Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-12
2002-02-19
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S007100, C435S007200, C435S069100, C435S320100, C435S325000, C435S358000, C436S501000, C530S300000, C530S350000, C536S023500
Reexamination Certificate
active
06348330
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention concerns a cloned human neurokinin-3 receptor (hereinafter identified as human NK3R).
Neurokinin B (NKB) is a naturally occuring peptide belonging to the neurokinin family of peptides, which also includes substance P (SP) and substance K (SK). NKB binds preferentially to the neurokinin-3 receptor (NK3R), although it also recognizes the other two receptor subtypes (NK1 and NK2) with lower affinity. As is well known in the art, neurokinin B and other tachykinins have been implicated in the pathophysiology of numerous diseases. Neurokinin peptides are reportedly involved in nociception and neurogenic inflammation. The physiological function of NK3R has been implicated in the regulation of enkephalin release, while the NK1 and NK2 receptor subtypes are involved in synaptic transmission (Laneuville et al.,
Life Sci.,
42:1295-1305 (1988)). Since the NKB genomic structure and subcellular distribution are different from those of SP and SK, the physiological function and regulatory mechanism of NKB may be different from SP and SK.
More specifically, neurokinin B is a pharmacologically-active neuropeptide that is produced in mammals and possesses a characteristic amino acid sequence that is illustrated below:
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2.
Several groups have reported the cloning of certain neurokinin receptors. T. M. Fong, et al.,
Mol. Pharmacol.,
41:24-30 (1991) have reported cloned human neurokinin-1 and neurokinin-1 short form receptor. J. Yokota, et al.,
J. Biol. Chem.,
264:17649 (1989) have reported cloned rat neurokinin-1 receptor. N. P. Gerard, et al.,
J. Biol. Chem.,
265:20455 (1990), have reported human neurokinin-2 receptor. Cloned rat and bovine neurokinin-2 receptor have likewise been reported. See respectively, Y. Sasi, and S. Nakanishi,
Biochem Biophys. Res. Comm.,
165:695 (1989), and Y. Masu, et al.,
Nature
329:836 (1987). Cloned rat neurokinin-3 receptor has been reported by R. Shigemoto, et al.,
J. Biol. Chem.,
265:623 (1990). The above references, however, neither disclose nor suggest the present invention.
The instant invention also concerns an assay protocol which can be used to determine the activity in body fluids of substances that bind human NK3R; these include neurokinin B. The assay can also be used for identifying and evaluating substances that bind NK3R. Thus, the assay can be used to identify neurokinin B antagonists and evaluate their binding affinity. Another method for an assay includes that described by M. A. Cascieri, et al.,
J. Biol. Chem.,
258:5158 (1983). See also, for example, R. M. Snider, et al.,
Science,
251:435 (1991) and S. McLean, et al.,
Science,
251:437 (1991). See also WIPO Patent Publications WO90/05525 and WO90/05729, published May 31, 1990. Methods to date have proven inferior, in part, for failure of the animal receptor (animal NK1R, NK2R or NK3R) activity to accurately reflect that of the human neurokinin-3 receptor. Furthermore, prior to this disclosure, human NK3R has not been available in a highly purified form or in substantial isolation from NK1R and/or NK2R. Use of such neurokinin receptor sources can not accurately depict the affinity of an agonist or an antagonist for a human NK3R.
SUMMARY OF THE INVENTION
A novel recombinant human neurokinin-3 receptor (hereinafter identified as human NK3R) is disclosed which has been prepared by polymerase chain reaction techniques. Also disclosed is the complete sequence of human NK3R complementary DNA; expression systems, including a CHO (chinese hamster ovarian cell line) stable expression system; and an assay using the CHO expression system.
Human NK3R can be used in an assay to identify and evaluate entities that bind neurokinin B receptor or NK3R. The assay can also be used in conjunction with diagnosis and therapy to determine the body fluid concentration of neurokinin-B related substances in patients. In addition, the complete sequence of the human NK3R is useful in the process of developing novel NK3 agonists and antagonists by computer modeling.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the invention concerns human neurokinin-3 receptor, said receptor being free of other human receptor proteins.
In one class this first embodiment concerns human neurokinin-3 receptor, said receptor being free of other human proteins.
Within this class, this first embodiment concerns human neurokinin-3 receptor from human cells such as glioblastoma, said receptor being free of other human proteins.
Also within this class, this first embodiment concerns human neurokinin-3 receptor, the receptor being recombinantly produced from non-human cells.
In a second class, this first embodiment concerns a protein corresponding to the amino acid sequence of human neurokinin-3 receptor, the protein comprising 465 amino acids. Within the second class this first embodiment concerns a protein comprising the following 465 amino acid sequence (SEQ ID NO:1:) depicted from the amino to the carboxy terminus:
Met Ala Thr Leu Pro Ala Ala Glu Thr Trp Ile Asp Gly Gly Gly Gly
1 5 10 15
Val Gly Ala Asp Ala Val Asn Leu Thr Ala Ser Leu Ala Ala Gly Ala
20 25 30
Ala Thr Gly Ala Val Glu Thr Gly Trp Leu Gln Leu Leu Asp Gln Ala
35 40 45
Gly Asn Leu Ser Ser Ser Pro Ser Ala Leu Gly Leu Pro Val Ala Ser
50 55 60
Pro Ala Pro Ser Gln Pro Trp Ala Asn Leu Thr Asn Gln Phe Val Gln
65 70 75 80
Pro Ser Trp Arg Ile Ala Leu Trp Ser Leu Ala Tyr Gly Val Val Val
85 90 95
Ala Val Ala Val Leu Gly Asn Leu Ile Val Ile Trp Ile Ile Leu Ala
100 105 110
His Lys Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Leu Ala
115 120 125
Phe Ser Asp Ala Ser Met Ala Ala Phe Asn Thr Leu Val Asn Phe Ile
130 135 &
Fong Tung Ming
Huang Ruey-Ruey C.
Strader Catherine D.
Bunner Bridget E.
Kemmerer Elizabeth
Merck & Co. , Inc.
Rose David L.
Thies J. Eric
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