Methods for identifying a compound that binds to a human 5-HT.sub.1E receptor

BACKGROUND OF THE INVENTION
Since the purification of a pressor substance in blood serum termed serotonin (Rapport et al., 1947) and later identified as 5-hydroxytryptamine (5-HT)(Rapport, 1949), there has been a plethora of reports demonstrating that this indoleamine not only plays a role in the functioning of peripheral tissues but, indeed, performs a key role in the brain as a neurotransmitter. Certainly, the anatomical localization of serotonin and serotonergic neurons in both the peripheral and central nervous systems supports its role in such diverse physiologic and behavioral functions as pain perception, sleep, aggression, sexual activity, hormone secretion, thermoregulation, motor activity, cardiovascular function, food intake and renal regulation (For review see Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka, 1991). Taken together, it appears that serotonin plays an important role in homeostasis and in modulating responsiveness to environmental stimuli. Accordingly, studies demonstrating that abnormalities in the serotonergic system may be associated with disease states has created a drug development effort towards agents which may selectively modulate the function of serotonin (Glennon, 1990).
In relation to the characterization of physiologic or biochemical responses resulting from the release of serotonin are simultaneous investigations examining the receptor sites responsible for the actions elicited by the indoleamine transmitter. Following early in vitro pharmacological assays describing the existence of two different serotonin receptors, designated as D and M, in the guinea pig ileum (Gaddum and Picarelli, 1957), the advent of receptor binding technique in the 1970's has brought to light during the last decade the diversity of 5-HT receptors existing in both the brain and peripheral tissues. Thus, although the concept of D and M receptors has not been invalidated, serotonin receptors not fitting either category have been identified using radioligand methods. To date using this technique, there appears to be four classes of serotonin receptors found in the brain: 5-HT.sub.1, 5-HT.sub.2, 5-HT.sub.3 and, 5-HT.sub.4 (Peroutka, 1991). Furthermore, 5-HT1 sites have been subclassified as: 5-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 5-HT.sub.1D (Hamon et al., 1990) and 5-HT.sub.1E (Leonhardt et al., 1989). Although a detailed characterization of the 5-HT.sub.1E binding site is lacking, extensive pharmacologic, biochemical and functional properties have clearly shown that the other four subtypes of 5-HT.sub.1 sites are receptors according to classical criteria. Interestingly, the 5-HT.sub.1E binding site was first observed in human cortical tissue using [.sup.3 H]5-HT as the radioligand probe in the presence of 5-carboxyamidotryptamine and mesulergine to mask other members of the 5-HT.sub.1 receptor class. The affinity constants of the nine drugs tested indicated a unique pharmacological profile. In particular, the low affinity of 5-CT and ergotamine seemed to clearly discriminate the pharmacologically defined 5-HT.sub.1D site from that of this novel serotonergic site. Importantly, it was demonstrated that 5-HT.sub.1E sites are saturable and exist in a density consistent with other known neurotransmitter receptors. Furthermore, this site appeared to interact with a GTP-binding protein. Overall, the data provided a framework suggesting that the 5-HT.sub.1E binding site may represent a functional receptor.
During the last few years, the field of molecular biology as provided an important facet to receptor research by cloning these proteins and allowing more precise characterizations in isolated systems (Hartig et al.,1990). This has been accomplished for the 5-HT.sub.1A (Fargin et al., 1988), 5-HT.sub.1C (Julius et al., 1988), 5-HT.sub.1D (Branchek et al., 1990) and 5-HT.sub.2 receptors (Pritchett et al., 1988). Thus, there is no doubt that these binding sites represent "true" functional receptors. Indeed, the pharmacological characterization of serotonin receptors involved in various physiological or biochemical functions is a key component of drug development for the serotonergic system. As one can deduce from the diversity of serotonin binding sites, many targets are available for advancement in selective drug design. The coupling of molecular biological methods to pharmacological characterization particularly for cloned human receptors will open new avenues for pharmaceutical development which have not been previously explored.
SUMMARY OF THE INVENTION
This invention provides an isolated nucleic acid molecule encoding a human 5-HT.sub.1E receptor.
This invention also provides an isolated protein which is a human 5-HT.sub.1E receptor.
This invention provides a vector comprising an isolated nucleic acid molecule encoding a human 5-HT.sub.1E receptor.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human 5-HT.sub.1E receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding the 5-HT.sub.1E receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT.sub.1E receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT.sub.1E receptor can bind to a human 5-HT.sub.1E receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT.sub.1E receptor with the ligand under conditions permitting binding of ligands known to bind to a 5-HT.sub.1E receptor, detecting the presence of any of the ligand bound to a human 5-HT.sub.1E receptor, and thereby determining whether the ligand binds to a human 5-HT.sub.1E receptor.
This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT.sub.1E receptor can functionally activate its activity or prevent the action of a ligand which does so. This comprises contacting a mammalian cell comprising an isolated DNA molecule which encodes a human 5-HT.sub.1E receptor with the ligand under conditions permitting the activation or blockade of a functional response, detected 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.sub.1E receptor functional output.
This invention further provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT.sub.1E receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT.sub.1E 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.sub.1E receptor.
This invention also provides a method of screening drugs to identify drugs which interact with, and activate or block the activation of, the human 5-HT.sub.1E receptor on the surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HT.sub.1E receptor 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.sub.1E receptor.
This invention provides 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.sub.1E receptor.
This invention also provides a method of detecting expression of the 5-HT.sub.1E receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT.sub.1E receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with 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.sub.1E receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HT.sub.1E receptor by the cell.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human 5-HT.sub.1E receptor so as to prevent translation of the mRNA molecule.
This invention provides an antibody directed to a human 5-HT.sub.1E receptor.
This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT.sub.1E receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT.sub.1E receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a 5-HT.sub.1E receptor and which hybridizes to mRNA encoding a 5-HT.sub.1E receptor thereby reducing its translation.
This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HT.sub.1E receptors which comprises producing a transgenic nonhuman animal whose levels of human 5-HT.sub.1E receptor expression are varied by use of an inducible promoter which regulates human 5-HT.sub.1E receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HT.sub.1E receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT.sub.1E receptor.
This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT.sub.1E 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.sub.1E receptor and labelled with a detectable marker; e. detecting labelled bands which have hybridized to the DNA encoding a human 5-HT.sub.1E 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.
This invention provides a method of preparing the isolated 5-HT.sub.1E receptor which comprises inducing cells to express 5-HT.sub.1E receptor, recovering the receptor from the resulting cells and purifying the receptor so recovered.
This invention also provides a method of preparing the isolated 5-HT.sub.1E receptor which comprises inserting nucleic acid encoding 5-HT.sub.1E receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a receptor so as to prevent translation of the mRNA molecule.
This invention also provides a transgenic nonhuman mammal expressing DNA encoding a receptor.
This invention further provides a transgenic nonhuman mammal expressing DNA encoding a receptor so mutated as to be incapable of normal receptor activity, and not expressing native receptor.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a transgenic nonhuman animal whose levels of receptor expression are varied by use of an inducible promoter which regulates receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of the receptor.
This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding the receptor and which hybridizes to mRNA encoding the receptor thereby preventing its translation.
This invention provides a method for determining whether a ligand not known to be capable of binding to a receptor can bind to a receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the receptor with the ligand under conditions permitting binding of ligands known to bind to a receptor, detecting the presence of any of the ligand bound to the receptor, and thereby determining whether the ligand binds to the receptor.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1(parts A through D). Nucleotide and deduced amino acid sequence of gene 5-HT.sub.1E. (Seq. I.D. Nos. 1 and 2).
Numbers above the nucleotide sequence indicate nucleotide position. DNA sequence was determined by the chain termination method of Sanger, et al., on denatured double-stranded plasmid templates using the enzyme Sequenase. Deduced amino acid sequence (single letter code) of a long open reading frame is shown.
FIG. 2(parts A through B). Comparison of the human 5-HT.sub.1E receptor primary structures with other serotonin receptors. (Seq. I.D. Nos. 3-5HT.sub.1A ; 4-5HT.sub.1C ; 5-5HT.sub.1D.alpha. ; 6-5HT.sub.1D.beta. ; 7-5HT.sub.2)
Amino acid sequences (single letter code) are aligned to optimize homology. The putative transmembrane spanning domains are indicated by stars and identified by Roman numerals (TM I-VII).
FIG. 3. Respective pKi values of various drugs for the inhibition of [.sup.3 H]5-HT specific binding to the cloned 5-HT.sub.1E receptor and human cortical membrane preparations containing the native 5-HT.sub.1E receptor.
Values for the native membrane preparation are taken from Leonhardt et al., 1989. "r" is the correlation coefficient between pKi values calculated for the two receptor preparations and clearly indicates the similarity in binding profile.
FIG. 4. 5-HT concentration-effect curve for the inhibition of forskolin-stimulated CAMP formation in Y1 cells transfected with the 5-HT.sub.1E receptor.
Values are expressed as a percentage of forskolin(Fsk)-stimulated cAMP production.
FIG. 5. 5-HT.sub.1E mRNA localization in the human brain.
cDNA was reverse transcribed from total RNA (5 .mu.g) using random hexamers (500 pmoles). One microgram of cDNA was subjected to 30 cycles of PCR amplification using 5'- and 3'-primers directed to the third cytoplasmic loop of the 5HT1E receptor gene. Amplified fragments were subjected to Southern blot analysis using an end-labeled oligonucleotide probe which was internal to the PCR primers. (+) control consisted of 5HT1E recombinant plasmid; (-) control consisted of all cDNA and PCR reagents without the addition of cDNA template. Intensity of signal is depicted as the number of plus signs as defined in the figure key.

DETAILED DESCRIPTION OF THE INVENTION
As used herein, the 5-HT receptor family is defined as the group of mammalian proteins that function as receptors for serotonin. A 5-HT receptor subfamily is defined as a subset of proteins belonging to the 5-HT receptor family which are encoded by genes which exhibit homology of greater than 72% or higher with each other in their deduced amino acid sequences within presumed transmembrane regions (linearly contiguous stretches of hydrophobic amino acids, bordered by charged or polar amino acids, that are long enough to form secondary protein structures that span a lipid bilayer). Four human 5-HT receptor subfamilies can be distinguished based on the information presently available: 5-HT.sub.1, 5-HT.sub.2, 5-HT.sub.3, and 5-HT.sub.4 (Peroutka, 1991). The 5-HT.sub.2 receptor subfamily contains the human 5-HT.sub.2 receptor. Although no other human members of this family have been described, the rat 5-HT.sub.2 receptor (Pritchett, et al. 1988; Julius, et al. Proc. Natl. Acad. Sci. USA 87:928-932, 1990) and the rat 5HT.sub.1C receptor (Julius, et al. 1988) constitute a rat 5-HT receptor subfamily. The 5-HT.sub.1 subfamily has been subdivided further as: 5-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 5-HT.sub.1D (Hamon et al., 1990) and 5-HT.sub.1E (Leonhardt et al., 1989). The 5-HT.sub.1A subfamily contains the human 5-HT.sub.1A receptor, also known as G-21 (Fargin, et al. 1988) The 5-HT.sub.1D receptor subfamily contains two members, the 5-HT.sub.1D-1 receptor (also termed 5-HT.sub.1D.alpha.) and the 5-HT.sub.1D-2 receptor (also termed 5-HT.sub.1D.beta.). The 5-HT.sub.1E subfamily contains the human 5-HT.sub.1E receptor (also termed clone hp75d). Although this definition differs from the pharmacological definition used earlier, there is significant overlap between the present definition and the pharmacological definition. Members of the 5-HT.sub.1E receptor subfamily so described include the 5-HT.sub.1E receptor and any other receptors which have a greater than 72% homology to the DNA and amino acid sequence shown in FIG. 1 according to the definition of "subfamily". This invention relates to the discovery of the first member of the human 5-HT.sup.1E receptor subfamily.
This invention provides an isolated nucleic acid molecule encoding a human 5-HT.sub.1E receptor. As used herein, the term "isolated nucleic acid molecule" means a nucleic acid molecule that is, a molecule in a form which does not occur in nature. Such a receptor is by definition a member of the 5-HT.sub.1E receptor subfamily. Therefore, any receptor which meets the defining criteria given above is a human 5-HT.sub.1E receptor. One means of isolating a human 5-HT.sub.1E receptor is to probe a human genomic library with a natural or artificially designed DNA probe, using methods well known in the art. DNA probes derived from the human receptor gene 5-HT.sub.1E are particularly useful probes for this purpose. DNA and cDNA molecules which encode human 5-HT.sub.1E receptors may be used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below. Transcriptional regulatory elements from the 5' untranslated region of the isolated clones, and other stability, processing, transcription, translation, and tissue specificity-determining regions from the 3' and 5' untranslated regions of the isolated genes are thereby obtained. Examples of a nucleic acid molecule are an RNA, cDNA, or isolated genomic DNA molecule encoding a human 5-HT.sub.1E receptor. Such molecules may have coding sequences substantially the same as the coding sequence shown in FIG. 1. The DNA molecule of FIG. 1 encodes the sequence of the human 5-HT.sub.1E receptor gene.
This invention further provides a cDNA molecule of encoding a human 5-HT.sub.1E receptor having a coding sequence substantially the same as the coding sequence shown in FIG. 1. This molecule is obtained by the means described above.
This invention also provides an isolated protein which is a human 5-HT.sub.1E receptor. As used herein, the term "isolated protein means a protein molecule free of other cellular components. An example of such protein is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1 which is a human 5-HT.sub.1E receptor. One means for obtaining isolated 5-HT.sub.1E receptor is to express DNA encoding the receptor in a suitable host, such as a bacterial, yeast, or mammalian cell, using methods well known in the art, and recovering the receptor protein after it has been expressed in such a host, again using methods well known in the art. The receptor may also be isolated from cells which express it, in particular from cells which have been transfected with the expression vectors described below in more detail.
This invention provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA encoding a human 5-HT.sub.1E receptor. Examples of vectors are viruses such as bacteriophages (such as phage lambda), cosmids, plasmids (such as pUC18, available from Pharmacia, Piscataway, N.J.), and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art. For example, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with a ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available. A specific example of such plasmids is a plasmid comprising cDNA having a coding sequence substantially the same as the coding sequence shown in FIG. 1 and designated clone hp75d (Seq. I.D. No. 1).
This invention also provides vectors comprising a DNA molecule encoding a human 5-HT.sub.1E receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding a human 5-HT.sub.1E receptor as to permit expression thereof. DNA having coding sequences substantially the same as the coding sequence shown in FIG. 1 may usefully be inserted into the vectors to express human 5-HT.sub.1E receptors. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982). Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general. Expression vectors are useful to produce cells that express the receptor. Certain uses for such cells are described in more detail below.
This invention further provides a plasmid adapted for expression in a bacterial, yeast, or, in particular, a mammalian cell which comprises a DNA molecule encoding a human 5-HT.sub.1E receptor and the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cell so located relative to the DNA encoding a human 5-HT.sub.1E receptor as to permit expression thereof. Some plasmids adapted for expression in a mammalian cell are pSVL (available from Pharmacia, Piscataway, N.J.) and pcEXV-3 (Miller J. and Germain R. N., J. Exp. Med. 164:1478 (1986)). A specific example of such plasmid is a plasmid adapted for expression in a mammalian cell comprising cDNA having coding sequences substantially the same as the coding sequence shown in FIG. 1 and the regulatory elements necessary for expression of the DNA in the mammalian cell which is designated pcEXV-hp75d and deposited under ATCC Accession No. 75138. Those skilled in the art will readily appreciate that numerous plasmids adapted for expression in a mammalian cell which comprise DNA of encoding human 5-HT.sub.1E receptors and the regulatory elements necessary to express such DNA in the mammalian cell may be constructed utilizing existing plasmids and adapted as appropriate to contain the regulatory elements necessary to express the DNA in the mammalian cell. The plasmids may be constructed by the methods described above for expression vectors and vectors in general, and by other methods well known in the art.
The deposit discussed supra, and the other deposits discussed herein, were made pursuant to, and in satisfaction of, the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.
This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT.sub.1E receptor, such as a mammalian cell comprising a plasmid adapted for expression in a mammalian cell, which comprises a DNA molecule encoding a human 5-HT.sub.1E receptor, the protein encoded thereby is expressed on the cell surface, and the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a human 5-HT.sub.1E receptor as to permit expression thereof. Numerous mammalian cells may be used as hosts, including, for example, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, Ltk.sup.- cells, Y1 cells, etc. A particular example of an Ltk.sup.- cell is a cell designated 5-HT.sub.1E -7 and deposited under ATCC Accession No. CRL 10913 and comprises the plasmid designated pcEXV-hp75d. Another example is the murine adrenal Y1 cell line designated Y-5-HT.sub.1E and deposited under ATCC Accession No. CRL 10914. Expression plasmids such as that described supra may be used to transfect mammalian cells by methods well known in the art such as calcium phosphate precipitation, or DNA encoding these 5-HT.sub.1E receptors may be otherwise introduced into mammalian cells, e.g., by microinjection, to obtain mammalian cells which comprise DNA, e.g., cDNA or a plasmid, encoding either human 5-HT.sub.1E receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT.sub.1E receptor can bind to a human 5-HT.sub.1E receptor which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT.sub.1E receptor, the protein encoded thereby is expressed on the cell surface, with the ligand under conditions permitting binding of ligands known to bind to the 5-HT.sub.1E receptor, detecting the presence of any of the ligand bound to the 5-HT.sub.1E receptor, and thereby determining whether the ligand binds to the 5-HT.sub.1E receptor. This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT.sub.1E receptor can functionally activate its activity or prevent the action of a ligand which does so. This comprises contacting a mammalian cell comprising an isolated DNA molecule which encodes a human 5-HT.sub.1E receptor with the ligand under conditions permitting the activation or blockade of a functional response, detected 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.sub.1E receptor functional output. The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1 preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is an Ltk.sup.- cell, in particular the Ltk.sup.- cell designated 5-HT.sub.1E -7. Another example of a non-neuronal mammalian cell to be used for functional assays is a Y1 murine adrenal cell, specifically the Y1 cell designated Y-5-HT.sub.1E. The preferred method for determining whether a ligand is capable of binding to the human 5-HT.sub.1E receptor comprises contacting a transfected nonneuronal mammalian cell (i.e. a cell that does not naturally express any type of 5-HT or G-protein coupled receptor, thus will only express such a receptor if it is transfected into the cell) expressing a 5-HT.sub.1E receptor on its surface, or contacting a membrane preparation derived from such a transfected cell, with the ligand under conditions which are known to prevail, and thus to be associated with, in vivo binding of the ligands to a 5-HT.sub.1E receptor, detecting the presence of any of the ligand being tested bound to the 5-HT.sub.1E receptor on the surface of the cell, and thereby determining whether the ligand binds to, activates or prevents the activation of the 5-HT.sub.1E receptor. This response system is obtained by transfection of isolated DNA into a suitable host cell containing the desired second messenger system such as phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase or ion channels. Such a host system is isolated from pre-existing cell lines, or can be generated by inserting appropriate components of second messenger systems into existing cell lines. Such a transfection system provides a complete response system for investigation or assay of the activity of human 5-HT.sub.1E receptors with ligands as described above. Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for these competitive binding assays. Functional assays of second messenger systems or their sequelae in transfection systems act as assays for binding affinity and efficacy in the activation of receptor function. A transfection system constitutes a "drug discovery system" useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the human 5-HT.sub.1E receptor. The transfection system is also useful for determining the affinity and efficacy of known drugs at the human 5-HT.sub.1E receptor sites.
This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT.sub.1E receptor on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT.sub.1E receptor on the surface of a cell 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, the human 5-HT.sub.1E receptor. This invention also provides a method of screening drugs to identify drugs which interact with, and activate or block the activation of, the human 5-HT.sub.1E receptor on the surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HT.sub.1E receptor 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.sub.1E receptor. The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1. Preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is an Ltk.sup.- cell, in particular the Ltk.sup.- cell designated 5-HT.sub.1E -7. Another example of a non-neuronal mammalian cell to be used for functional assays is a Y1 murine adrenal cell, specifically the Y1 cell designated Y-5-HT.sub.1E. Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed 5-HT.sub.1E receptor protein in transfected cells, using radioligand binding methods well known in the art, examples of which are shown in the binding assays described herein. Drug candidates are also screened for selectivity by identifying compounds which bind with high affinity to one particular 5-HT.sub.1E receptor subtype but do not bind with high affinity to any other serotonin receptor subtype or to any other known receptor site. Because selective, high affinity compounds interact primarily with the target 5-HT.sub.1E receptor site after administration to the patient, the chances of producing a drug with unwanted side effects are minimized by this approach. This invention provides a pharmaceutical composition comprising a drug identified by the method described above and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. Once the candidate drug has been shown to be adequately bio-available following a particular route of administration, for example orally or by injection (adequate therapeutic concentrations must be maintained at the site of action for an adequate period to gain the desired therapeutic benefit), and has been shown to be non-toxic and therapeutically effective in appropriate disease models, the drug may be administered to patients by that route of administration determined to make the drug bio-available, in an appropriate solid or solution formulation, to gain the desired therapeutic benefit.
This invention provides 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.sub.1E receptor, for example with a coding sequence included within the sequence shown in FIG. 1. As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. Detection of nucleic acid encoding human 5-HT.sub.1E receptors is useful as a diagnostic test for any disease process in which levels of expression of the corresponding 5-HT.sub.1E receptor is altered. DNA probe molecules are produced by insertion of a DNA molecule which encodes human 5-HT.sub.1E receptor or fragments thereof into suitable vectors, such as plasmids or bacteriophages, followed by insertion into suitable bacterial host cells and replication and harvesting of the DNA probes, all using methods well known in the art. For example, the DNA may be extracted from a cell lysate using phenol and ethanol, digested with restriction enzymes corresponding to the insertion sites of the DNA into the vector (discussed above), electrophoresed, and cut out of the resulting gel. An example of such DNA molecule is shown in FIG. 1. The probes are useful for `in situ` hybridization or in order to locate tissues which express this gene family, or for other hybridization assays for the presence of these genes or their mRNA in various biological tissues. In addition, synthesized oligonucleotides (produced by a DNA synthesizer) complementary to the sequence of a DNA molecule which encodes human 5-HT.sub.1E receptor of are useful as probes for these genes, for their associated mRNA, or for the isolation of related genes by homology screening of genomic or cDNA libraries, or by the use of amplification techniques such as the Polymerase Chain Reaction. Synthesized oligonucleotides as described may also be used to determine the cellular localization of the mRNA produced by the, 5-HT.sub.1E gene by in situ hybridization. An example of such an oligonucleotide is: GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT (Seq. I.D. No. 8).
This invention also provides a method of detecting expression of a 5-HT.sub.1E receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT.sub.1E receptor which comprises obtaining total mRNA from the cell using methods well known in the art and contacting the mRNA so obtained with 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.sub.1E receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HT.sub.1E receptor by the cell. Hybridization of probes to target nucleic acid molecules such as mRNA molecules employs techniques well known in the art. In one possible means of performing this method, nucleic acids are extracted by precipitation from lysed cells and the mRNA is isolated from the extract using a column which binds the poly-A tails of the mRNA molecules. The mRNA is then exposed to radioactively labelled probe on a nitrocellulose membrane, and the probe hybridizes to and thereby labels complementary mRNA sequences. Binding may be detected by autoradiography or scintillation counting.
However, other methods for performing these steps are well known to those skilled in the art, and the discussion above is merely an example.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human 5-HT.sub.1E receptor so as to prevent translation of the mRNA molecule. The antisense oligonucleotide may have a sequence capable of binding specifically with any sequences of the cDNA molecule whose sequence is shown in FIG. 1. As used herein, the phrase "binding specifically" means the ability of a nucleic acid sequence to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. A particular example of an antisense oligonucleotide is an antisense oligonucleotide comprising chemical analogues of nucleotides.
This invention also provides a pharmaceutical composition comprising an amount of the oligonucleotide described above effective to reduce expression of a human 5-HT.sub.1E receptor by passing through a cell membrane and binding specifically with mRNA encoding a human 5-HT.sub.1E receptor in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. The oligonucleotide may be coupled to a substance which inactivates mRNA, such as a ribozyme. The pharmaceutically acceptable hydrophobic carrier capable of passing through cell membranes may also comprise 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. The structure may be part of a protein known to bind a cell-type specific receptor, for example an insulin molecule, which would target pancreatic cells. DNA molecules having coding sequences substantially the same as the coding sequence shown in FIG. 1 may be used as the oligonucleotides of the pharmaceutical composition.
This invention also provides a method of treating abnormalities which are alleviated by reduction of expression of a 5-HT.sub.1E receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to reduce expression of the 5-HT.sub.1E receptor by the subject. This invention further provides a method of treating an abnormal condition related to 5-HT.sub.1E receptor activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to reduce expression of the 5-HT.sub.1E receptor by the subject. Several examples of such abnormal conditions are dementia, Parkinson's disease, feeding disorders, pathological anxiety, schizophrenia, or a migraine headache.
Antisense oligonucleotide drugs inhibit translation of mRNA encoding these receptors. Synthetic oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding the 5-HT.sub.1E receptor and inhibit translation of mRNA and are useful as drugs to inhibit expression of 5-HT.sub.1E receptor genes in patients. This invention provides a means to therapeutically alter levels of expression of human 5-HT.sub.1E receptors by the use of a synthetic antisense oligonucleotide drug (SAOD) which inhibits translation of mRNA encoding these receptors. Synthetic oligonucleotides, or other antisense chemical structures designed to recognize and selectively bind to mRNA, are constructed to be complementary to portions of the nucleotide sequences shown in FIG. 1 of DNA, RNA or of chemically modified, artificial nucleic acids. The SAOD is designed to be stable in the blood stream for administration to patients by injection, or in laboratory cell culture conditions, for administration to cells removed from the patient. The SAOD is designed to be capable of passing through cell membranes in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SAOD which render it capable of passing through cell membranes (e.g. by designing small, hydrophobic SAOD chemical structures) or by virtue of specific transport systems in the cell which recognize and transport the SAOD into the cell. In addition, the SAOD can be designed for administration only to certain selected cell populations by targeting the SAOD to be recognized by specific cellular uptake mechanisms which binds and takes up the SAOD only within certain selected cell populations. For example, the SAOD may be designed to bind to a receptor found only in a certain cell type, as discussed above. The SAOD is also designed to recognize and selectively bind to the target mRNA sequence, which may correspond to a sequence contained within the sequence shown in FIG. 1 by virtue of complementary base pairing to the mRNA. Finally, the SAOD is designed to inactivate the target mRNA sequence by any of three mechanisms: 1) by binding to the target mRNA and thus inducing degradation of the mRNA by intrinsic cellular mechanisms such as RNAse I digestion, 2) by inhibiting translation of the mRNA target by interfering with the binding of translation-regulating factors or of ribosomes, or 3) by inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups, which either degrade or chemically modify the target mRNA. Synthetic antisense oligonucleotide drugs have been shown to be capable of the properties described above when directed against mRNA targets (J. S. Cohen, Trends in Pharm. Sci. 10, 435 (1989); H. M. Weintraub, Sci. Am. January (1990) p. 40). In addition, coupling of ribozymes to antisense oligonucleotides is a promising strategy for inactivating target mRNA (N. Sarver et al., Science 247, 1222 (1990)). An SAOD serves as an effective therapeutic agent if it is designed to be administered to a patient by injection, or if the patient's target cells are removed, treated with the SAOD in the laboratory, and replaced in the patient. In this manner, an SAOD serves as a therapy to reduce receptor expression in particular target cells of a patient, in any clinical condition which may benefit from reduced expression of 5-HT.sub.1E receptors.
This invention provides an antibody directed to the human 5-HT.sub.1E receptor, for example a monoclonal antibody directed to an epitope of a human 5-HT.sub.1E 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.sub.1E receptor included in the amino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). Amino acid sequences may be analyzed by methods well known in the art to determine whether they produce hydrophobic or hydrophilic regions in the proteins which they build. In the case of cell membrane proteins, hydrophobic regions are well known to form the part of the protein that is inserted into the lipid bilayer which forms the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Therefore antibodies to the hydrophilic amino acid sequences shown in FIG. 1 will bind to a surface epitope of a human 5-HT.sub.1E receptor, as described. Antibodies directed to human 5-HT.sub.1E receptors may be serum-derived or monoclonal and are prepared using methods well known in the art. For example, monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody. Cells such as NIH3T3 cells or Ltk.sup.- cells may be used as immunogens to raise such an antibody. Alternatively, synthetic peptides may be prepared using commercially available machines and the amino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). As a still further alternative, DNA, such as a cDNA or a fragment thereof, may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen. These antibodies are useful to detect the presence of human 5-HT.sub.1E receptors encoded by the isolated DNA, or to inhibit the function of the receptors in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.
This invention provides a pharmaceutical composition which comprises an amount of an antibody directed to the human 5-HT.sub.1E receptor effective to block binding of naturally occurring ligands to the 5-HT.sub.1E receptor, and a pharmaceutically acceptable carrier. A monoclonal antibody directed to an epitope of a human 5-HT.sub.1E 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.sub.1E receptor included in the amino acid sequence shown in FIG. 1 is useful for this purpose.
This invention also provides a method of treating abnormalities which are alleviated by reduction of expression of a human 5-HT.sub.1E receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to block binding of naturally occurring ligands to the 5-HT.sub.1E receptor and thereby alleviate abnormalities resulting from overexpression of a human 5-HT.sub.1E receptor. Binding of the antibody to the receptor prevents the receptor from functioning, thereby neutralizing the effects of overexpression. The monoclonal antibodies described above are both useful for this purpose. This invention additionally provides a method of treating an abnormal condition related to an excess of 5-HT.sub.1E receptor activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to block binding of naturally occurring ligands to the 5-HT.sub.1E receptor and thereby alleviate the abnormal condition. Some examples of abnormal conditions are dementia, Parkinson's disease, feeding disorders, pathological anxiety, schizophrenia, and a migraine headache.
This invention provides a method of detecting the presence of a 5-HT.sub.1E receptor on the surface of a cell which comprises contacting the cell with an antibody directed to the human 5-HT.sub.1E receptor, under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby the presence of the human 5-HT.sub.1E receptor on the surface of the cell. Such a method is useful for determining whether a given cell is defective in expression of 5-HT.sub.1E receptors on the surface of the cell. Bound antibodies are detected by methods well known in the art, for example by binding fluorescent markers to the antibodies and examining the cell sample under a fluorescence microscope to detect fluorescence on a cell indicative of antibody binding. The monoclonal antibodies described above are useful for this purpose.
This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT.sub.1E receptor. This invention also provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT.sub.1E receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a 5-HT.sub.1E receptor and which hybridizes to mRMA encoding a 5-HT.sub.1E receptor thereby reducing its translation. The DNA may additionally comprise an inducible promoter or additionally comprise tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of DNA are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequence shown in FIG. 1. An example of a transgenic animal is a transgenic mouse. Examples of tissue specificity-determining regions are the metallothionein promotor (Low, M. J., Lechan, R. M., Hammer, R. E. et al. Science 231:1002-1004 (1986)) and the L7 promotor (Oberdick, J., Smeyne, R. J., Mann, J. R., Jackson, S. and Morgan, J. I. Science 248:223-226 (1990)).
Animal model systems which elucidate the physiological and behavioral roles of human 5-HT.sub.1E receptors are produced by creating transgenic animals in which the expression of a 5-HT.sub.1E receptor is either increased or decreased, or the amino acid sequence of the expressed 5-HT.sub.1E receptor protein is altered, by a variety of techniques. Examples of these techniques include: 1) Insertion of normal or mutant versions of DNA encoding a human 5-HT.sub.1E receptor or homologous animal versions of these genes, by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). 2) Homologous recombination (Capecchi M. R. Science 244:1288-1292 (1989); Zimmer, A. and Gruss, P. Nature 338:150-153 (1989)) of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these 5-HT.sub.1E receptors. The technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native receptor but does express, for example, an inserted mutant receptor, which has replaced the native receptor in the animal's genome by recombination, resulting in underexpression of the receptor. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added receptors, resulting in overexpression of the receptor. One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding a human 5-HT.sub.1E receptor is purified from a vector (such as plasmid pCEXV-hp75d described above) by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.
Since the normal action of receptor-specific drugs is to activate or to inhibit the receptor, the transgenic animal model systems described above are useful for testing the biological activity of drugs directed against these 5-HT.sub.1E receptors even before such drugs become available. These animal model systems are useful for predicting or evaluating possible therapeutic applications of drugs which activate or inhibit these 5-HT.sub.1E receptors by inducing or inhibiting expression of the native or trans-gene and thus increasing or decreasing expression of normal or mutant 5-HT.sub.1E receptors in the living animal. Thus, a model system is produced in which the biological activity of drugs directed against these 5-HT.sub.1E receptors are evaluated before such drugs become available. The transgenic animals which over or under produce the 5-HT.sub.1E receptor indicate by their physiological state whether over or under production of the 5-HT.sub.1E receptor is therapeutically useful. It is therefore useful to evaluate drug action based on the transgenic model system. One use is based on the fact that it is well known in the art that a drug such as an antidepressant acts by blocking neurotransmitter uptake, and thereby increases the amount of neurotransmitter in the synaptic cleft. The physiological result of this action is to stimulate the production of less receptor by the affected cells, leading eventually to underexpression. Therefore, an animal which underexpresses receptor is useful as a test system to investigate whether the actions of such drugs which result in under expression are in fact therapeutic. Another use is that if overexpression is found to lead to abnormalities, then a drug which down-regulates or acts as an antagonist to 5-HT.sub.1E receptor is indicated as worth developing, and if a promising therapeutic application is uncovered by these animal model systems, activation or inhibition of the 5-HT.sub.1E receptor is achieved therapeutically either by producing agonist or antagonist drugs directed against these 5-HT.sub.1E receptors or by any method which increases or decreases the expression of these 5-HT.sub.1E receptors in man.
This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HT.sub.1E receptors which comprises producing a transgenic nonhuman animal whose levels of human 5-HT.sub.1E receptor expression are varied by use of an inducible promoter which regulates human 5-HT.sub.1E receptor expression. This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HT.sub.1E receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT.sub.1E receptor. Such animals may be produced by introducing different amounts of DNA encoding a human 5-HT.sub.1E receptor into the oocytes from which the transgenic animals are developed.
This invention also provides a method for identifying a substance capable of alleviating abnormalities resulting from overexpression of a human 5-HT.sub.1E receptor comprising administering the substance to a transgenic nonhuman mammal expressing at least one artificially introduced DNA molecule encoding a human 5-HT.sub.1E receptor 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.sub.1E receptor. As used herein, the term "substance" means a compound or composition which may be natural, synthetic, or a product derived from screening. Examples of DNA molecules are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequence shown in FIG. 1.
This invention provides a pharmaceutical composition comprising an amount of the substance described supra effective to alleviate the abnormalities resulting from overexpression of 5-HT.sub.1E receptor and a pharmaceutically acceptable carrier.
This invention further provides a method for treating the abnormalities resulting from overexpression of a human 5-HT.sub.1E receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from overexpression of a human 5-HT.sub.1E receptor.
This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human 5-HT.sub.1E receptor comprising administering the substance to the transgenic nonhuman mammal described above which expresses only nonfunctional human 5-HT.sub.1E receptor 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.sub.1E receptor.
This invention also provides a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of 5-HT.sub.1E receptor and a pharmaceutically acceptable carrier.
This invention further provides a method for treating the abnormalities resulting from underexpression of a human 5-HT.sub.1E receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from underexpression of a human 5-HT.sub.1E receptor.
This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT.sub.1E 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.sub.1E receptor and labelled with a detectable marker: e) detecting labelled bands which have hybridized to the DNA encoding a human 5-HT.sub.1E 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 thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific human 5-HT.sub.1E receptor allele.
This invention provides a method of preparing the isolated 5-HT.sub.1E receptor which comprises inducing cells to express 5-HT.sub.1E receptor, recovering the receptor from the resulting cells, and purifying the receptor so recovered. An example of an isolated 5-HT.sub.1E receptor is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1. For example, cells can be induced to express receptors by exposure to substances such as hormones. The cells can then be homogenized and the receptor isolated from the homogenate using an affinity column comprising, for example, serotonin or another substance which is known to bind to the receptor. The resulting fractions can then be purified by contacting them with an ion exchange column, and determining which fraction contains receptor activity or binds anti-receptor antibodies.
This invention provides a method of preparing the isolated 5-HT.sub.1E receptor which comprises inserting nucleic acid encoding 5-HT.sub.1E receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered. An example of an isolated 5-HT.sub.1E receptor is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1. This method for preparing 5-HT.sub.1E receptor uses recombinant DNA technology methods well known in the art. For example, isolated nucleic acid encoding 5-HT.sub.1E receptor is inserted in a suitable vector, such as an expression vector. A suitable host cell, such as a bacterial cell, or a eukaryotic cell such as a yeast cell, is transfected with the vector. 5-HT.sub.1E receptor is isolated from the culture medium by affinity purification or by chromatography or by other methods well known in the art.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a receptor so as to prevent translation of the mRNA molecule.
This invention also provides a transgenic nonhuman mammal expressing DNA encoding a receptor.
This invention further provides a transgenic nonhuman mammal expressing DNA encoding a receptor so mutated as to be incapable of normal receptor activity, and not expressing native receptor.
This invention provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a transgenic nonhuman animal whose levels of receptor expression are varied by use of an inducible promoter which regulates receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of the receptor.
This invention further provides transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding the receptor and which hybridizes to mRNA encoding the receptor thereby preventing its translation.
This invention provides a method for determining whether a ligand not known to be capable of binding to a receptor can bind to a receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the receptor with the ligand under conditions permitting binding of ligands known to bind to a receptor, detecting the presence of any of the ligand bound to the receptor, and thereby determining whether the ligand binds to the receptor.
Applicants have identified individual receptor subtype proteins and have described methods for the identification of pharmacological compounds for therapeutic treatments. Pharmacological compounds which are directed against specific receptor subtypes provide effective new therapies with minimal side effects.
This invention identifies for the first time a new receptor protein, its amino acid sequence, and its human gene. Furthermore, this invention describes a previously unrecognized group of receptors within the definition of a 5-HT.sub.1E receptor. The information and experimental tools provided by this discovery are useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for this new receptor protein, its associated mRNA molecule or its associated genomic DNA. The information and experimental tools provided by this discovery will be useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for this new receptor protein, its associated mRNA molecule, or its associated genomic DNA.
Specifically, this invention relates to the first isolation of a human cDNA and genomic clone encoding a 5-HT.sub.1E receptor. A new human gene for the receptor identified herein as 5-HT.sub.1E has been identified and characterized, and a series of related cDNA and genomic clones have been isolated. In addition, the human 5-HT.sub.1E receptor has been expressed in Ltk.sup.- cells and Y1 cells by transfecting the cells with the plasmid pcEXV-hp75d. The pharmacological binding properties of the protein encoded have been determined, and these binding properties classify this protein as a serotonin 5-HT.sub.1E receptor. Mammalian cell lines expressing this human 5-HT.sub.1E receptor at the cell surface have been constructed, thus establishing the first well-defined, cultured cell lines with which to study this 5-HT.sub.1E receptor.
The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Materials and Methods
Cloning and Sequencing:
A human placental genomic library (Stratagene) was screened using oligonucleotides derived from the human 5-HT.sub.1D.beta. receptor gene (U.S. Ser. No. 520,716), as a probe. Overlapping oligomers complementary to the 5-HT.sub.1D.beta. sequence in transmembrane domains III, V and VI were labeled with .sup.32 P-dATP and .sup.32 P-dCTP by synthesis with the large fragment of DNA Polymerase (Maniatis et al., 1982). Hybridization was performed at 40.degree. C. in a solution containing 25% formamide, 10% dextran sulfate, 5.times. SSC (1.times. SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 1.times. Denhardt's (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and 200 .mu.g/ml of sonicated salmon sperm DNA. The filters were washed at 40.degree. C. in 0.1.times. SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposed at -70.degree. C. to Kodak XAR film in the presence of an intensifying screen. Lambda phage hybridizing to the probe were plaque purified and DNA was prepared for Southern blot analysis (Southern, 1975; Maniatis et al., 1982). For subcloning and further Southern blot analysis DNA was inserted into pUC18 (Pharmacia, Piscataway, N.J.). Nucleotide sequence analysis was done by the Sanger dideoxy nucleotide chain-termination method (Sanger 1977) on denatured double-stranded plasmid templates using Sequenase (U.S. Biochemical Corp., Cleveland, Ohio).
Expression:
The entire coding region of clone hp75d was cloned into the eukaryotic expression vector pcEXV-3 (Miller, 1986). Stable cell lines were obtained by cotransfection with the plasmid pcEXV-3 (containing the 5-HT.sub.1E receptor gene) and the plasmid pGCcos3neo (containing the aminoglycoside transferase gene) into Ltk.sup.- cells and Y1 cells using calcium phosphate (reagents obtained from Specialty Media, Lavellette, N.J.). The cells were grown in a controlled environment (37.degree. C., 5% CO.sub.2) as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island, N.Y.) or Flo Medium Specialty Media, Inc., Lavallette, N.J.) containing 25 mM glucose and supplemented with 10% bovine calf serum, 100 U/ml penicillin G and 100 .mu.g/ml streptomycin sulfate. Stable clones were then selected for resistance to the antibiotic G-418 and harvested membranes were screened for their ability to bind [.sup.3 H] serotonin.
Detection of 5-HT.sub.1E Receptor mRNA in Brain Using PCR:
Total RNA from human brain tissue was extracted using the RNasol protocol as described by the manufacturer. cDNA was prepared from 5 .mu.g of total RNA with random hexanucleotide primers (500 pmoles) using Superscript reverse transcriptase (BRL, MD.) in 50 mM Tris-HCL pH8.3 buffer containing 40 u RNasin, 2.5 mM MgCl.sub.2, 50 mM KCL and 1 mM dNTPs, at 42.degree. C. for 1 hr. The reaction was stopped by heating at 95.degree. C. for 5 min. and chilled on ice. After terminating the reaction, RNase H (2 u) was added and incubated at 37.degree. C. for 20 min. An aliquot (1 .mu.g) of the first strand cDNA was diluted (1:5) into a 50 .mu.l PCR reaction mixture (200 .mu.M dNTPs final concentration) containing 1.25 U of Taq polymerase (Cetus Corp., CA) in the buffer supplied by the manufacturer, and 1 .mu.M of 5' primer=5'TACCACGCGGCCAAGAGCCTTTACCA 3' (Seq. I.D. No. 9) and 3' primer=5'TGGTGCTAGAGATCTGCTGACGTTC 3' (Seq. I.D. No. 10), oligonucleotides derived from the third cytoplasmic loop region. The PCR amplification reaction was carried out on a Perkin Elmer Cetus thermal cycler by first a 5 min. incubation at 95.degree. C. followed by 30 rounds of the following cycle: 2 min. at 94.degree. C., 2 min. at 42.degree. C., 3 min. at 72.degree. C., with a 3" extension, followed at the end by a 15 min. incubation at 72.degree. C. In order to control for the amplification of DNA (carried over during the RNA extraction), control PCR reactions were run in parallel with RNA diluted in the same manner as the cDNA samples. If necessary, RNA samples were pretreated with RNase-free DNase to eliminate any contaminating DNA. Positive controls were included in all experiments, consisting of plasmid containing 5HT1E receptor gene sequences. The products of the PCR amplification were separated by electrophoresis in 1.5% agarose. Amplified fragments were identified by blotting the gel to nitrocellulose and probing with an oligonucleotide internal to the 5'- and 3'-primers (5'GAGAAGTCAGACACACAGAAAGTCTGTGTAAGTTTTACAACTTGC 3') (Seq. I.D. No. 11) and end-labeled with [c-.sup.32 P]ATP using T.sub.4 polynucleotide kinase. Hybridization was performed at 40.degree. C. in a solution containing 50% formamide, 10% dextran sulfate, 5.times. SSC (1.times. SSC is 0.15M sodium chloride, 0.015M sodium citrate), 1.times. Denhardt's (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and 200 .mu.g/ml of sonicated salmon sperm DNA. The filters were washed at 50.degree. C. in 0.1.times. SSC containing 0.1% sodium dodecyl sulfate and exposed at -70.degree. C. to Kodak XAR film in the presence of an intensifying screen.
Membrane Preparation:
Membranes were prepared from transfected Ltk.sup.- cells which were grown to 100% confluency. The cells were washed twice with phosphate-buffered saline, scraped from the culture dishes into 5 ml of ice-cold phosphate-buffered saline, and centrifuged at 200.times.g for 5 min at 4.degree. C. The pellet was resuspended in 2.5 ml of ice-cold Tris buffer (20 mM Tris-HCl, pH 7.4 at 23.degree. C., 5 mM EDTA) and homogenized by a Wheaton tissue grinder. The lysate was subsequently centrifuged at 200.times.g for 5 min at 4.degree. C. to pellet large fragments which were discarded. The supernatant was collected and centrifuged at 40,000.times.g for 20 min at 4.degree. C. The pellet resulting from this centrifugation was washed once in ice-cold Tris buffer and finally resuspended in a final buffer containing 50 mM Tris-HCl and 0.5 mM EDTA, pH 7.4 at 23.degree. C. Membrane preparations were kept on ice and utilized within two hours for the radioligand binding assays. Protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin as the standard.
Radioligand Binding:
[.sup.3 H]5HT binding was performed using slight modifications of the 5-HT.sub.1E assay conditions reported by Leonhardt et al. (1989) with the omission of masking ligands. Radioligand binding studies were achieved at 37.degree. C. in a total volume of 250 .mu.l of buffer (50 mM Tris, 10 mM MgCl.sub.2, 0.2 mM EDTA, 10 .mu.M pargyline, 0.1% ascorbate, pH 7.4 at 37.degree. C.) in 96 well microtiter plates. Saturation studies were conducted using [.sup.3 H]5-HT at 12 different concentrations ranging from 0.5 nM to 100 nM. Displacement studies were performed using 4.5-5.5 nM [.sup.3 H]5-HT. The binding profile of drugs in competition experiments was accomplished using 10-12 concentrations of compound. Incubation times were 30 min for both saturation and displacement studies based upon initial investigations which determined equilibrium binding conditions. Nonspecific binding was defined in the presence of 10 .mu.M 5-HT. Binding was initiated by the addition of 50 .mu.l membrane homogenates (10-20 .mu.g). The reaction was terminated by rapid filtration through presoaked (0.5% polyethyleneimine) filters using 48R Cell Brandel Harvester (Gaithersburg, Md.). Subsequently, filters were washed for 5 seconds with ice cold buffer (50 mM Tris HCL, pH 7.4 at 4.degree. C.), dried and placed into vials containing 2.5. ml of Readi-Safe (Beckman, Fullerton, Calif.) and radioactivity was measured using a Beckman LS 5000TA liquid scintillation counter. The efficiency of counting of [.sup.3 H]5HT averaged between 45-50%. Binding data was analyzed by computer-assisted nonlinear regression analysis (Accufit and Accucomp, Lundon Software, Chagrin Falls, Ohio). IC.sub.50 values were converted to Ki values using the Cheng-Prusoff equation (1973). All experiments were performed in triplicate.
Measurement of cAMP Formation:
Transfected Y1 cells (expression level=688 fmol/mg of protein) were incubated in DMEM, 5 mM theophylline, 10 mM Hepes (4-[2-Hydroxyethyl]-1-piperazineethanesulfonic acid), 10 .mu.M pargyline, for 20 minutes at 37.degree. C., 5% CO.sub.2. A 5-HT dose-effect curve was then conducted by adding 12 different final concentrations of drug ranging from 10 .mu.M to 0.1 nM, followed immediately by the addition of forskolin (10 .mu.M). Subsequently, the cells were incubated for an additional 10 minutes at 37.degree. C., 5% CO.sub.2. The media was aspirated and the reaction terminated by the addition of 100 mM HCl. The plates were stored at 4.degree. C. for 15 minutes and centrifuged for 5 minutes (500.times.g at 4.degree. C.) to pellet cellular debris. Aliquots of the supernatant fraction were then stored at -20.degree. C. prior to assessment of cAMP formation by radioimmunoassay (cAMP Radioimmunoassay kit. Advanced Magnetics, Cambridge, Mass.).
Detection of 5-HT.sub.1E Receptor mRNA in Brain Using In Situ Hybridization Histochemistry
Labeling of 5-HT.sub.1E receptor oligo probe with digoxigenin-11-dUTP: The 5-HT.sub.1E receptor oligo probe (45 mer sequence: GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT) (Seq. I.D. No. 12) was synthesized in Molecular Biology Department of Neurogenetic Corp. The 3' end tailing reaction was as follows: To a sterile 1.5 ml Eppendorf tube add (1) 8 .mu.l of 5-HT.sub.1E receptor oligo probe (520 ng, denatured at 95.degree. C., then chilled on ice); (2) 8 .mu.l of 5.times. tailing buffer (BRL); (3) 5 .mu.l of digoxigenin-11-dUTP (Boehringer); (4) 4 .mu.l of dATP at 1/50 dilution; (5) 7 .mu.l of terminal transferase (TdT, BRL); (6) 7 .mu.l of distilled water. The above reaction mixture was incubated at 37.degree. C. for 5 min. The tailed oligo probe was purified by ethanol precipitation, vacuum dried and reconstituted in 40 .mu.l of distilled water.
Tissue Preparation:
Guinea pig brains were dissected and frozen, first in methylbutane, then on dry ice. Brain sections were cut at 11 .mu.m on a cryostat, thaw-mounted on gelatin-coated slides, and stored at -80.degree. C. On the day of the experiment, the brain sections were quickly brought to room temperature using a cool air stream, and fixed in 3% paraformaldehyde made up in 0.1 M PBS containing 0.02% diethylpyrocarbonate(DEPC). After rinsed in 0.1M PBS and 2.times. SSC, the tissue sections were dehydrated in graded ethanol and air dried.
Prehybridization:
All the tissue sections were pre-incubated with hybridization buffer in a humid chamber at 25.degree. C. for 1 hr. Twenty ml of the hybridization buffer contained (1). 10 ml of 100% formamide; (2) 4 ml of 20.times.SSC; (3) 0.4 ml of 50.times. Denhardts; (4) 1.0 ml of 10 mg/ml salmon sperm DNA; (5) 0.5 ml of 10 mg/ml yeast tRNA; (6) 4 ml of Dextran sulfate.
Hybridization:
The digoxigenin labeled 5-HT.sub.1E oligo probe (40 .mu.l) was diluted in 1 ml of hybridization buffer. Each tissue section was then covered with 0.1 ml of the hybridization buffer with probe and incubated at 40.degree. C. for 82 hr. After incubation all the sections were washed in 2.times. SSC (25.degree. C., 1 hr), in 1.times. SSC (25.degree. C., 1 hr), finally in 0.5.times. SSC, first 37.degree. C. 30 min, then 25.degree. C. 30 min.
Immunological Detection:
Following the post-hybridization washes slides were rinsed in Buffer #1 (100 mM Tris-HCl; 150 mM NaCl; pH 7.5) for 1 min, then incubated with 2% normal sheep serum plus 0.3% triton X-100 in Buffer #1 for 30 min. After this preparation the sections were incubated with anti-digoxigenin conjugated with alkaline phosphatase at dilution 1:500 in Buffer #1 containing 1% normal sheep serum and 0.3% triton X-100 at RT for 1.5 hr, then at 4.degree. C. overnight. The next day the tissue sections were rinsed in Buffer #1 for 10 min, then in Buffer #2 (100 mM Tris-HCl; 100 mM NaCl; 50 mM MgCl.sub.2 ; pH9.5) for 10 min. The chromagen solution was prepared immediately before the colorimetric reaction: i.e. to 10 ml of Buffer #2, (1) 45 .mu.l of 4-nitro blue tetrazolium chloride (NBT, 75 mg/ml in 70% dimethylformamide); (2) 35 .mu.l of 5-bromo-4-chloro-3-indolyl-phosphate (x-phosphate, 50 mg/ml in 100% dimethylformamide); and (3) 2.5 mg of levamisole were added. In order to carry out the colorimetric reaction sections were incubated with the chromogen solution in a humid, light-tight box overnight. The chromogen reaction was halted by rinsing the slides in Buffer #3 (10 mM Tris-HCl; 1 mM EDTA; pH 8.0). The sections were then rinsed in PBS, covered with Aquamount, and examined under a light microscope.
Drugs:
[.sup.3 H]5-HT (specific activity=28 Ci/mmole) was obtained from New England Nuclear, Boston, Mass. All other chemicals were obtained from commercial sources and were of the highest grade purity available.
Result
Isolation of a Genomic Clone Encoding a 5HT.sub.1E Receptor
A human genomic placental library was screened with oligonucleotide probes derived from transmembrane domains III, V and VI of the 5-HT.sub.1D.beta. receptor gene. The hybridization of these probes with the library was performed at low stringency and the result was the appearance of several hundred positive signals. Subsequently, approximately 350 of these clones were purified and categorized into various groups, based upon which of the three probes were responsible for the hybridization signal associated with a given clone. One group of clones exhibited hybridization signals with both transmembrane domain probes III and V. A number of these clones were subject to Southern blot analysis and determined to be identical or overlapping clones. A representative of this group, hp75d, was further characterized by nucleic acid sequence analysis and encoded what appeared to be a new serotonin receptor based upon its deduced amino acid sequence.
Nucleotide Sequence and Deduced Amino Acid Sequence of hp75d
DNA sequence information obtained from clone hp75d is shown in FIG. 1. An open reading frame extending from an ATG start codon at position 1 to a stop codon at position 1095 can encode a protein 365 amino acids in length, having a relative molecular mass (M.sub.f) of 41,633. A comparison of this protein sequence with previously characterized neurotransmitter receptors indicates that hp75d encodes a receptor which is a new member of a family of molecules which span the lipid bilayer seven times and couple to guanine nucleotide regulatory proteins (the G protein-coupled receptor family). A variety of structural features which are invariant in this family were present including the aspartic acid residues of transmembrane regions II and III, the DRY sequence at the end of transmembrane region III, and the conserved proline residues of transmembrane regions IV, V, VI and VII (Hartig et al. and references therein), were present in clone hp75d. A comparison of the transmembrane homology of hp75d to the other cloned serotonin receptors is shown in FIG. 2 and exhibits the following order of identity: 5-HT.sub.1D.alpha. (65%), 5-HT.sub.1D.beta. (64%), 5-HT.sub.1A (52%), 5-HT.sub.1C (40%) and 5-HT.sub.2 (39%).
Receptor Expression in Transfected Mammalian Cells
Saturation analysis of membranes prepared from stably transfected Ltk.sup.- cells demonstrated that the receptor expressed was saturable and of high affinity. Scatchard plot analysis by non-linear regression revealed a Kd of 10.3.+-.1.2 nM (mean.+-.S.E.M., n=7) and a B max consistent with a high level of expression, 10.9.+-.2.6 picomoles/mg of protein (mean.+-.S.E.M., n=7). The percent specific binding determined at the measured Kd value for [.sup.3 H]5-HT was greater than 85% of total binding. Furthermore, evidence that the receptor is coupled to a G-protein was demonstrated by the ability of Gpp(NH)p, a non-hydrolyzable analog of GTP, to inhibit the specific binding of [.sup.3 H]5-HT (IC.sub.50 =1885.+-.556, n.sub.N =0.87.+-.0.04, I.sub.max =26.4.+-.5.6%).
Pharmacological analysis of the receptor was accomplished by testing the ability of drugs from different chemical classes to displace [.sup.3 H]5-HT specific binding (Table 1). Of the compounds investigated, 5-HT and 5-hydroxylated tryptamine derivatives possessed the highest affinity which according to the classification system of Peroutka Table 1 Ki (nM) values of various drugs for the inhibition of [.sup.3 H]5-HT specific binding to clonal 5-HT.sub.1E cell membranes. Binding assays were performed with 4.5-5.5 nM of [.sup.3 H]5-HT and 10-12 different concentrations of each inhibitory drug. Ki values were calculated from the IC.sub.50 values using the Cheng-Prusoff equation. Each value is the mean.+-.S.E.M. of 3-4 independent determinations.
TABLE 1______________________________________Ki (nM) values of various drugs for the inhibition of [.sup.3 H]5-HT specific binding to clonal 5-HT.sub.1E cell membranes. Binding assayswere performed with 4.5-5.5 nM of [.sup.3 H]5-HT and 10-12 different concentrations of each inhibitory drug. Ki values were calculated fromthe IC.sub.50 values using the Cheng-Prusoff equation. Each value is the mean .+-. S.E.M. of 3-4 independent determinations. COMPOUND Ki (nM)______________________________________5-NT 10.9 .+-. 1.0 Lysergol 42.8 .+-. 5.3 Ergonovine 87.7 .+-. 7.6 Methylergonovine 89.4 .+-. 4.2 a-Methyl-5-NT 121 .+-. 13 Methiothepin 194 .+-. 4 1-Napthylpiperazine 207 .+-. 69 Methysergide 228 .+-. 16 Oxymetazoline 419 .+-. 49 5-Methoxy-N,N-DMT 528 .+-. 32 Ergotamine 599 .+-. 39 2-Methyl-5-NT 817 .+-. 203 1270 .+-. 233 Sumatriptan 2520 .+-. 135 Tryptamine 2559 .+-. 827 DOI 2970 .+-. 592 5-Methoxytryptamine 3151 .+-. 1041 8-OM-DPAT 3333 .+-. 310 3434 .+-. 102 Spiperone 5051 .+-. 689 TFMPP 6293 .+-. 259 5-CT 7875 .+-. 284 Ketanserin >10,000 >10,000 >10,000 LY-165163 (PAPP) >10,000 DP-5-CT >10,000______________________________________
and Snyder (1979) makes this site a member of the 5-HT.sub.1 class. Interestingly, 5-CT possessed low affinity and, thus, discriminates this receptor from that of the 5-HT.sub.1D receptor as well as other members of this class. Various ergoline compounds also bound with high affinity including agents which have potent hallucinogenic activity. Excluding methiothepin and 1-napthylpiperazine (Ki values=194 and 207 nM, respectively), piperazine derivatives had low affinity and displayed Ki values greater than 700 nM. Furthermore, rauwolfia alkaloids and serotonergic agents that possess high affinity for various subtypes of receptor within the serotonin family including ketanserin (5-HT.sub.2), 8-OH-DPAT (5-HT.sub.1A), DOI (5-HT.sub.1C /5-HT.sub.2), spiperone (5-HT.sub.1A /5-HT.sub.2), pindolol (5-HT.sub.1A /5-HT.sub.1B) and zacopride (5-HT.sub.3) had very poor affinity. In all cases, the Hill Coefficients did not differ significantly from unity. Taken together, the pharmacological profile of the 5-HT.sub.1E receptor is unique and contrasts to that of other known serotonin receptors. Accordingly, the probability of developing selective drugs for this receptor subtype is increased. Additional supporting evidence that the 5-HT.sub.1E receptor is functionally coupled to a G-protein was obtained by testing the ability of 5-HT to inhibit forskolin-stimulated cAMP production in Y1 cells transfected with the 5-HT.sub.1E receptor. FIG. 4 demonstrates that the endogenous indoleamine, 5-HT, produced a concentration-related decrease in forskolin-stimulated cAMP production with an EC.sub.50 of 23.1 nM. The maximum inhibition of cAMP production was 32%.
The tissue distribution of 5HT.sub.1E receptor mRNA was detected using PCR technology on cDNA from tissue-derived total RNA. The mRNA localization in human brain tissues demonstrates the presence of 5HT.sub.1E in: frontal cortex, cerebellar cortex, temporal cortex, choroid plexus, hippocampus, brain stem and cortex. We have not identified an area of the brain which does not contain 5HT.sub.1E. We conclude that the 5HT.sub.1E mRNA is abundant in human brain. These findings suggest that the 5HT.sub.1E receptor is not restricted to any one region of the brain and possibly cell type, but is expressed in numerous neuronal cell groups in many distinct regions of the human brain, as has been described for 5HT.sub.1C.
The presence of 5HT.sub.1E receptor mRNA in various regions of the human brain suggest that 5HT.sub.1E may modulate a number of the central actions attributed to serotonin. The abundance of 5HT.sub.1E receptors (mRNA) in the hippocampus may affect mood, behavior and hallucinogenesis. A greater understanding of possible physiological roles of this receptor subtype may be realized by the development of more specific 5HT.sub.1E receptor drugs as well as physiological manipulations of 5HT.sub.1E mRNAs/receptors.
The cellular localization of 5-HT.sub.1E receptor mRNA was detected with digoxigenin-11-dUTP labeled oligo probes employing in situ hybridization(ISHH) technology. Digoxigenin-11-dUTP labeled oxytocin(OT) oligo probes were used as a positive control for the experiment since the distribution of OT neurons in the central nervous system is well known. OT cells were intensely stained in the Guinea Pig's hypothalamus. Tissue sections pre-treated with RNase A were used as a negative control. Guinea pig brain sections were examined under a light microscope. 5-HT.sub.1E receptor mRNA was found in cells located in the frontal cortex, piriform cortex, hippocampus (CA1, CA2 and CA3), lateral septal nucleus, triangular septal nucleus, septofimbrial nucleus and the basal ganglia (caudate-putamen and globus pallidus). It was detected in the amygdaloid complex, the bed nucleus stria terminalis and the hypothalamic area including anterior hypothalamus, periventricular nucleus, paraventricular nucleus (magnocellular and parvocellular populations), supraoptic nucleus which seemed to include both vasopressin and oxytocin cell populations, and the lateral hypothalamus. The 1E receptor mRNA was also detected in the thalamic area including anteroventral thalamic, anterodorsal thalamic, mediodorsal thalamic, ventrolateral thalamic, reticular thalamic paracentral thalamic, paratenial thalamic nuclei and the nucleus stria medullaris. Control sections pre-treated with RNase A did not exhibit any staining pattern.
Experimental Discussion
The deduced amino acid sequence of hp75d was analyzed to uncover relationships between it and the other cloned serotonin receptor sequences. Although the homology within the membrane spanning domains was greatest with the 5-HT.sub.1D.alpha. receptor (FIG. 2), the nature of this newly cloned receptor could not be clearly predicted. The rational for this ambiguity is the interpretation of the transmembrane domain homology (approximately 65%) to the 5-HT.sub.1D.alpha. and 5-HT.sub.1D.beta. receptor subfamily. Closely related members of a "subfamily" of serotonin receptors (i.e. "subtypes") generally share a common transmitter and also have similar pharmacological profiles and physiological roles (for example, 5-HT.sub.2 and 5-HT.sub.1C or 5-HT.sub.1D.alpha. and 5-HT.sub.1D.beta.). Such "subtypes" display an amino acid identity of approximately 75-80% in their transmembrane domains. Serotonin receptors which are not members of the same "subfamily", but are members of the serotonin "family" (in which the receptors use the same neurotransmitter; i.e. 5-HT.sub.2 and 5-HT.sub.1D.alpha.) generally show much lower transmembrane homology (approximately 45%). Such transmembrane amino acid homologies can, therefore, give insight into the relationship between receptors and be used as predictors of receptor pharmacology. According to this type of analysis, although the newly cloned receptor appears to be more related to the 5-HT.sub.1D subfamily, it is likely to be in a subfamily distinct from all the other serotonin receptors.
The present pharmacological evidence substantiates the existence of a novel serotonin receptor in the human brain as first suggested by Leonhardt et al. (1989). Comparison of the pharmacological profile observed in native cortical membranes to that revealed for the cloned 5-HT.sub.1E receptor yielded a correlation coefficient of 0.987 (FIG. 3). Indeed, there were some differences in measured affinity constants but the relative values of selected drugs performed in both studies was similar. In this regard, a close examination of the Scatchard plot analysis performed in the study by Leonhardt et al. (1989) reveals that the reported Kd value may have been underestimated since the radioligand concentrations did not exceed 10 nM. In order to have accurately determined the dissociation constant, the radioligand concentration should have been extended to at least 50 nM or 10 times the estimated Kd (Yamamura et al., 1985). The initial study, however, was limited in tissue supply and lacked a comprehensive pharmacological characterization. The cloning of the 5-HT.sub.1E site will now allow more extensive investigations into the nature of this unique receptor.
The structure-activity relationships observed in the present study suggest that there are important requirements for high affinity binding to the 5-HT.sub.1E receptor. Substitution or removal of the 5-hydroxy group on serotonin decreases the affinity 300 fold for the receptor (egs., tryptamine, 5-methoxytryptamine and 5-carboxyamidotryptamine). Additionally, 2-methylation and .alpha.-methylation of 5-HT essentially abolishes its affinity for the 5-HT.sub.1E site. In contrast to these substitutions, N,N-dimethylation of the aliphatic side chain of the indole ring increases the affinity approximately 20 fold (unpublished observations). Basic structural requirements of the ergoline derivatives demonstrate that N-methylation of the indole ring decreases affinity as does bulky substitutions. Furthermore, piperazine derivatives are not bound at high affinity.
Notably, the application of the human 5-HT.sub.1E receptor clone to pharmaceutical research can lead to new drug design and development. The localization of this receptor in the cerebral cortex (Leonhardt et al., 1989) and parts of the basal ganglia such as the putamen and globus pallidus (Lowther et al., 1991) suggests a putative link to limbic, cognitive and/or motor function (Nieuwenhuys et al., 1988; Kandel and Schwartz, 1985) and, thus, may be involved in such abnormal conditions as dementia, Parkinson's disease, feeding disorders, anxiety and schizophrenia. Notably, the ergot compounds that possess affinity for this site have been demonstrated to affect these type of behaviors in humans as well as animals (Wilkinson and Dourish, 1991). In relation to this, it appears that the 5-HT.sub.1E binding site is also present in rat and bovine brain (Leonhardt et al., 1989) as well as guinea-pig, rabbit and dog where the data was initially interpreted as evidence for subtypes of the 5-HT.sub.1D receptor (Middlemiss, 1990). Nonetheless, it must be taken into consideration that this novel site can possibly lead to selective drug therapy devoid of side effects. In regard to this, serotonin uptake blockers are effective in treating neuropsychiatric disorders such as depression and obsessive-compulsive illness (Blier et al., 1987; Asberg et al., 1986; Insel et al., 1985). However, these agents have side effects and, in fact, the mechanism of action for these compounds are not linked to any particular serotonergic receptor. The possibility that agents selective for the 5-HT.sub.1E receptor may have clinical utility as antidepressants, for example, without the side effects attributed to current treatment modalities can have significant implications for drug therapy. Furthermore, it should be noted that ergoline derivatives have had clinical usefulness as drugs capable of relieving migraines and, thus, the involvement of the 5-HT.sub.1E receptor in this disorder deserves future attention. Ultimately, in depth investigations into the localization of the 5-HT.sub.1E receptor in brain and peripheral tissue will target new sites that can lead to specific functional roles for this serotonergic receptor.
Another consideration for therapeutic application of this site may be related to the treatment of feeding disorders such as obesity, bulimia nervosa and/or anorexia nervosa. The involvement of serotonin and feeding behavior has received much attention during the last decade. It is now known that many of the identified and well-characterized serotonergic receptors are capable of modulating feeding (Blundell and Lawton, 1990). Notably, serotonin uptake blockers which have been used to treat feeding disorders act nonselectively and as such have side-effect potential (Jimerson et al., 1990). Although many different serotonergic receptors are involved in feeding, the search for the one site that can be exploited for selective drug development has yet to be found. There is no doubt that interest exists in finding drugs that interact with the serotonin system for the treatment of feeding disorders (Cooper, 1989).
Thus, the pharmacological profile of the cloned human 5-HT.sub.1E receptor is unique and contrasts to other known serotonergic receptors. The utility of this site expressed in a cellular system and, thus, isolated for study will create excellent opportunities in drug development directed towards a novel serotonergic receptor that may have wide-range implications for drug therapy. Indeed, the potential therapeutic applications may extend to neuropsychiatric disorders including depression, anxiety, schizophrenia, dementia and obsessive-compulsive illness as well as obesity and migraine. The localization of 5-HT.sub.1E receptor mRNA by in situ hybridization makes it possible to predict its physiological and pathological functions. 5-HT.sub.1E receptor mRNA is detected in the limbic structures, such as the hippocampus, septal nuclei, piriform cortex (olfactory system), amygdaloid complex and the bed nucleus stria terminalis. The olfactory system sends afferent fibers to the hippocampus through the subiculum, and to the amygdaloid complex (Kupfermann, 1985). In turn the outputs of the hippocampus project to the septal area and hypothalamus while the amygdaloid complex projects to the hypothalamus via the stria terminalis (Kupfermann, 1985). The involvement of the limbic system in emotional behavior (e.g., fear, pleasure, sexual activities) and memory is well known (Kupfermann, 1985). Therefore, the finding of 5-HT.sub.1E receptor mRNA in these structures indicates a potential role in neuropsychiatric disorders such as depression, obsessive-compulsive illness, anxiety, schizophrenia and dementia.
The hypothalamus regulates the body adjustments to the external and internal environments. It can control hunger (the ventromedial nucleus and the lateral hypothalamus), endocrine functions (e.g. the supraoptic nucleus, the paraventricular nucleus and the periventricular nucleus), affective (emotional) behavior (the ventromedial and dorsomedial nuclei) and the activity of the visceral nervous system (the anterior hypothalamus) (Diamond et al., 1985). The discovery of 5-HT.sub.1E receptor mRNA in these nuclei indicates physiological and pathological roles of this receptor subtype in cardiovascular, gastrointestinal, endocrine, neurological and psychiatric systems.
The thalamus is a relay station where the sensory and motor-related pathways passing up the brain stem synapse before proceeding on to the cerebral cortex for more elaborate integration and analysis. The 5-HT.sub.1E receptor mRNA was found in the anterior thalamic nucleus which receives the input from the mammillothalamic tract and sends fibers to the cingulate gyrus. Thus, this nucleus is an important part of the circuit connecting the hypothalamus, the thalamus, and the limbic lobe (Diamond et al., 1985). The 5-HT.sub.1E receptor mRNA was also found in the mediodorsal thalamic nucleus which is involved in emotional behavior and in the ventral lateral thalamic nucleus which connects the basal ganglia and premotor area. Furthermore, 5-HT.sub.1E receptor mRNA was found in the nucleus reticularis thalami through which pass most of the thalamocortical and corticothalamic fibers. The axons of corticothalamic and thalamocortical neurons provide collaterals for synapse with reticular cells, exerting a facilitatory effect, while the axons of the GABA-rich reticularis cells project back on the specific thalamic neurons, continuously modulating (by inhibition) the ascending flow of thalamocortical impulses (Diamond et al., 1985). The reticularis neurons play a significant role in the interactions between the thalamus and the frontal cortex. In addition, the 5-HT.sub.1E receptor mRNA was also visualized in the basal ganglia including the caudate-putamen and globus pallidus both of which control motor activity (Alheid et al., 1990). These findings indicate important roles of the 5-HT.sub.1E receptors in emotional behavior, sensation (e.g., pain) and motor activity.
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Middlemiss, D. N., Suman-Chauhan, N., Smith, S. M., Picton, C., Shaw, D. and Bevan, Y.: Subpopulations of 5-HT1D recognition sites in guinea-pig, rabbit, dog and human cortex. The Second IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, Jul. 11-13, 1990, Abstract #P-30.
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Peroutka, S. J.: Serotonin receptor subtypes: Basic and clinical aspects. New York: Wiley-Liss, Inc., 1991.
Peroutka, S. J. and Snyder, S. H.: Multiple serotonin receptors, differential binding of [.sup.3 H]5-hydroxytryptamine, [.sup.3 H]lysergic acid diethylamide and [.sup.3 H]spiroperidol. Mol. Pharmacol. 16:687-699, 1979.
Pritchett, D. B., Bach, A. W. J., Wozny, M., Taleb, O., Dal Toso, R., Shih, J. and Seeburg, P. H.: Structure and functional expression of cloned rat serotonin 5-HT2 receptor. EMBO J. 7:4135-4140, 1988.
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Yamamura, H. I., Enna, S. J. and Kuhar, M. J.: Neurotransmitter receptor binding. New York: Raven Press, 1985.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 12 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2463 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: human plac - #ental genomic (B) CLONE: hp75d - - (viii) POSITION IN GENOME: (C) UNITS: bp - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 736..1830 - - (ix) FEATURE: (A) NAME/KEY: mat.sub.-- - #peptide (B) LOCATION: 736..1830 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATATACATCA TGGAATACTA TGCAGCCCCC CCCAAGGATG CGTTCCATGT CC -#TTTGCAGG 60 - - GACATGGATG AGTTGCAAAC CATATTCTCA ACAAACTAAC ACAGCCACAG AA -#AACCAAAC 120 - - ACCACATGCT CTCACTCACG AGTGGAGTTG AACAATGAGA ACACATGGCA CA -#GGGCCGGG 180 - - AACATGACAC ACCAGGGCCT GTTGGGGGGT GGAGGGCTAG GGGAGGGATG GC -#ATTAGGAG 240 - - AAGTACCTAA TGTAGATGAT TGGTTGTTGG GTGCAGCAAA CCACCATGGC AC -#ATGTATAC 300 - - CTATGTAGCA AACCTGCAAG TTCTGCACAT GTATCCCAGG ACTTAAAGTA TA -#ATTTAAAA 360 - - AAAAACAGTT TGAAAACTTC CCTGAAGTAA AAAAAGTATC CTTTGAGGAA CA -#ATGTAACG 420 - - ATGAGCTCAA GTTCCACAGG AAAGAGAAAA TTAAAATTTA TAAAGAATTT AT -#AAATATCA 480 - - AACTATTTTC ATGTTTTCCA GGAAAAGTGT GGCTTTCTCA TTCATTAACC AA -#TAGCATAA 540 - - TATTTTCCAG GAACCTTCAC TCAGAAGAAA TGCTGTGGCC CTTCCCTTTA CC -#AACAGAAA 600 - - ATGGAACACA AGAGACCACA TAGCTGAACA AATTATAGCC TCCTTACAAG TG -#AGAAACCT 660 - - TCGAGGCTAC ATAGTTTTCA GCCAAAGGAA AATAACCAAC AGCTTCTCCA CA -#GTGTAGAC 720 - - TGAAACAAGG GAAAC ATG AAC ATC ACA AAC TGT ACC - #ACA GAG GCC AGCATG 771 Met - #Asn Ile Thr Asn Cys Thr Thr Glu Ala Ser M - #et - # 1 5 - # 10 - - GCT ATA AGA CCC AAG ACC ATC ACT GAG AAG AT - #G CTC ATT TGC ATG ACT 819 Ala Ile Arg Pro Lys Thr Ile Thr Glu Lys Me - #t Leu Ile Cys Met Thr 15 - # 20 - # 25 - - CTG GTG GTC ATC ACC ACC CTC ACC ACG TTG CT - #G AAC TTG GCT GTG ATC 867 Leu Val Val Ile Thr Thr Leu Thr Thr Leu Le - #u Asn Leu Ala Val Ile 30 - # 35 - # 40 - - ATG GCT ATT GGC ACC ACC AAG AAG CTC CAC CA - #G CCT GCC AAC TAC CTA 915 Met Ala Ile Gly Thr Thr Lys Lys Leu His Gl - #n Pro Ala Asn Tyr Leu 45 - # 50 - # 55 - # 60 - - ATC TGT TCT CTG GCC GTG ACG GAC CTC CTG GT - #G GCA GTG CTC GTC ATG 963 Ile Cys Ser Leu Ala Val Thr Asp Leu Leu Va - #l Ala Val Leu Val Met 65 - # 70 - # 75 - - CCC CTG AGC ATC ATC TAC ATT GTC ATG GAT CG - #C TGG AAG CTT GGG TAC 1011 Pro Leu Ser Ile Ile Tyr Ile Val Met Asp Ar - #g Trp Lys Leu Gly Tyr 80 - # 85 - # 90 - - TTC CTC TGT GAG GTG TGG CTG AGT GTG GAC AT - #G ACC TGC TGC ACC TGC 1059 Phe Leu Cys Glu Val Trp Leu Ser Val Asp Me - #t Thr Cys Cys Thr Cys 95 - # 100 - # 105 - - TCC ATC CTC CAC CTC TGT GTC ATT GCC CTG GA - #C AGG TAC TGG GCC ATC 1107 Ser Ile Leu His Leu Cys Val Ile Ala Leu As - #p Arg Tyr Trp Ala Ile 110 - # 115 - # 120 - - ACC AAT GCT ATT GAA TAC GCC AGG AAG AGG AC - #G GCC AAG AGG GCC GCG 1155 Thr Asn Ala Ile Glu Tyr Ala Arg Lys Arg Th - #r Ala Lys Arg Ala Ala 125 1 - #30 1 - #35 1 -#40 - - CTG ATG ATC CTT ACC GTC TGG ACC ATC TCC AT - #T TTC ATC TCC ATGCCC 1203 Leu Met Ile Leu Thr Val Trp Thr Ile Ser Il - #e Phe Ile Ser Met Pro 145 - # 150 - # 155 - - CCT CTG TTC TGG AGA AGC CAC CGC CGC CTA AG - #C CCT CCC CCT AGT CAG 1251 Pro Leu Phe Trp Arg Ser His Arg Arg Leu Se - #r Pro Pro Pro Ser Gln 160 - # 165 - # 170 - - TGC ACC ATC CAG CAC GAC CAT GTT ATC TAC AC - #C ATT TAC TCC ACG CTG 1299 Cys Thr Ile Gln His Asp His Val Ile Tyr Th - #r Ile Tyr Ser Thr Leu 175 - # 180 - # 185 - - GGT GCG TTT TAT ATC CCC TTG ACT TTG ATA CT - #G ATT CTC TAT TAC CGG 1347 Gly Ala Phe Tyr Ile Pro Leu Thr Leu Ile Le - #u Ile Leu Tyr Tyr Arg 190 - # 195 - # 200 - - ATT TAC CAC GCG GCC AAG AGC CTT TAC CAG AA - #A AGG GGA TCA AGT CGG 1395 Ile Tyr His Ala Ala Lys Ser Leu Tyr Gln Ly - #s Arg Gly Ser Ser Arg 205 2 - #10 2 - #15 2 -#20 - - CAC TTA AGC AAC AGA AGC ACA GAT AGC CAG AA - #T TCT TTT GCA AGTTGT 1443 His Leu Ser Asn Arg Ser Thr Asp Ser Gln As - #n Ser Phe Ala Ser Cys 225 - # 230 - # 235 - - AAA CTT ACA CAG ACT TTC TGT GTG TCT GAC TT - #C TCC ACC TCA GAC CCT 1491 Lys Leu Thr Gln Thr Phe Cys Val Ser Asp Ph - #e Ser Thr Ser Asp Pro 240 - # 245 - # 250 - - ACC ACA GAG TTT GAA AAG TTC CAT GCC TCC AT - #C AGG ATC CCC CCC TTC 1539 Thr Thr Glu Phe Glu Lys Phe His Ala Ser Il - #e Arg Ile Pro Pro Phe 255 - # 260 - # 265 - - GAC AAT GAT CTA GAT CAC CCA GGA GAA CGT CA - #G CAG ATC TCT AGC ACC 1587 Asp Asn Asp Leu Asp His Pro Gly Glu Arg Gl - #n Gln Ile Ser Ser Thr 270 - # 275 - # 280 - - AGG GAA CGG AAG GCA GCA CGC ATC CTG GGG CT - #G ATT CTG GGT GCA TTC 1635 Arg Glu Arg Lys Ala Ala Arg Ile Leu Gly Le - #u Ile Leu Gly Ala Phe 285 2 - #90 2 - #95 3 -#00 - - ATT TTA TCC TGG CTG CCA TTT TTC ATC AAA GA - #G TTG ATT GTG GGTCTG 1683 Ile Leu Ser Trp Leu Pro Phe Phe Ile Lys Gl - #u Leu Ile Val Gly Leu 305 - # 310 - # 315 - - AGC ATC TAC ACC GTG TCC TCG GAA GTG GCC GA - #C TTT CTG ACG TGG CTC 1731 Ser Ile Tyr Thr Val Ser Ser Glu Val Ala As - #p Phe Leu Thr Trp Leu 320 - # 325 - # 330 - - GGT TAT GTG AAT TCT CTG ATC AAC CCT CTG CT - #C TAT ACG AGT TTT AAT 1779 Gly Tyr Val Asn Ser Leu Ile Asn Pro Leu Le - #u Tyr Thr Ser Phe Asn 335 - # 340 - # 345 - - GAA GAC TTT AAG CTG GCT TTT AAA AAG CTC AT - #T AGA TGC CGA GAG CAT 1827 Glu Asp Phe Lys Leu Ala Phe Lys Lys Leu Il - #e Arg Cys Arg Glu His 350 - # 355 - # 360 - - ACT TAGACTGTAA AAAGCTAAAA GGCACGACTT TTTCCAGAGC CTCATGAGT - #G 1880 Thr 365 - - GATGGGGGTA AGGGGTGCAA CTTATTAATT CTTGAACATA CTTGGTTCAG GA -#GAGTTTGT 1940 - - AAGTATGTGT GGTCTTGTTT CCTTGTTTGT TTGTTTGTTT TGTTCTGTTT TG -#TTTGAGGA 2000 - - TTGTTATTTG GCCTCCTGTT TTCTACCTCT GGTCTTATCT GTGATACATA AT -#TTCAAATA 2060 - - AACATTATCA TACAAAAACA GAAATTTTGC CAGAAGTAAT AATAAGATGA AA -#TACTAAAT 2120 - - ACCTTTTATG GGTTTTTTTT TTTTAGCCAT TTCAGTTACC CTGCAATTAA AG -#AATGCCAA 2180 - - AAATATCTTT ATTTGCAGAA TTTCTTATTA CTTATAAATT AAATACCTGA TA -#ATGCCCTC 2240 - - CATGGCATTA AATCTGAGAT TATGGCTCTA TCTGCGTACA TATTCCAGTG GG -#AATTGCAT 2300 - - GACTACATAA AGAATTAAAA GAAAGTGATG TGCTGTCATC TACGGCTTGC GA -#CCTGAGCT 2360 - - AAAGTCGGGG GCTGTAGCAC TGTGACTACG TAGCCTATCA TTTCAGGTAA AA -#ATAGTACA 2420 - - GCTGGCTTGT CTTGTTAGTT CATGATTAAA TAAACTTCTC TTT - # 246 - #3 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 365 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Asn Ile Thr Asn Cys Thr Thr Glu Ala Se - #r Met Ala Ile Arg Pro 1 5 - # 10 - # 15 - - Lys Thr Ile Thr Glu Lys Met Leu Ile Cys Me - #t Thr Leu Val Val Ile 20 - # 25 - # 30 - - Thr Thr Leu Thr Thr Leu Leu Asn Leu Ala Va - #l Ile Met Ala Ile Gly 35 - # 40 - # 45 - - Thr Thr Lys Lys Leu His Gln Pro Ala Asn Ty - #r Leu Ile Cys Ser Leu 50 - # 55 - # 60 - - Ala Val Thr Asp Leu Leu Val Ala Val Leu Va - #l Met Pro Leu Ser Ile 65 - # 70 - # 75 - # 80 - - Ile Tyr Ile Val Met Asp Arg Trp Lys Leu Gl - #y Tyr Phe Leu Cys Glu 85 - # 90 - # 95 - - Val Trp Leu Ser Val Asp Met Thr Cys Cys Th - #r Cys Ser Ile Leu His 100 - # 105 - # 110 - - Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Al - #a Ile Thr Asn Ala Ile 115 - # 120 - # 125 - - Glu Tyr Ala Arg Lys Arg Thr Ala Lys Arg Al - #a Ala Leu Met Ile Leu 130 - # 135 - # 140 - - Thr Val Trp Thr Ile Ser Ile Phe Ile Ser Me - #t Pro Pro Leu Phe Trp 145 1 - #50 1 - #55 1 -#60 - - Arg Ser His Arg Arg Leu Ser Pro Pro Pro Se - #r Gln Cys Thr IleGln 165 - # 170 - # 175 - - His Asp His Val Ile Tyr Thr Ile Tyr Ser Th - #r Leu Gly Ala Phe Tyr 180 - # 185 - # 190 - - Ile Pro Leu Thr Leu Ile Leu Ile Leu Tyr Ty - #r Arg Ile Tyr His Ala 195 - # 200 - # 205 - - Ala Lys Ser Leu Tyr Gln Lys Arg Gly Ser Se - #r Arg His Leu Ser Asn 210 - # 215 - # 220 - - Arg Ser Thr Asp Ser Gln Asn Ser Phe Ala Se - #r Cys Lys Leu Thr Gln 225 2 - #30 2 - #35 2 -#40 - - Thr Phe Cys Val Ser Asp Phe Ser Thr Ser As - #p Pro Thr Thr GluPhe 245 - # 250 - # 255 - - Glu Lys Phe His Ala Ser Ile Arg Ile Pro Pr - #o Phe Asp Asn Asp Leu 260 - # 265 - # 270 - - Asp His Pro Gly Glu Arg Gln Gln Ile Ser Se - #r Thr Arg Glu Arg Lys 275 - # 280 - # 285 - - Ala Ala Arg Ile Leu Gly Leu Ile Leu Gly Al - #a Phe Ile Leu Ser Trp 290 - # 295 - # 300 - - Leu Pro Phe Phe Ile Lys Glu Leu Ile Val Gl - #y Leu Ser Ile Tyr Thr 305 3 - #10 3 - #15 3 -#20 - - Val Ser Ser Glu Val Ala Asp Phe Leu Thr Tr - #p Leu Gly Tyr ValAsn 325 - # 330 - # 335 - - Ser Leu Ile Asn Pro Leu Leu Tyr Thr Ser Ph - #e Asn Glu Asp Phe Lys 340 - # 345 - # 350 - - Leu Ala Phe Lys Lys Leu Ile Arg Cys Arg Gl - #u His Thr 355 - # 360 - # 365 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 422 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - Met Asp Val Leu Ser Pro Gly Gln Gly Asn As - #n Thr Thr Ser Pro Pro 1 5 - # 10 - # 15 - - Ala Pro Phe Glu Thr Gly Gly Asn Thr Thr Gl - #y Ile Ser Asp Val Thr 20 - # 25 - # 30 - - Val Ser Tyr Gln Val Ile Thr Ser Leu Leu Le - #u Gly Thr Leu Ile Phe 35 - # 40 - # 45 - - Cys Ala Val Leu Gly Asn Ala Cys Val Val Al - #a Ala Ile Ala Leu Glu 50 - # 55 - # 60 - - Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Il - #e Gly Ser Leu Ala Val 65 - #70 - #75 - #80 - - Thr Asp Leu Met Val Ser Val Leu Val Leu Pr - #o Met Ala Ala Leu Tyr 85 - # 90 - # 95 - - Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Va - #l Thr Cys Asp Leu Phe 100 - # 105 - # 110 - - Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Se - #r Ile Leu His Leu Cys 115 - # 120 - # 125 - - Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Th - #r Asp Pro Ile Asp Tyr 130 - # 135 - # 140 - - Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Al - #a Leu Ile Ser Leu Thr 145 1 - #50 1 - #55 1 -#60 - - Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro Pr - #o Met Leu Gly TrpArg 165 - # 170 - # 175 - - Thr Pro Glu Asp Arg Ser Asp Pro Asp Ala Cy - #s Thr Ile Ser Lys Asp 180 - # 185 - # 190 - - His Gly Tyr Thr Ile Tyr Ser Thr Phe Gly Al - #a Phe Tyr Ile Pro Leu 195 - # 200 - # 205 - - Leu Leu Met Leu Val Leu Tyr Gly Arg Ile Ph - #e Arg Ala Ala Arg Phe 210 - # 215 - # 220 - - Arg Ile Arg Lys Thr Val Lys Lys Val Glu Ly - #s Thr Gly Ala Asp Thr 225 2 - #30 2 - #35 2 -#40 - - Arg His Gly Ala Ser Pro Ala Pro Gln Pro Ly - #s Lys Ser Val AsnGly 245 - # 250 - # 255 - - Glu Ser Gly Ser Arg Asn Trp Arg Leu Gly Va - #l Glu Ser Lys Ala Gly 260 - # 265 - # 270 - - Gly Ala Leu Cys Ala Asn Gly Ala Val Arg Gl - #n Gly Asp Asp Gly Ala 275 - # 280 - # 285 - - Ala Leu Glu Val Ile Glu Val His Arg Val Gl - #y Asn Ser Lys Glu His 290 - # 295 - # 300 - - Leu Pro Leu Pro Ser Glu Ala Gly Pro Thr Pr - #o Cys Ala Pro Ala Ser 305 3 - #10 3 - #15 3 -#20 - - Phe Glu Arg Lys Asn Glu Arg Asn Ala Glu Al - #a Lys Arg Lys MetAla 325 - # 330 - # 335 - - Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Le - #u Gly Ile Ile Met Gly 340 - # 345 - # 350 - - Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Il - #e Val Ala Leu Val Leu 355 - # 360 - # 365 - - Pro Phe Cys Glu Ser Ser Cys His Met Pro Th - #r Leu Leu Gly Ala Ile 370 - # 375 - # 380 - - Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Le - #u Asn Pro Val Ile Tyr 385 3 - #90 3 - #95 4 -#00 - - Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala Ph - #e Lys Lys Ile IleLys 405 - # 410 - # 415 - - Cys Leu Phe Cys Arg Gln 420 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 460 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - Met Val Asn Leu Gly Asn Ala Val Arg Ser Le - #u Leu Met His Leu Ile 1 5 - # 10 - # 15 - - Gly Leu Leu Val Trp Gln Phe Asp Ile Ser Il - #e Ser Pro Val Ala Ala 20 - # 25 - # 30 - - Ile Val Thr Asp Thr Phe Asn Ser Ser Asp Gl - #y Gly Arg Leu Phe Gln 35 - # 40 - # 45 - - Phe Pro Asp Gly Val Gln Asn Trp Pro Ala Le - #u Ser Ile Val Val Ile 50 - # 55 - # 60 - - Ile Ile Met Thr Ile Gly Gly Asn Ile Leu Va - #l Ile Met Ala Val Ser 65 - #70 - #75 - #80 - - Met Glu Lys Lys Leu His Asn Ala Thr Asn Ty - #r Phe Leu Met Ser Leu 85 - # 90 - # 95 - - Ala Ile Ala Asp Met Leu Val Gly Leu Leu Va - #l Met Pro Leu Ser Leu 100 - # 105 - # 110 - - Leu Ala Ile Leu Tyr Asp Tyr Val Trp Pro Le - #u Pro Arg Tyr Leu Cys 115 - # 120 - # 125 - - Pro Val Trp Ile Ser Leu Asp Val Leu Phe Se - #r Thr Ala Ser Ile Met 130 - # 135 - # 140 - - His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Va - #l Ala Ile Arg Asn Pro 145 1 - #50 1 - #55 1 -#60 - - Ile Glu His Ser Arg Phe Asn Ser Arg Thr Ly - #s Ala Ile Met LysIle 165 - # 170 - # 175 - - Ala Ile Val Trp Ala Ile Ser Ile Gly Val Se - #r Val Pro Ile Pro Val 180 - # 185 - # 190 - - Ile Gly Leu Arg Asp Glu Ser Lys Val Phe Va - #l Asn Asn Thr Thr Cys 195 - # 200 - # 205 - - Val Leu Asn Asp Pro Asn Phe Val Leu Ile Gl - #y Ser Phe Val Ala Phe 210 - # 215 - # 220 - - Phe Ile Pro Leu Thr Ile Met Val Ile Thr Ty - #r Phe Leu Thr Ile Tyr 225 2 - #30 2 - #35 2 -#40 - - Val Leu Arg Arg Gln Thr Leu Met Leu Leu Ar - #g Gly His Thr GluGlu 245 - # 250 - # 255 - - Glu Leu Ala Asn Met Ser Leu Asn Phe Leu As - #n Cys Cys Cys Lys Lys 260 - # 265 - # 270 - - Asn Gly Gly Glu Glu Glu Asn Ala Pro Asn Pr - #o Asn Pro Asp Gln Lys 275 - # 280 - # 285 - - Pro Arg Arg Lys Lys Lys Glu Lys Arg Pro Ar - #g Gly Thr Met Gln Ala 290 - # 295 - # 300 - - Ile Asn Asn Glu Lys Lys Ala Ser Lys Val Le - #u Gly Ile Val Phe Phe 305 3 - #10 3 - #15 3 -#20 - - Val Phe Leu Ile Met Trp Cys Pro Phe Phe Il - #e Thr Asn Ile LeuSer 325 - # 330 - # 335 - - Val Leu Cys Gly Lys Ala Cys Asn Gln Lys Le - #u Met Glu Lys Leu Leu 340 - # 345 - # 350 - - Asn Val Phe Val Trp Ile Gly Tyr Val Cys Se - #r Gly Ile Asn Pro Leu 355 - # 360 - # 365 - - Val Tyr Thr Leu Phe Asn Lys Ile Tyr Arg Ar - #g Ala Phe Ser Lys Tyr 370 - # 375 - # 380 - - Leu Arg Cys Asp Tyr Lys Pro Asp Lys Lys Pr - #o Pro Val Arg Gln Ile 385 3 - #90 3 - #95 4 -#00 - - Pro Arg Val Ala Ala Thr Ala Leu Ser Gly Ar - #g Glu Leu Asn ValAsn 405 - # 410 - # 415 - - Ile Tyr Arg His Thr Asn Glu Arg Val Ala Ar - #g Lys Ala Asn Asp Pro 420 - # 425 - # 430 - - Glu Pro Gly Ile Glu Met Gln Val Glu Asn Le - #u Glu Leu Pro Val Asn 435 - # 440 - # 445 - - Pro Ser Asn Val Val Ser Glu Arg Ile Ser Se - #r Val 450 - # 455 - # 460 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 375 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Ser Pro Leu Asn Gln Ser Ala Glu Gly Le - #u Pro Gln Glu Ala Ser 1 5 - # 10 - # 15 - - Asn Arg Ser Leu Asn Ala Thr Glu Thr Ser Gl - #u Ala Trp Asp Pro Arg 20 - # 25 - # 30 - - Thr Leu Gln Ala Leu Lys Ile Ser Leu Pro Va - #l Leu Leu Ser Val Ile 35 - # 40 - # 45 - - Thr Leu Ala Thr Val Leu Ser Asn Ala Phe Va - #l Leu Thr Thr Ile Leu 50 - # 55 - # 60 - - Leu Thr Arg Lys Leu His Thr Pro Ala Asn Ty - #r Leu Ile Gly Ser Leu 65 - #70 - #75 - #80 - - Ala Thr Thr Asp Leu Leu Val Ser Ile Leu Va - #l Met Pro Ile Ser Met 85 - # 90 - # 95 - - Ala Tyr Thr Ile Thr His Thr Trp Asn Phe Gl - #y Gln Ile Leu Cys Asp 100 - # 105 - # 110 - - Ile Trp Leu Ser Ser Asp Ile Thr Cys Cys Th - #r Ala Ser Ile Leu His 115 - # 120 - # 125 - - Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Al - #a Ile Thr Asp Ala Leu 130 - # 135 - # 140 - - Glu Tyr Ser Lys Arg Arg Thr Ala Gly His Al - #a Ala Thr Met Ile Ala 145 1 - #50 1 - #55 1 -#60 - - Ile Val Trp Ala Ile Ser Ile Cys Ile Ser Il - #e Pro Pro Leu PheTrp 165 - # 170 - # 175 - - Arg Gln Glu Lys Ala Gln Glu Glu Met Ser As - #p Cys Leu Val Asn Thr 180 - # 185 - # 190 - - Ser Gln Ile Ser Tyr Thr Ile Tyr Ser Thr Cy - #s Gly Ala Phe Tyr Ile 195 - # 200 - # 205 - - Pro Ser Val Leu Leu Ile Ile Leu Tyr Gly Ar - #g Ile Tyr Arg Ala Ala 210 - # 215 - # 220 - - Arg Asn Arg Ile Leu Asn Pro Pro Ser Leu Se - #r Gly Lys Arg Phe Thr 225 2 - #30 2 - #35 2 -#40 - - Thr Ala His Leu Ile Thr Gly Ser Ala Gly Se - #r Val Cys Ser LeuAsn 245 - # 250 - # 255 - - Ser Ser Leu His Glu Gly His Ser His Ser Al - #a Gly Ser Pro Leu Phe 260 - # 265 - # 270 - - Phe Asn His Val Lys Ile Lys Leu Ala Asp Se - #r Ala Leu Glu Arg Lys 275 - # 280 - # 285 - - Arg Ile Ser Ala Ala Arg Glu Arg Lys Ala Th - #r Lys Ile Leu Gly Ile 290 - # 295 - # 300 - - Ile Leu Gly Ala Phe Ile Ile Cys Trp Leu Pr - #o Phe Phe Val Val Ser 305 3 - #10 3 - #15 3 -#20 - - Leu Val Leu Pro Ile Cys Arg Asp Ser Cys Tr - #p Ile His Pro GlyLeu 325 - # 330 - # 335 - - Phe Asp Phe Phe Thr Trp Leu Gly Tyr Leu As - #n Ser Leu Ile Asn Pro 340 - # 345 - # 350 - - Ile Ile Tyr Thr Val Phe Asn Glu Glu Phe Ar - #g Gln Ala Phe Gln Lys 355 - # 360 - # 365 - - Ile Val Pro Phe Arg Lys Ala 370 - # 375 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 398 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pr - #o Ala Pro Ala Gly Ser 1 5 - # 10 - # 15 - - Glu Thr Trp Val Pro Gln Ala Asn Leu Ser Se - #r Ala Pro Ser Gln Asn 20 - # 25 - # 30 - - Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp Se - #r Ile Ser Leu Pro Trp 35 - # 40 - # 45 - - Lys Val Leu Leu Val Met Leu Leu Ala Leu Il - #e Thr Leu Ala Thr Thr 50 - # 55 - # 60 - - Leu Ser Asn Ala Phe Val Ile Ala Thr Val Ty - #r Arg Thr Arg Lys Leu 65 - #70 - #75 - #80 - - His Thr Pro Ala Asn Tyr Leu Ile Ala Ser Le - #u Asp Val Thr Asp Leu 85 - # 90 - # 95 - - Leu Val Ser Ile Leu Val Ile Pro Ile Ser Th - #r Met Tyr Thr Val Thr 100 - # 105 - # 110 - - Asp Arg Trp Thr Leu Ser Gln Val Val Cys As - #p Phe Trp Leu Ser Ser 115 - # 120 - # 125 - - Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu Hi - #s Leu Cys Val Ile Ala 130 - # 135 - # 140 - - Leu Asp Arg Tyr Trp Ala Ile Thr Asp Ala Va - #l Glu Tyr Ser Ala Lys 145 1 - #50 1 - #55 1 -#60 - - Arg Thr Pro Lys Arg Ala Ala Val Met Ile Al - #a Leu Val Trp ValPhe 165 - # 170 - # 175 - - Ser Ile Ser Ile Ser Leu Pro Pro Phe Phe Tr - #p Arg Gln Ala Lys Ala 180 - # 185 - # 190 - - Glu Glu Glu Val Ser Glu Cys Val Val Asn Th - #r Asp His Ile Leu Tyr 195 - # 200 - # 205 - - Thr Val Tyr Ser Thr Val Gly Ala Phe Tyr Ph - #e Pro Thr Leu Leu Leu 210 - # 215 - # 220 - - Ile Ala Leu Tyr Gly Arg Ile Tyr Val Glu Al - #a Arg Ser Arg Ile Leu 225 2 - #30 2 - #35 2 -#40 - - Lys Gln Thr Pro Asn Arg Thr Gly Lys Arg Le - #u Thr Arg Ala GlnLeu 245 - # 250 - # 255 - - Ile Thr Asp Ser Pro Gly Ser Thr Ser Ser Va - #l Thr Ser Ile Asn Ser 260 - # 265 - # 270 - - Arg Val Pro Asp Val Pro Ser Glu Ser Gly Se - #r Pro Val Tyr Val Asn 275 - # 280 - # 285 - - Gln Val Lys Val Arg Val Ser Asp Ala Leu Le - #u Glu Lys Lys Lys Leu 290 - # 295 - # 300 - - Met Ala Ala Arg Glu Arg Lys Ala Thr Lys Th - #r Leu Gly Ile Ile Leu 305 3 - #10 3 - #15 3 -#20 - - Gly Ala Phe Ile Val Cys Trp Leu Pro Phe Ph - #e Ile Ile Ser LeuVal 325 - # 330 - # 335 - - Met Pro Ile Cys Lys Asp Ala Cys Trp Phe Hi - #s Leu Ala Ile Phe Asp 340 - # 345 - # 350 - - Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Le - #u Ile Asn Pro Ile Ile 355 - # 360 - # 365 - - Tyr Thr Met Ser Asn Glu Asp Phe Lys Gln Al - #a Phe His Lys Leu Ile 370 - # 375 - # 380 - - Arg Leu Ser Ala Gln Val Asp Leu Pro Phe Al - #a Val Gly Pro 385 3 - #90 3 - #95 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 471 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - Met Asp Ile Leu Cys Glu Glu Asn Thr Ser Le - #u Ser Ser Thr Thr Asn 1 5 - # 10 - # 15 - - Ser Leu Met Gln Leu Asn Asp Asp Thr Arg Le - #u Tyr Ser Asn Asp Phe 20 - # 25 - # 30 - - Asn Ser Gly Glu Ala Asn Thr Ser Asp Ala Ph - #e Asn Trp Thr Val Asp 35 - # 40 - # 45 - - Ser Glu Asn Arg Thr Asn Leu Ser Cys Glu Gl - #y Cys Leu Ser Pro Ser 50 - # 55 - # 60 - - Cys Leu Ser Leu Leu His Leu Gln Glu Lys As - #n Trp Ser Ala Leu Leu 65 - #70 - #75 - #80 - - Thr Ala Val Val Ile Ile Leu Thr Ile Ala Gl - #y Asn Ile Leu Val Ile 85 - # 90 - # 95 - - Met Ala Val Ser Leu Glu Lys Lys Leu Gln As - #n Ala Thr Asn Tyr Phe 100 - # 105 - # 110 - - Leu Met Ser Leu Ala Ile Ala Asp Met Leu Le - #u Gly Phe Leu Val Met 115 - # 120 - # 125 - - Pro Val Ser Met Leu Thr Ile Leu Tyr Gly Ty - #r Arg Trp Pro Leu Pro 130 - # 135 - # 140 - - Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu As - #p Val Leu Phe Ser Thr 145 1 - #50 1 - #55 1 -#60 - - Ala Ser Ile Met His Leu Cys Ala Ile Ser Le - #u Asp Arg Tyr ValAla 165 - # 170 - # 175 - - Ile Gln Asn Pro Ile His His Ser Arg Phe As - #n Ser Arg Thr Lys Ala 180 - # 185 - # 190 - - Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Se - #r Val Gly Ile Ser Met 195 - # 200 - # 205 - - Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Se - #r Lys Val Phe Lys Glu 210 - # 215 - # 220 - - Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Va - #l Leu Ile Gly Ser Phe 225 2 - #30 2 - #35 2 -#40 - - Val Ser Phe Phe Ile Pro Leu Thr Ile Met Va - #l Ile Thr Tyr PheLeu 245 - # 250 - # 255 - - Thr Ile Lys Ser Leu Gln Lys Glu Ala Thr Le - #u Cys Val Ser Asp Leu 260 - # 265 - # 270 - - Gly Thr Arg Ala Lys Leu Ala Ser Phe Ser Ph - #e Leu Pro Gln Ser Ser 275 - # 280 - # 285 - - Leu Ser Ser Glu Lys Leu Phe Gln Arg Ser Il - #e His Arg Glu Pro Gly 290 - # 295 - # 300 - - Ser Tyr Thr Gly Arg Arg Thr Met Gln Ser Il - #e Ser Asn Glu Gln Lys 305 3 - #10 3 - #15 3 -#20 - - Ala Cys Lys Val Leu Gly Ile Val Phe Phe Le - #u Phe Val Val MetTrp 325 - # 330 - # 335 - - Cys Pro Phe Phe Ile Thr Asn Ile Met Ala Va - #l Ile Cys Lys Glu Ser 340 - # 345 - # 350 - - Cys Asn Glu Asp Val Ile Gly Ala Leu Leu As - #n Val Phe Val Trp Ile 355 - # 360 - # 365 - - Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Va - #l Tyr Thr Leu Phe Asn 370 - # 375 - # 380 - - Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Il - #e Gln Cys Gln Tyr Lys 385 3 - #90 3 - #95 4 -#00 - - Glu Asn Lys Lys Pro Leu Gln Leu Ile Leu Va - #l Asn Thr Ile ProAla 405 - # 410 - # 415 - - Leu Ala Tyr Lys Ser Ser Gln Leu Gln Met Gl - #y Gln Lys Lys Asn Ser 420 - # 425 - # 430 - - Lys Gln Asp Ala Lys Thr Thr Asp Asn Asp Cy - #s Ser Met Val Ala Leu 435 - # 440 - # 445 - - Gly Lys Gln His Ser Glu Glu Ala Ser Lys As - #p Asn Ser Asp Gly Val 450 - # 455 - # 460 - - Asn Glu Lys Val Ser Cys Val 465 4 - #70 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (v) FRAGMENT TYPE: N-terminal - - (vi) ORIGINAL SOURCE: (A) ORGANISM: oligonucleot - #ide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - GATGGTACAC TGGCTGGGGG GTGGGCTGAG TTGACGGTGG CT - # - # 42 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: oligonucleot - #ide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - TACCACGCGG CCAAGAGCCT TTACCA - # - # 26 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: oligonucleot - #ide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - TGGTGCTAGA GATCTGCTGA CGTTC - # - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: oligonucleot - #ide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - GAGAAGTCAG ACACACAGAA AGTCTGTGTA AGTTTTACAA CTTGC - # - #45 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: OLIGONUCLEOT - #IDE - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - GATGGTACAC TGGCTGGGGG GTGGGCTGAG TTGACGGTGG CT - # - # 42__________________________________________________________________________

Number	Name	Date
4675285	Clark	Jun 1987
4904582	Tullis	Feb 1990
4985352	Julius et al.	Jan 1991
5155218	Weinshank et al.	Oct 1992
5242822	Marullo et al.	Sep 1993
5360735	Weinshank et al.	Nov 1994
5472866	Gerald et al.	Dec 1995
5476782	Weinshank et al.	Dec 1995
5786155	Weinshank et al.	Jul 1998

Number	Date	Country
2267906	Dec 1993	GBX
9117174	Nov 1991	WOX
9311147	Jun 1993	WOX

	Number	Date	Country
Parent	542358	Oct 1995
Parent	370542	Jan 1995

	Number	Date	Country
Parent	194113	Feb 1994
Parent	803626	Dec 1991

Methods for identifying a compound that binds to a human 5-HT.sub.1E receptor

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US Referenced Citations (9)

Foreign Referenced Citations (3)

Non-Patent Literature Citations (21)

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Entry
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