Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
2000-05-23
2004-09-21
Romeo, David S. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S326000, C530S345000, C530S402000, C530S408000
Reexamination Certificate
active
06794491
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel ligand polypeptide for the G protein-coupled receptor protein and a DNA comprising a DNA encoding the ligand polypeptide.
BACKGROUND
Many hormones and neurotransmitters mediate Of biological functions through specific receptors present on the cell membrane. Many of these receptors engage themselves in the intracellular transduction of signals through activation of the coupled guanine nucleotide-binding protein (hereinafter sometimes referred to briefly as G protein) and have the common structure comprising 7 transmembrane domains. Therefore, these receptors are collectively referred to as G protein-coupled receptor or 7-transmembrane receptor.
One of the pathways to modulate biological functions mediated by such hormones or neurotransmitters through G protein-coupled receptors is the hypothalamo-pituitary system. Thus, the secretion of pituitary hormone from the hypophysis is controlled by hypothalamic hormones (pituitatropic releasing factor) and the functions of the target cells or organs are regulated through the pituitary hormones released into the circulation. This pathway carries out functional modulations of importance to the living body, such as homeostasis and regulation of the reproduction, development, metabolism and growth of individuals. The secretion of pituitary hormones is controlled by a positive feedback or a negative feedback mechanism involving hypothalamic hormone and the peripheral hormone secreted from the target endocrine gland. The various receptor proteins present in the hypophysis are playing a central role in the regulation of the hypothalamus-pituitary system.
Meanwhile, it is known that these hormones and factors as well as their receptors are not localized in the hypothalamus-pituitary system but are broadly distributed in the brain. Therefore, it is suspected that, in the central nervous system, this substance called hypothalamus hormone is functioning as a neurotransmitter or a neuromodulator. Moreover, the substance is distributed in peripheral tissues as well and thought to be playing important roles in the respective tissue.
The pancreas is playing a crucial role in the carbohydrate metabolism by secreting glucagon and insulin as well as digestive juice. While insulin is secreted from the pancreatic &bgr; cells, its secretion is mainly stimulated by glucose. However, it is known that &bgr; cells have a variety of receptors and the secretion of insulin is controlled by a number of factors in addition to glucose as well as peptide hormones, e.g. galanine, somatostatin, gastric inhibitory polypeptide, glucagon, amyrin, etc.; sugars, e.g. mannose etc.; amino acids, and neurotransmitters, among others.
The means only heretofore available for identifying ligands for said G protein-coupled receptor proteins is estimation from the homology in primary structure of G protein-coupled receptor proteins.
Recently, investigation for novel opioid peptides by introducing a cDNA coding for a receptor protein which a ligand is unknown, i.e. an orphan G protein-coupled receptor protein, into animal cells have been reported (Reinsheid, R. K. et al., Science, 270, 792-35 794, 1995, Menular, J.-C., et al., Nature 377, 532-535, 1995). However, in view of similarities to known G protein-coupled receptor proteins and tissue distributions, it could be easily anticipated in these cases that the ligand would be belonging to the family of opioid peptides. The history of research and development in the realm of substances acting on the living body through the opioid receptor dates back to many years ago and various antagonists and agonists had been developed. Therefore, among the compounds artificially synthesized, an agonist of the receptor was picked out and, using it as a probe, expression of the receptor in the receptor cDNA-transfected cells was verified. Then, a search was made for an activator of the intracellular signal transduction which was similar to the agonist, the activator so found was purified, and the structure of the ligand was determined. However, when the homology of an orphan receptor to known G protein-coupled receptor proteins is low, it was very difficult to predict its ligand.
Ligands for orphan G protein-coupled receptors expressed in the hypophysis, central nervous system, and pancreatic &bgr; cells are considered to be useful for developing medicines, but their structures and functions have not been elucidated as yet.
DISCLOSURE OF INVENTION
Employing a cell in which a cDNA coding for orphan G protein-coupled receptor protein hare been expressed by a suitable means and using measurement of a specific cell stimulation activity exemplified by a signal transduction activity as an indicator, the inventors of the present invention succeeded in screening a polypeptide which said receptor protein recognizes as a ligand.
Furthermore, the inventors found that a compound can be screened which is capable of changing the binding activity of this ligand which is an activating factor to said receptor protein.
The present invention, therefore, relates to
(1) A polypeptide which comprises an amino acid sequence represented by SEQ ID NO:73 or its substantial equivalent thereto, or its amide or ester, or a salt thereof.
(2) The polypeptide as described in (1) above, which comprises the amino acid sequence represented by SEQ ID NO:3, SEC ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.
(3) The polypeptide as described in (1) above, which comprises the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:59.
(4) A partial peptide of the polypeptide as described in (1) above its amide or ester, or a salt thereof.
(5) A DNA which comprises a DNA having a nucleotide sequence coding for the polypeptide as described in (1) above or the partial peptide as described in (4) above.
(6) The DNA as described in (5) above which comprises a nucleotide sequence represented by SEQ ID NO:2, SEQ ID NO:1l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.
(7) A recombinant vector comprising the DNA as described in (5) above.
(8) A transformant carrying the DNA as described in (5) above or the recombinant vector as described (7) above.
(9) A method for producing the polypeptide as described in (1) above or the partial peptide as described in (4) above, which comprises culturing the transformant as described in (8) above.
(10) A pharmaceutical composition containing the polypeptide, its amide or ester as described in (1) above, or a pharmaceutically acceptable salt thereof.
(11) A pharmaceutical composition containing the partial peptide peptide, its amide or ester as described in (4) above, or a pharmaceutically acceptable salt thereof.
(12) A pharmaceutical composition containing the DNA as described in (5) above.
(13) The pharmaceutical composition as described in (10), (11), or (12) above, which is a pituitary function modulator.
(14) The pharmaceutical composition as described in (10), (11), or (12) above, which is a central nervous system function modulator.
(15) The pharmaceutical composition as described in (10), (11), or (12) above, which is a pancreatic function modulator.
(16) An antibody against the polypeptide as described in (1) above or against the partial peptide as described in (4) above.
(17) A screening method for a compound capable of changing the binding activity of the polypeptide as described in (1) above or the partial peptide as described in (4) above, with a receptor protein comprising an amino acid sequence represented by SEQ ID NO:21 or its partial, peptide or its substantial equivalent thereto, or a
Fujii Ryo
Fukusumi Shoji
Habata Yugo
Hinuma Shuji
Hosoya Masaki
Alexander John B.
Conlin David C.
Edwards & Angell LLP
Romeo David S.
Takeda Chemical Industries Ltd.
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