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.
This is a Continuation-In-Part application of U.S. application Ser. No. 07/947,672, filed Sep. 18, 1992, which is a Continuation-In-Part application of U.S. application Ser. No. 07/902,826, filed Jun. 23, 1992, the entire contents of both applications are hereby incorporated by reference.
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.
A method and recombinant assay cell for detecting growth hormone releasing hormone (GHRH) in a sample, the method includes exposing a recombinant cell to the sample and measuring transcription of a reporter gene. A suitable recombinant cell includes a reporter gene operatively connected to a cAMP-responsive promoter and a GHRH-responsive protein whose binding to GHRH induces the production of cAMP. GHRH present in an assay sample results in the GHRH-responsive protein activating the production of cAMP, which then activates the c-AMP-responsive promoter to express the reporter protein. The amount of reporter protein produced is quantitatively correlated to the amount of GHRH in the sample. In one embodiment, the protein is a cell surface receptor for GHRH.
