Corticotropin-releasing factor2 receptors

Details Corticotropin-releasing factor2 receptors Corticotropin-releasing factor2 receptors

2001-06-13

2004-04-20

Ulm, John (Department: 1646)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023500

Reexamination Certificate

active

06723841

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to cell surface receptors, and more specifically, to Corticotropin-Releasing Factor
2
receptors.
BACKGROUND OF THE INVENTION
Corticotropin-releasing factor (“CRF”) is a 41-amino acid peptide originally isolated from the hypothalamus by virtue of its ability to stimulate the production of adrenocorticotropic hormone (“ACTH”) and other proopiomelanocortin (“POMC”) products of the anterior pituitary (Vale et al.,
Science
213:1394-1397, 1981). Briefly, CRF is believed to initiate its biological effects by binding to a plasma membrane receptor which is distributed throughout the brain (DeSouza et al.,
Science
224:1449-1451, 1984), pituitary (Wynn et al.,
Biochem. Biophys. Res. Comm
. 110:602-608, 1983), adrenals (Udelsman et al.,
Nature
319:147-150, 1986) and spleen (Webster, E. L., and E. B. DeSouza,
Endocrinology
122:609-617, 1988). This receptor is coupled to a GTP-binding protein (Perrin et al.,
Endocrinology
118:1171-1179, 1986) which mediates CRF-stimulated increase in intracellular cAMP (Bilezikjian, L. M., and W. W. Vale,
Endocrinology
113:657-662, 1983).
In addition to its role in stimulating the production of ACTH and POMC, CRF is also believed to coordinate many of the endocrine, autonomic, and behavioral responses to stress, and may be involved in the pathophysiology of affective disorders. Moreover, CRF is believed to be a key intermediary in communication between the immune, central nervous, endocrine and cardiovascular systems (Crofford et al.,
J. Clin. Invest
. 90:2555-2564, 1992; Sapolsky et al.,
Science
238:522-524, 1987; Tilders et al.,
Regul. Peptides
5:77-84, 1982; Fisher et al.,
Reg. Peptide
5:153-161, 1983).
A receptor for CRF has been cloned from rat (Perrin et al.,
Endo
133(6):3058-3061, 1993), and human brain (Chen et al.,
PNAS
90(19):8967-8971, 1993; Vita et al.,
FEBS
335(1):1-5, 1993). This receptor is a 415 amino acid protein comprising seven membrane spanning domains, and has a predicted molecular weight of 44,000 daltons. A comparison of identity between rat and human sequences shows a high degree of homology (97%) at the amino acid level. In addition, Scatchard analysis of recombinantly produced human receptor demonstrates a single component site, with a high affinity (K
d
of 1.6±0.3 nM) for CRF.
The present invention provides new, previously unidentified CRF receptors, designated as the “Corticotropin-Releasing Factor-
2
(“CRF
2
”), Corticotropin-Releasing Factor receptors. In addition, the present invention provides compositions and methods which utilize such CRF
2
receptors, as well as other, related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods which utilize CRF
2
receptors (also termed “CRF
2
Corticotropin-Releasing Factor receptors” or “CRF
2
R”). Within one aspect of the present invention, isolated nucleic acid molecules are provided which encode CRF
2
receptors. Within one embodiment, nucleic acid molecules are provided which encode a CRF
2
receptor such as that disclosed in Sequence I.D. No. 4, from amino acid number 1 to amino acid number 411. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number 1449. Within another embodiment, nucleic acid molecules are provided which encode a CRF
2
receptor such as that disclosed in Sequence ID No. 2 from amino acid number 1 to amino acid number 431. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 1336. Nucleic acid molecules which encode CRF
2
receptors of the present invention may be isolated from virtually any warm-blooded animal, including for example, humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats and mice.
Within another aspect of the present invention, isolated nucleic acid molecules are provided which encode portions of a CRF
2
receptor, such as the N-terminal extracellular domain. Within one embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence ID. No. 3, from nucleotide number 216 to nucleotide number 570. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid number 1 to amino acid number 118. Within another embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 451. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence ID No. 2 from amino acid number 1 to amino acid number 138.
Within other aspects of the invention, expression vectors are provided which are capable of expressing the above-described nucleic acid molecules. Within other aspects, recombinant viral vectors are provided which are capable of directing the expression of the above-described nucleic acid molecules. Representative examples of such viral vectors include retroviral vectors, adenoviral vectors, and herpes simplex virus vectors. Also provided by the present invention are host cells which contain the above-described expression vectors, as well as the receptor or portions thereof which are encoded by the above-described nucleic acid molecules. Within other embodiments, isolated portions of CRF
2
receptors are provided, including for example, isolated portions of extracellular domains such as the N-terminal extracellular domain.
Within other aspects of the invention, isolated antibodies are provided which are capable of specifically binding to the above-described CRF
2
receptors. Within one embodiment, the antibodies may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and antibody fragments. Within other embodiments, antibodies are provided which are capable of blocking the binding of CRF (or other substrates such as sauvagine or urotensin I) to a CRF
2
receptor. Within preferred embodiments, the antibodies may be selected from the group consisting of murine and human antibodies. Within preferred aspects of the invention, the above-noted antibodies are produced by hybridomas.
Within yet another aspect of the present invention, nucleic acid molecules are provided which are capable of specifically hybridizing to a nucleic acid molecule encoding any of the CRF
2
receptors described above. Such molecules may be between at least “y” nucleotides long, wherein “y” is any integer between 14 and 1230, and furthermore, may be selected suitable for use as probes or primers described below. Particularly preferred probes of the present invention are at least 18 nucleotides in length.
Within other aspects of the present invention, methods for detecting the presence of a compound which binds to a CRF
2
receptor are provided, comprising the steps of (a) exposing one or more compounds to cells that express CRF
2
receptors under conditions and for a time sufficient to allow binding of the compounds to the receptors, and (b) isolating compounds which bind to the receptors, such that the presence of a compound which binds to a CRF
2
receptor may be detected. Within another aspect, methods for detecting the presence of a compound which binds to a CRF
2
receptor are provided, comprising the steps of (a) exposing one or more compounds to a CRF
2
receptor N-terminal extracellular domain under conditions and for a time sufficient to allow binding of a compound to the N-terminal extracellular domain, and (b) isolating compounds which bind to the CRF
2
receptor N-terminal extracellular domain, such that the presence of a compound which binds to a CRF
2
receptor may be detected. Within one embodiment, the compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes, and radionuclides.
Wi

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