Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-12-14
2003-03-25
Pak, Michael (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06538107
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel DNAs which are useful as DNA primers for a polymerase chain reaction (PCR); methods for amplifying DNAs each coding for a G protein coupled receptor protein via PCR techniques using said DNA; screening methods for DNAs each encoding a G protein coupled receptor protein via PCR techniques using said DNA; G protein coupled receptor protein-encoding DNAs obtained by said screening method; G protein coupled receptor proteins which are encoded by the DNA obtained via said screening method, peptide fragments or segments thereof, and modified peptide derivatives thereof; etc.
The present invention also relates to novel G protein coupled receptor proteins; novel G protein coupled receptor protein-encoding DNAs; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
The present invention also relates to novel human amygdaloid nucleus-derived G protein coupled receptor proteins;
novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
The present invention also relates to novel mouse pancreatic &bgr; cell line MIN6-derived G protein coupled receptor proteins; novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and aid protein-encoding DNA; etc. Further, the present invention relates to novel human-derived G protein coupled receptor proteins (human prinoceptors); novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
BACKGROUND OF THE INVENTION
A variety of hormones, neurotransmitters and the like control, regulate or adjust the functions of living bodies via specific receptors located in cell membranes. Many of these receptors mediate the transmission of intracellular signals via activation of guanine nucleotide-binding proteins (hereinafter, sometimes referred to as G proteins) with which the receptor is coupled and possess the common (homologous) structure, i.e. seven transmembranes (membrane-spanning regions (domains)). Therefore, such receptors are generically referred to as G protein coupled receptors or seven transmembrane (membrane-spanning) receptors.
G protein coupled receptor proteins have a very important role as targets for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living bodies. Each molecule has its own receptor protein which is specific thereto, whereby the specificities of individual physiologically active substances, including specific target cells and organs, specific pharmacological actions, specific action strength, action time, etc., are decided. Accordingly, it has been believed that, if G protein coupled receptor genes or cDNA can be cloned, those will be helpful not only for the clarification of structure, function, physiological action, etc. of the G protein coupled receptor but also for the development of pharmaceuticals by investigating the substances which act on the receptor. Until now, only several G protein coupled receptor genes or cDNAs have been cloned but it is believed that there are many unknown G protein coupled receptor genes which have not been recognized yet.
The characteristic feature of the G protein coupled receptor proteins which have been known up to now is that seven clusters of hydrophobic amino acid residues are located in the primary structure and pass through (span) the cell membrane at each region thereof. It has been known that such a structure is common among all of the known G protein coupled receptor proteins and further that the amino acid sequences corresponding to the area where the protein passes through the membrane (membrane-spanning region or transmembrane region) and the amino acid sequences near the membrane-spanning region are often highly conserved among the receptors. When an unknown protein has such a structure, it is strongly suggested that said protein is within a category of the G protein coupled receptor proteins. In addition, some amino acid residue alinements are common (homologous) and, by taking it as a characteristic feature, it is further strongly suggested that said protein is a G protein coupled receptor protein.
Libert, F, et al. (Science, 244:569-571; 1989) reported a method for cloning novel receptor genes by means of a polymerase chain reaction (hereinafter, sometimes referred to as PCR or a PCR technique) for a synthetic DNA primer which was synthesized based upon the information of common amino acid sequences obtained from a comparison among known G protein coupled receptor proteins. Libert, F. et al. used a pair of synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions. However, in general, the design of primers used for the PCR regulates the molecular species of DNAs which are to be amplified. In addition, when a similarity (homology) in the amino acid sequence level is used as a basis, the use of different codons affects on the binding (hybridization) of the primer thereby resulting in a decrease in the amplifying efficiency. Accordingly, although various novel receptor protein DNAs have been obtained using said DNA primers, it is not possible to succeed in amplifying DNAs for all receptor proteins in the prior art.
Further, the amino acid sequence which is common to from the first to the seventh membrane-spanning regions among 74 G protein coupled receptor proteins was reported by William C. Probst, et al. (DNA and Cell Biology, Vol. 11, No. 1, 1992, pp. 1-20). In this report, however, there is no suggestion for a method in which DNA coding for a novel G protein coupled receptor protein is screened by means of PCR using DNA primers which are complementary to the DNA coding for those amino acid sequences.
It would be desirable to develop DNA primers for PCR techniques which allow selective and efficient screenings of DNAs coding for the areas (regions) more nearer the full length of novel G protein coupled receptor proteins by utilizing the common (homologous) sequence(s) of the G protein coupled receptor protein or the DNA coding therefor.
It would also be desirable to develop synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions, said primer being useful in screening for DNA coding for G protein coupled receptor proteins in more selective and efficient manner as compared with a series of the synthetic DNA primers corresponding to the sequences of the third to the sixth membrane-spanning regions as reported by Libert, F. et al.
G protein coupled receptor proteins are important for investigating substances which control the function of living organisms and proceeding developments thereof as pharmaceuticals. Finding and development of candidate compounds for new pharmaceuticals can be efficiently proceeded by using G protein coupled receptor proteins and by conducting receptor binding experiments and evaluating experiments on agonists/antagonists using intracellular information transmittance systems as indexes. Especially when the presence of a novel G protein coupled receptor protein can be clarified, the presence of a substance having a specific action thereon can be suggested.
If a novel DNA which codes for a novel G protein coupled receptor protein can be efficiently screened and isolated, it will now be possible to proceed with the isolation of DNA having an entire coding region, the construction of an expression system therefor and the screening of an acting ligand.
A hypothalamo-hypophysial system is one of the passages for controlling, regulating or adjusting the functions of organisms relying upon interactions of hormones and neurotransmitters with G protein coupled r
Fujii Ryo
Hinuma Shuji
Ito Yasuaki
Conlin David G.
Edwards & Angell LLP
Intellectual Property Practice Group
Pak Michael
Takeda Chemical Industries Ltd.
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