DNA encoding a human 5-HT1F receptor and uses thereof

Abstract

This invention provides an isolated nucleic acid molecule encoding a human 5-HT 1F receptor, an isolated protein which is a human 5-HT 1F receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HT 1F receptors. mammalian cells comprising such vectors, antibodies directed to the human 5-HT 1F receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-HT 1F receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HT 1F receptor, pharmaceutical compounds related to human 5-HT 1F receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human 5-HT 1F receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HT 1F receptor.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule encoding a human 5-HT 1F  receptor.  
     
     
         2 . An isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule is a DNA molecule.  
     
     
         3 . An isolated DNA molecule of  claim 2 , wherein the DNA molecule is a cDNA molecule encoding a human 5-HT 1F  receptor.  
     
     
         4 . An isolated human 5-HT 1F  receptor protein.  
     
     
         5 . A vector comprising the DNA molecule of  claim 2 .  
     
     
         6 . A plasmid comprising the vector of  claim 5 .  
     
     
         7 . A vector of  claim 5  adapted for expression in a bacterial cell which comprises the regulatory elements necessary for expression of the DNA in the bacterial cell so located relative to the DNA encoding the 5-HT 1F  receptor as to permit expression thereof.  
     
     
         8 . A vector of  claim 5  adapted for expression in a yeast cell which comprises the regulatory elements necessary for expression of the DNA in the yeast cell so located relative to the DNA encoding the 5-HT 1F  receptor as to permit expression thereof.  
     
     
         9 . A vector of  claim 5  adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding the 5-HT 1F  receptor as to permit expression thereof.  
     
     
         10 . A plasmid of  claim 6  adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding the 5-HT 1F  receptor as to permit expression thereof.  
     
     
         11 . A plasmid comprising the cDNA molecule of clam  3  adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the cDNA molecule as to permit expression thereof, designated pMO5-h116a (ATCC Accession No. 75175).  
     
     
         12 . A mammalian cell comprising the plasmid of  claim 6 .  
     
     
         13 . An Ltk −  cell comprising the plasmid of  claim 6 .  
     
     
         14 . An NIH3T3 cell comprising the plasmid of  claim 6 .  
     
     
         15 . An Ltk −  cell comprising the plasmid of  claim 11 , designated L-5-HT 1F  (ATCC Accession No. CRL 10957).  
     
     
         16 . An NIL3T3 cell comprising the plasmid of  claim 11  designated N-5-HT 1F  (ATCC Accession No. CRL 10956).  
     
     
         17 . A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT 1F  receptor.  
     
     
         18 . The nucleic acid probe of  claim 17  wherein the nucleic acid is DNA.  
     
     
         19 . An antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a human 5-HT 1F  receptor so as to prevent translation of the mRNA molecule.  
     
     
         20 . An antisense oligonucleotide having a sequence capable of binding specifically to the cDNA molecule of  claim 3 .  
     
     
         21 . An antisense oligonucleotide of  claim 19  comprising chemical analogues of nucleotides.  
     
     
         22 . An antibody directed to a human 5-HT 1F  receptor.  
     
     
         23 . A monoclonal antibody directed to an epitope of a human 5-HT 1F  receptor present on the surface of a cell and having an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the human 5-HT 1F  receptor.  
     
     
         24 . A pharmaceutical composition comprising an amount of the oligonucleotide of  claim 19  effective to reduce expression of a human 5-HT 1F  receptor by passing through a cell membrane and binding specifically with mRNA encoding a human 5-HT 1F  receptor in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane.  
     
     
         25 . A pharmaceutical composition of  claim 24 , wherein the oligonucleotide is coupled to a substance which inactivates mRNA.  
     
     
         26 . A pharmaceutical composition of  claim 25 , wherein the substance which inactivates mRNA is a ribozyme.  
     
     
         27 . A pharmaceutical composition of  claim 24 , wherein the pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane comprises a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type.  
     
     
         28 . A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human 5-HT 1F  receptor and a pharmaceutically acceptable carrier.  
     
     
         29 . A pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpressior of 5-HT 1F  receptor and a pharmaceutically acceptable carrier.  
     
     
         30 . A pharmaceutical composition which comprises an amount of the antibody of  claim 22  effective to block binding of naturally occurring ligands to the 5-HT 1F  receptor and a pharmaceutically acceptable carrier.  
     
     
         31 . A transgenic nonhuman mammal expressing DNA encoding a human 5-HT 1F  receptor.  
     
     
         32 . A transgenic nonhuman mammal expressing DNA encoding a human 5-HT 1F  receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT 1F  receptor.  
     
     
         33 . A transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT 1F  receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a 5-HT 1F  receptor and which hybridizes to mRNA encoding a 5-HT 1F  receptor thereby reducing its translation.  
     
     
         34 . The transgenic nonhuman mammal of any of claims  31 ,  32  or  33 , wherein the DNA encoding a human 5-HT 1F  receptor additionally comprises an inducible promoter.  
     
     
         35 . The transgenic nonhuman mammal of any of claims  3 :,  32  or  33 , wherein the DNA encoding a human 5-HT 1F  receptor additionally comprises tissue specific regulatory elements.  
     
     
         36 . A transgenic nonhuman mammal of any of claims  31 ,  32  or  33 , wherein the transgenic nonhuman mammal is a mouse.  
     
     
         37 . A method for determining whether a ligand not known to be capable of binding to a human 5-HT 1F  receptor can bind to a human 5-HT 1F  receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT 1F  receptor with the ligand under conditions permitting binding of ligands known to bind to a 5-HT 1F  receptor, detecting the presence of any of the ligand bound to a human 5-HT 1F  receptor, and thereby determining whether the ligand binds to a human 5-HT 1F  receptor.  
     
     
         38 . A method for determining whether a ligand not known to be capable of binding to the human 5-HT 1F  receptor can functionally activate receptor activity or prevent the action of a ligand which does so comprising contacting a mammalian cell of  claim 12  with the ligand under conditions permitting the activation or blockade of a functional response, and detecting by means of a bioassay from the mammalian cell such as a second messenger response, and thereby determining whether the ligand activates or prevents the activation of the human 5-HT 1 F  receptor functional output.  
     
     
         39 . The method of  claim 37  or  38  wherein the mammalian cell is nonneuronal in origin.  
     
     
         40 . A method of  claim 39 , wherein the mammalian cell nonneuronal in origin is an Ltk −  cell.  
     
     
         41 . A method of  claim 39 , wherein the mammalian cell nonneuronal in origin is an NIH3T3 cell.  
     
     
         42 . A ligand detected by the method of  claim 37  or  38 .  
     
     
         43 . A method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT 1F  receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT 1F  receptor with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human 5-HT 1F  receptor.  
     
     
         44 . A method of screening drugs to identify drugs which interact with, and activate or block the activation of, the human 5-HT 1F  receptor on the surface of a cell which comprises contacting the mammalian cell of  claim 12  with a plurality of drugs, determining those drugs which activate or block the activation of the receptor in the mammalian cell using a bioassay such as a second messenger assays, and thereby identifying drugs which specifically interact with, and activate or block the activation of, a human 5-HT 1F  receptor.  
     
     
         45 . The method of  claim 43  or  44  wherein the mammalian cell is nonneuronal in origin.  
     
     
         46 . The method of  claim 45  wherein the mammalian cell nonneuronal in origin is an Ltk −  cell.  
     
     
         47 . The method of  claim 45  wherein the mammalian cell nonneuronal in origin is an NIH3T3 cell.  
     
     
         48 . A pharmaceutical composition comprising a drug identified by the method of  claim 43  or  44  and a pharmaceutically acceptable carrier.  
     
     
         49 . A method of detecting expression of the 5-HT 1F  receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT 1F  receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with the nucleic acid probe of  claim 17  under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HT 1F  receptor by the cell.  
     
     
         50 . A method of treating abnormalities in a subject, wherein the abnormality is alleviated by the reduced expression of a 5-HT 1F  receptor which comprises administering to a subject an effective amount of the pharmaceutical composition of  claim 24  effective to reduce expression of the 5-HT 1F  receptor by the subject.  
     
     
         51 . A method of treating an abnormal condition related to an excess of 5-HT 1F  receptor activity which comprises administering to a subject an effective amount of the pharmaceutical composition of  claim 24  effective to reduce expression of the 5-HT 1F  receptor in the subject.  
     
     
         52 . The method of  claim 51  wherein the abnormal condition is dementia.  
     
     
         53 . The method of  claim 51  wherein the abnormal condition Parkinson's disease.  
     
     
         54 . The method of  claim 51  wherein the abnormal condition is a feeding disorder.  
     
     
         55 . The method of  claim 51  wherein the abnormal condition is pathological anxiety.  
     
     
         56 . The method of  claim 51  wherein the abnormal condition is schizophrenia.  
     
     
         57 . The method of  claim 51  wherein the abnormal condition is a migraine headache.  
     
     
         58 . A method of treating abnormalities which are alleviated by reduction of expression of a human 5-HT 1F  receptor which comprises administering to a subject an amount of the pharmaceutical composition of  claim 30  effective to block binding of naturally occurring ligands to the 5-HT 1F  receptor and thereby alleviate abnormalities resulting from overexpression of a human 5-HT 1F  receptor.  
     
     
         59 . A method of treating an abnormal condition related to an excess of 5-HT 1F  receptor activity which comprises administering to a subject an amount of the pharmaceutical composition of  claim 30  effective to block binding of naturally occurring ligands to the 5-HT 1F  receptor and thereby alleviate the abnormal condition.  
     
     
         60 . The method of  claim 59  wherein the abnormal condition is dementia.  
     
     
         61 . The method of  claim 59  wherein the abnormal condition is Parkinson's disease.  
     
     
         62 . The method of  claim 59  wherein the abnormal condition is a feeding disorder.  
     
     
         63 . The method of  claim 59  wherein the abnormal condition is a pathological anxiety.  
     
     
         64 . The method of  claim 59  wherein the abnormal condition is schizophrenia.  
     
     
         65 . The method of  claim 59  wherein the abnormal condition is a migraine headache.  
     
     
         66 . A method of detecting the presence of a human 5-HT 1F  receptor on the surface of a cell which comprises contacting the cell with the antibody of  claim 22  under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of a human 5-HT 1F  receptor on the surface of the cell.  
     
     
         67 . A method of determining the physiological effects of expressing varying levels of human 5-HT 1F  receptors which comprises producing a transgenic nonhuman mammal whose levels of human S-HT 1F  receptor expression are varied by use of an inducible promoter which regulates human 5-HT 1F  receptor expression.  
     
     
         68 . A method of determining the physiological effects of expressing varying levels of human 5-HT 1F  receptors which comprises producing a panel of transgenic nonhuman mammals each expressing a different amount of human 5-HT 1F  receptor.  
     
     
         69 . A method for identifying a substance capable of alleviating the abnormalities resulting from overexpression of a human 5-HT 1F  receptor comprising administering a substance to the transgenic nonhuman mammal of  claim 31  and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of a human 5-HT 1F  receptor.  
     
     
         70 . A method for treating the abnormalities resulting from overexpression of a human 5-HT 1F  receptor comprises administering to a subject an amount of the pharmaceutical composition of  claim 28  effective to alleviate the abnormalities resulting from overexpression of a human 5-HT 1F  receptor.  
     
     
         71 . A method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human 5-HT 1F  receptor comprising administering the substance to the transgenic nonhuman mammal of either of claims  32  or  33  and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of a human 5-HT 1F  receptor.  
     
     
         72 . A method for treating the abnormalities resulting from underexpression of a human 5-HT 1F  receptor which comprises administering to a subject an amount of the pharmaceutical composition of  claim 29  effective to alleviate the abnormalities resulting from underexpression of a human 5-HT 1F  receptor.  
     
     
         73 . A method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT 1F  receptor allele which comprises: 
 a. obtaining DNA of subjects suffering from the disorder;    b. performing a restriction digest of the DNA with a panel of restriction enzymes;    c. electrophoretically separating the resulting DNA fragments on a sizing gel;    d. contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human 5-HT 1F  receptor and labelled with a detectable marker;    e. detecting labelled bands which have hybridized to the DNA encoding a human 5-HT 1F  receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder;    f. preparing DNA obtained for diagnosis by steps a-e; and    g. comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.    
     
     
         74 . The method of claim  73  wherein a disorder associated with the expression of a specific human 5-HT 1F  receptor allele is diagnosed.  
     
     
         75 . A method of preparing the isolated 5-HT 1F  receptor of  claim 4  which comprises: 
 a. inducing cells to express 5-HT 1F  receptor;  
 b. recovering the receptor from the resulting cells; and  
 c. purifying the receptor so recovered.  
 
     
     
         76 . A method of preparing the isolated 5-HT 1F  receptor of  claim 4  which comprises: 
 a. inserting nucleic acid encoding 5-HT 1F  receptor in a suitable vector;  
 b. inserting the resulting vector in a suitable host cell;  
 c. recovering the receptor produced by the resulting cell; and  
 d. purifying the receptor so recovered.