Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-06-25
2004-06-15
Ulm, John D. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S007210, C435S069100, C536S023500
Reexamination Certificate
active
06750322
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel guanosine triphosphate binding protein-coupled receptor proteins, DNA encoding the proteins, and methods of screening for drug-candidate compounds using them.
BACKGROUND ART
Many hormones and neurotransmitters regulate physiological functions through specific receptor proteins located on the cell membrane. Many of these receptor-proteins transduce signals into the cell by activating a guanosine triphosphate binding protein (occasionally referred to as “G protein” below) that is coupled to them. These receptor proteins are thereby named as G protein-coupled receptors. Since they have a common structure, composed of seven transmembrane regions, they are also generally called “seven-transmembrane receptor proteins.”
G protein-coupled receptors, which are expressed on the surface of cells in vivo and functioning cells of tissues, play an extremely important role as a target of molecules such as hormones, neurotransmitters, and biologically active compounds, which regulate the functions of these cells and tissues. Therefore, G protein-coupled receptor proteins have received great attention as targets in drug-development.
G protein-coupled receptors reported so far include: muscarinic acetylcholine receptors M1, M2, M3, and M4 (Peralta et al., EMBO J., 6:3923-3929 (1987)), muscarinic acetylcholine receptor M5 (Bonner et al., Neuron, 1:403-410 (1988)), adenosine receptor A1 (Libert et al., Science, 244:569-572 (1989)), &agr;1A adrenoreceptor (Bruno et al., Biochem. Biophys. Res. Commun., 179:1485-1490 (1991)), &bgr;1 adrenoreceptor (Frielle et al., Proc. Natl. Acad. Sci. USA, 84:7920-7924 (1987)), angiotensin receptor AT
1
(Takayanagi et al., Biochem. Biophys. Res. Commun., 183:910-916 (1992)), endothelin receptor ETA (Adachi et al., Biochem. Biophys. Res. Commun., 180:1265-1272 (1991)), gonadotropin releasing factor receptor (Kaker et al., Biochem. Biophys. Res. Commun., 189:289-295 (1992)), histamine receptor H
2
(Ruat et al., Proc. Natl. Acad. Sci. USA, 87:1658-1672 (1992)), neuropeptide Y receptor Y1 (Larhammar et al., J. Biol. Chem., 267:10935-10938 (1992)), interleukin-8 receptor IL8RA (Holmes et al., Science, 2563:1278-1280 (1991)), dopamine receptor D
1
(Mahan et al., Proc. Natl. Acad. Sci. USA, 87:2196-2200 (1990)), metabolic glutamate receptor mGluR/1 (Masu et al., Nature, 349:760-765 (1991)), and somatostatin receptor SS
1
(Yamada et al., Proc. Natl. Acad. Sci. USA, 89:251-255) (for reference, Watson S. and Arkinstall S., The G protein Linked Receptor FactsBook, Academic Press (1994)). Examples of developed medicines aimed at G protein-coupled receptors are: terazosine hydrochloride (antihypertensive agent, &agr;1 adrenoreceptor antagonist), atenolol (antiarrhythmia, &bgr;1 adrenoreceptor antagonist), dicyclomine hydrochloride (antispasmodic agent, acetylcholine receptor antagonist), ranitidine hydrochloride (drug for peptic ulcers, histamine receptor H2 antagonist), trazodone hydrochloride (antidepressant, serotonin receptor 5-HT1B antagonist), and buprenorphine hydrochloride (analgesic agent, opioid receptor &kgr; agonist) (for reference, Stadel et al., Trends Pharm. Sci., 18:430-437 (1997); Medicine Handbook 5
th
edition, Yakugyo-Jiho).
The hypothalamus, a part of the brain which governs a number of programs that trigger a particular response, contributes to the homeostasis of the internal environment by means of a variety of outputs, as the center of the autonomic nervous system. For instance, it releases hormones such as thyrotropic hormone-releasing hormone, gonadotropic hormone-releasing hormone, and growth hormone-releasing hormone, and thereby regulates the entire endocrine system through the actions of these hormones on the specific receptors expressed in target cells. These outputs in the hypothalamus are thought to be mediated by receptors expressed in the hypothalamus and compounds reacting with them. Therefore, elucidation of the relationship between the compounds regulating the hypothalamus outputs and their specific receptors expressed in the hypothalamus is extremely important in developing novel medicines for the treatment of diseases arising from endocrine disorders.
DISCLOSURE OF THE INVENTION
The present invention provides a novel human-derived G protein-coupled receptor protein and rat-derived one corresponding thereto, both of which are expressed in the brain (in particular, thalamus and hypothalamus, etc.). It also provides a method of screening for ligands and drug-candidate compounds using these receptor proteins.
The inventors first selected a region highly conserved in known G protein-coupled receptor proteins, then designed primers corresponding to the region, and performed reverse transcriptase-polymerase chain reaction (RT-PCR) using mRNA obtained from rat thalamus and hypothalamus. Next, amplified clones were randomly selected, and their partial nucleotide sequences were determined. To remove known clones from the nucleotide sequence determined-clones, colony-hybridization was performed using, as a probe, cDNA clones judged by homology search to be encoding a known G protein-coupled receptor protein. Negative clones that failed to hybridize with any probe were selected. Using probes prepared based on the nucleotide sequence of the negative clones, the inventors screened cDNA libraries from rat thalamus and hypothalamus, and succeeded in isolating a full-length cDNA (rat BG2 cDNA) encoding a rat G protein-coupled receptor.
Moreover, the present inventors screened human hippocampus libraries using specific probes and successfully isolated a human cDNA (human BG2 cDNA) corresponding to the rat cDNA.
To identify a ligand for the G protein-coupled receptor protein encoded by the isolated human BG2 cDNA, the present inventors prepared cells expressing the protein and stimulated the protein to screen compounds which changed the concentration of cAMP in the cells. As a result, histamine was found to have an activity of stimulating the human G protein-coupled receptor protein expressed on the cell surface and lowering the intracellular cAMP concentration. In addition, histamine was found to have an activity of actually binding to the protein.
The present inventors also isolated a cDNA encoding an alternative splicing variant for rat BG2 (rat BG2-2 cDNA), expressed the protein encoded by the cDNA on a cell surface, and detected the response against the histamine stimulation, to find that the protein had an activity of reducing the intracellular cAMP concentration in response to the histamine stimulation in the same manner as in human BG2.
The G protein-coupled receptor protein found by the present inventors is a very useful tool in screening for agonists and antagonists thereof, and the agonists and antagonists isolated by the screening are expected to be used as pharmaceuticals.
The present invention relates to novel human- and rat-derived G protein-coupled receptor proteins, DNAs encoding them, and screening of ligands and drug-candidate compounds using the proteins.
Specifically, the invention relates to:
(1) a guanosine triphosphate-binding protein-coupled receptor protein comprising the amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20 or 25, and
(b) the amino acid sequence of SEQ ID NO:20 or 25, in which one or more amino acids are replaced, deleted, or added;
(2) the protein of (1), wherein the protein has an activity of binding to histamine;
(3) the protein of (1), wherein the protein has an activity of changing the intracellular cAMP concentration or calcium concentration in response to histamine stimulation;
(4) a partial peptide of the receptor protein of any one of (1) to (3);
(5) a DNA encoding the receptor protein of any one of (1) to (3) or the partial peptide of (4);
(6) the DNA of (5), wherein the DNA comprises a coding region of the nucleotide sequence of SEQ ID NO:21 or 26;
(7) a vector containing the DNA of (5) or (6);
(8) a transformant carrying the DNA of (5) or (6) or the vector of (7);
(9) a method for producing the
Hidaka Yusuke
Itadani Hiraku
Kobayashi Masahiko
Nakamura Takao
Ohta Masataka
Banyu Pharmaceutical Co. Ltd.
Fish & Richardson P.C.
Ulm John D.
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