PCR reactions generally following manufacture instructions. Two exceptions were the addition of 5% DMSO to the PCR reactions and PCR cycling as follows: 1) 94°C for 1 minute, 2) 5 cycles of 94°C for 30 seconds, 72°C for 3 minutes, 3) 5 cycles of 94°C for 30 seconds, 70°C for 3 minutes, 4) 20 cycles of 94°C for 30 seconds, 68°C for 3 minutes. The rabbit motilin ORF fragment was digested with EcoRI and Notl, gel-purified, ligated into pcDNA3 vector and transformed into SCSI E. coli (Stratagene, La Jolla, CA).

Example 2: Cloning of the Dog Motilin Receptor Exon 1 A dog motilin receptor exon was identified and cloned by screening the canine lambda FixII genomic library (Stratagene, La Jolla , CA) with the human MTLR probe (see Example 1). Using techniques illustrated herein, such as those described in Example 1, the full-length clone can readily be obtained.

Hybridization was performed using reduced-stringency conditions. 1.2 x 10⁶ phage plaques of the once amplified library were plated onto E. coli XLl-Blue MRA at

30,000 pfu per 150 mm plate. The phage were transferred onto nylon hybridization transfer membranes (NEN, Boston, MA) in duplicate, denatured, neutralized, washed and probed with random primed (Prime-It II kit, Stratagene, La Jolla, CA) P³²dCTP labeled human MTLR exon I and exon II probes. The membranes were prehybridized (50% formamide, 2X Denhardt's, 5X SSPE, 0.1% SDS, 100 μg/mL salmon sperm DNA) for two hours followed by overnight hybridization (50% formamide, 2X Denhardt's, 5X SSPE, 10% dextran sulfate, 0.1% SDS, 100 μg/mL salmon sperm DNA), shaking in a 32°C incubator with probe at 1 x 10⁶ cpm/mL. The filters were washed in 4X SSC, 0.1% SDS solution at 23°C followed by 2X SSC, 0.1% SDS at 42°C and finally IX SSC, 0.1% SDS at 55°C. After two rounds of plaque purification seven clones were isolated. Lamba

DNA was isolated from the seven clones using a liquid lysate preparation. The indicator strain XLl-Blue MRA was lysed with eluted phage and cell debris spun down. The liquid phage stock was treated with RNaseA at 38 μg/mL, 37°C for 30 minutes and PEG- precipitated (10% PEG8000/1M NaCl in SM buffer) overnight at 4°C. Pelleted phage DNA was proteinase K treated (50 μg/mL, 68°C, 15 minutes). This was followed by phenol/chloroform and chloroform extractions and ethanol precipitation.

Lambda DNA was spooled out with a sterile pipet tip, washed with 70% ethanol and resuspended in sterile water. Each DNA was digested with a band of restriction enzymes (BamHI, EcoRI, Notl, Pstl, Smal and Xbal), electrophoresed on 1% Seakem GTG IX TAE agarose gel, southern blotted and probed with human MTLR exon I and II probes as described above. Hybridizing bands were subcloned and sequenced on ABI 377 automated sequencer using Big Dye terminator premix (Perkin Elmer, Foster City, CA). Sequence information obtained was then analyzed using the Sequencer program. Of these, a 2kB Notl fragment from lambda DNA 35 contained the largest fragment of dog MTLR encoding exon I, the splice junction, and intronic sequence.

Example 3: Dog and Rabbit Motilin Receptor Sequences

The nucleotide and amino acid sequences for SEQ. XD. NOs. 1, 2, 3, and 4 are provided as follows:

SEP. XD. NO. 1 MGGPGNSSDGAEGAQLPCDERLCSPFPLGALVPVTAVCLGLFAVGVSGNLVTVLLIG RYRDMRTTTNLYLGSMAVSDLLILLGLPLDLYRLWRSRPWVFGQLLCRLSLYLGEG CTYATLLHVTALSVERYLAVCRPLRARALLSRRRARALIAALWAVALLSAAPFFFLV GVEQDAGGPGLNGSARLARAPSPPPGPEAALFSRECRPSPSQLGALRVMLWVTTAYF FLPFLCLCVLYGRIGRELRRRRGPLRGRAASGRERGHRQAVRVL

SEP. ID. NO. 2 MGSPWNGSDGPEDAREPPWAALPPCDERRCSPFPLGTLVPVTAVCLGLFAVGVSGN VVTVLLIGRYRDMRTTTNLYLGSMAVSDLLILLGLPFDLYRLWRSRPWVFGQLLCRL SLYVGEGCTYASLLHMTALSVERYLAICRPLRARVLVTRRRVRALIAALWAVALLS AGPFFFLVGVEQDPAVFAAPDRNGTVPLDPSSPAPASPPSGPGAEAAALFSRECRPSR AQLGLLRVMLWVTTAYFFLPFLCLSILYGLIARQLWRGRGPLRGPAATGRERGHRQT VRVLLVVVLAFIVCWLPFHVGRIIYINTQDSRMMYFSQYFNIVALQLFYLSASENPILY NLISKKYRAAARRLLRESRAGPSGVCGSRGPEQDVAGDTGGDTAGCTETSANTKTA A

SEP. ED. NO. 3 ATGGGCGGCCCCGGGAACAGCAGCGACGGCGCGGAGGGCGCGCAGCTGCCGTG CGACGAGCGCCTGTGCTCGCCCTTCCCCCTGGGGGCGCTGGTGCCGGTGACGGCC GTGTGCCTGGGCCTGTTCGCCGTCGGCGTGAGCGGCAACCTGGTGACGGTGCTGC TGATCGGCCGCTACCGCGACATGCGCACCACCACCAACCTGTACCTGGGCAGCA TGGCCGTGTCCGACCTGCTCATCCTGCTGGGGCTGCCCCTCGACCTGTACCGCCT GTGGCGCTCGCGGCCCTGGGTGTTCGGGCAGCTGCTGTGCCGCCTGTCGCTGTAC CTGGGCGAGGGCTGCACCTACGCCACGCTGCTGCACGTGACGGCGCTGAGCGTC GAGCGCTACCTGGCCGTGTGCCGCCCGCTCCGCGCCCGCGCGCTGCTGTCCCGGC GCCGCGCCCGCGCGCTCATCGCGGCGCTCTGGGCCGTGGCGCTGCTGTCGGCCGC GCCCTTCTTCTTCCTGGTGGGCGTCGAGCAGGACGCGGGCGGCCCCGGCCTCAAC GGCAGCGCGCGGCTGGCGCGGGCGCCCTCCCCGCCGCCGGGGCCCGAGGCGGCG CTCTTCAGCCGGGAGTGCCGGCCCAGCCCGTCGCAGCTGGGCGCGCTGCGCGTC ATGCTCTGGGTCACCACCGCCTACTTCTTCCTGCCCTTCCTGTGCCTCTGCGTCCT GTACGGGCGCATCGGCCGCGAGCTGCGGAGGCGCCGGGGGCCGCTGCGGGGCC GGGCCGCCTCGGGGCGCGAGCGGGGCCACCGCCAGGCCGTCCGCGTGCTG

SEQ. ID. NO. 4 ATGGGCAGCCCCTGGAACGGCAGCGACGGCCCCGAGGACGCGCGGGAGCCGCC GTGGGCCGCGCTGCCGCCGTGCGATGAGCGCCGCTGCTCGCCCTTCCCCTTGGGC ACGCTGGTGCCTGTGACGGCCGTGTGCCTGGGCCTGTTCGCCGTCGGGGTGAGCG GCAACGTGGTGACCGTGCTGCTGATCGGGCGCTACCGGGACATGCGGACCACCA CCAACCTGTACCTGGGCAGCATGGCCGTGTCCGACCTGCTCATCCTGCTCGGGCT GCCCTTCGACCTGTACCGCCTGTGGCGCTCGAGGCCCTGGGTGTTCGGACAGCTG CTCTGCCGCCTGTCGCTGTACGTGGGCGAGGGCTGCACCTACGCCTCGCTGCTGC ACATGACGGCGCTCAGCGTGGAGCGCTACCTGGCCATCTGCCGCCCGCTGCGTG CCCGCGTCTTGGTCACCCGCCGCCGGGTCCGCGCGCTCATCGCCGCGCTCTGGGC CGTGGCGCTGCTTTCGGCCGGGCCCTTCTTCTTTCTGGTGGGCGTCGAGCAGGAC CCCGCGGTCTTCGCGGCCCCGGACCGCAACGGTACTGTGCCGCTGGACCCCTCGT CGCCCGCCCCGGCGTCCCCGCCGTCGGGGCCCGGAGCGGAGGCCGCGGCTCTGT TCAGCCGCGAGTGCCGGCCGAGCCGCGCGCAGCTGGGCTTGCTGCGCGTCATGC TGTGGGTTACCACCGCCTACTTTTTCCTGCCCTTCCTCTGCCTCAGCATCCTCTAC GGGCTCATCGCGCGGCAGCTGTGGCGGGGTCGGGGCCCGCTGCGAGGCCCGGCG GCCACGGGTCGGGAGAGGGGCCACCGGCAGACCGTCCGCGTCCTGCTGGTGGTG GTTCTGGCCTTTATAGTGTGCTGGCTGCCTTTCCACGTTGGCAGGATCATTTACAT AAACACCCAAGACTCGCGGATGATGTACTTCTCCCAGTACTTTAACATTGTCGCG CTGCAGCTTTTCTACCTGAGTGCGTCCATCAACCCAATCCTCTACAACCTCATCTC CAAGAAGTACAGAGCGGCTGCCCGCAGACTGCTGCGCGAAAGCCGAGCGGGGC CCAGTGGTGTGTGCGGAAGCAGGGGCCCTGAGCAGGACGTTGCAGGGGACACTG GCGGAGACACAGCTGGCTGCACCGAGACCAGCGCTAACACAAAGACGGCTGCAT AG

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 purified polypeptide comprising a unique amino acid region of SEQ. ED. NO. 1 or SEQ. ED. NO. 2 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ID. NO. 5 or SEQ. ED. NO. 6.

2. The polypeptide of claim 1, wherein said unique region is from SEQ. ED. NO. 1.

3. The polypeptide of claim 2, wherein said unique region comprises an amino acid sequence selected from the group consisting of:

GPGNSSDGA (SEQ. ID. NO. 9);

VCLGLFAVGV (SEQ. ID. NO. 10);

ALLSSRRRA (SEQ. ID. NO. 11); APFFFLVGVEQDAGG (SEQ. ID. NO. 12); and

CLCVLYGRI (SEQ. ID. NO. 13).

4. The polypeptide of claim 2, wherein said polypeptide comprises the amino acid sequence of SEQ. ED. NO. 1.

5. The polypeptide of claim 4, wherein said polypeptide consists of the amino acid sequence of SEQ. XD. NO. 1.

6. The polypeptide of claim 1, wherein said unique region is from SEQ. ID. NO. 2.

7. The polypeptide of claim 6, wherein said unique region comprises an amino acid sequence selected from the group consisting of:

DPAVFAAPDR (SEQ. XD. NO. 14); NGTVPLDPS (SEQ. ID. NO. 15);

SPAPASPPSGPG (SEQ. ID. NO. 16); RRLLRESRAG (SEQ. ID. NO. 17); and SGVCGSRGPEQD (SEQ. ID. NO. 18).

8. The polypeptide of claim 6, wherein said polypeptide comprises the amino acid sequence of SEQ. ED. NO. 2.

9. The polypeptide of claim 8, wherein said polypeptide consists of the amino acid sequence of SEQ. ED. NO. 2.

10. A purified nucleic acid comprising a nucleotide sequence encoding for the polypeptide of any one of claims 1-9.

11. A purified nucleic acid comprising a unique nucleotide sequence region of SEQ. XD. NO. 3 or SEQ. ED. NO. 4 that is at least 18 contiguous nucleotides in length or the complement thereof, wherein said unique region is not present in SEQ. ED. NO. 7 or SEQ. ED. NO. 8.

12. The purified nucleic acid of claim 11, wherein said unique sequence region is from SEQ. ED. NO. 3.

13. The purified nucleic acid of claim 12, wherein said unique region comprises a nucleotide sequence selected from the group consisting of: GGCCCCGGGAACAGCAGCGACGGCGCG (SEQ. ED. NO.19);

GGCCGTGTGCCTGGGCCT (SEQ. ED. NO.20);

CGCGCGCTGCTGTCCCGG (SEQ. ED. NO.21);

AGGACGCGGGCGGCCCCG (SEQ. ED. NO.22); and

CCGCGAGCTGCGGAGGCG (SEQ. ED. NG.23).

14. The purified nucleic acid of claim 12, wherein said nucleic acid comprises the nucleotide sequence of SEQ. XD. NP. 3.

15. The purified nucleic acid of claim 14, wherein said nucleic acid consists of the nucleotide sequence of SEQ. ID. NO. 3.

16. The purified nucleic acid of claim 11, wherein said unique sequence region is from SEQ. XD. NO. 4.

17. The purified nucleic acid of claim 16, wherein said unique region comprises a sequence selected from the group consisting of: TTCGGCCGGGCCCTTCTTCTTT (SEQ. ED. NO. 24); GGTCTTCGCGGCCCCGGA (SEQ. ED. NO. 25); CGGTACTGTGCCGCTGGA (SEQ. ED. NO. 26);

GCTTTTCTACCTGAGTGCGTCC (SEQ. ID. NO. 27); and CGAGCGGGGCCCAGTGGTG (SEQ. ID. NO. 28).

18. The purified nucleic acid of claim 16, wherein said nucleic acid comprises the nucleotide sequence of SEQ. XD. NO. 4.

19. The purified nucleic acid of claim 18, wherein said nucleic acid consists of the nucleotide sequence of SEQ. XD. NO. 4.

20. An expression vector comprising a recombinant nucleotide sequence encoding for a unique amino acid region of SEQ. ED. NO. 1 or SEQ. ED. NO. 2 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ED. NO. 5 or SEQ. ID. NO. 6.

21. A recombinant cell comprising an expression vector encoding for a unique amino acid region of SEQ. XD. NO. 1 or SEQ. ED. NO. 2, that is at least 9 contiguous amino acids in length, functionally coupled to a promoter recognized by said cell, wherein said unique region is not present in SEQ. ED. NO. 5 or SEQ. ED. NO. 6.

22. A recombinant cell made by a process comprising the step of introducing into said cell an expression vector encoding for a unique amino acid region of SEQ. ID. NO. 1 or SEQ. ID. NO. 2, that is at least 9 contiguous amino acids in length wherein said unique region is not present in SEQ. ED. NO. 5 or 6.

23. A method of measuring the ability of a compound to effect motilin receptor activity comprising the steps of: a) contacting a recombinant cell with said compound, wherein said recombinant cell comprises a recombinant nucleic acid expressing a functional motilin receptor that comprises a unique amino acid region of SEQ. XD. NO. 1 or SEQ. ED. NO. 2 that is at least 9 contiguous amino acids in length, provided that said unique region is not present in SEQ. ID. NO. 5 or 6; and b) measuring motilin receptor activity.

24. A method of preparing a motilin receptor polypeptide comprising the step of growing the recombinant cell of claim 21 under conditions wherein said polypeptide is expressed from said expression vector.