Molecular cloning of the ovine pituitary growth hormone releasing hormone receptor

• US 5,852,187 A
• Filed: 06/12/1996
• Issued: 12/22/1998
• Est. Priority Date: 06/23/1992
• Status: Expired due to Term

• Current Assignee: University of Virginia Patent Foundation (University of Virginia)
• Original Assignee: University of Virginia Patent Foundation (University of Virginia)
• Inventors: Horikawa, Reiko, Thorner, Michael O., Gaylinn, Bruce D., Lyons, Charles E. Jr.
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US08/660,963
• Time in Patent Office: 923 Days
• Field of Search: 536/23.5, 536/23.1, 435/6, 435/69.1, 435/91.2, 530/350, 530/399
• US Class Current: 536/23.5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/60 Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: G01N 2333/60 Growth-hormone releasing factors (GH-RF) (Somatoliberin)
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

Growth hormone releasing hormone (GHRH) receptor binding has been characterized using a unique binding assay utilizing iodinated GHRH probes. Photoaffinity GHRH probes have been constructed which allow for photolabeling and characterization of the receptor. In addition, high affinity biotinylated GHRH analogs have been constructed. Solubilization of GHRH-R/GHRH complexes and extraction of specifically bound GHRH using a mild detergent solution, followed by affinity chromatography, leads to a substantially purified GHRH-R isolate. Electrophoretic treatment of the GHRH-R isolate produces GHRH-R of sufficient purity to conduct sequencing of the receptor. Cloning of a gene encoding for polypeptides (protein or fragments thereof) having GHRH-R activity is accomplished using a bacterial host, and the cloned gene is expressed in a mammalian cell line. In addition, molecular cloning of the ovine GHRH-R is provided.

Claims

1. A purified and isolated nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:

9 of the ovine growth hormone receptor.

2. A purified and isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:

10 of the ovine growth hormone releasing hormone receptor.

3. A nucleic acid molecule having the nucleic acid sequence SEQ ID NO:

9 encoding an ovine growth hormone releasing hormone receptor, prepared according to the following process;

(a) preparing an ovine pituitary library;

(b) subdividing the library into pools;

(c) performing PCR on each pool to identify a pool that exhibits a growth hormone releasing hormone receptor signal; and

(d) performing hybridization screening on the pool identified in step (c) using a hybridization buffer and a detectable labeled growth hormone releasing hormone receptor DNA oligonucleotide probe to obtain the nucleic acid molecule encoding the ovine growth hormone releasing hormone receptor.