MOLECULAR CHARACTERIZATION OF GNRH RECEPTORS

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Gonadotropin-releasing hormone (GnRH), a hypothalamic decapeptide, is the key neural regulator of the reproductive process. Its receptor is located in the cell membrane of anterior pituitary gonadotropes where it mediates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In addition to anterior pituitary, GnRH receptors are reported to be present in the ovary, testis, placenta and brain. In recent years, numerous investigators also have found GnRH receptors in various human tumors (breast, colon, prostate, ovarian, uterine and pancreatic), whereas under normal conditions GnRH receptors are not detected in these tissues. However, the role of GnRH receptors in cancer is unclear. The broad, long- term objective of the proposed studies is to investigate the mechanism of LH release from gonadotropes, and the role of GnRH receptors in cancer. More specifically, for this grant application our efforts will be directed toward the molecular characterization of GnRH receptors(s) cloned from the human anterior pituitary gland and their regulation in normal and malignant tissues. We have isolated a clone from a human anterior pituitary cDNA library which is composed of 1557 nucleotides. It encodes a protein with topology similar to that of other G-protein coupled 7- transmembrane receptors. Binding studies of the cloned receptor demonstrate a high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot analysis of human pituitary RNA reveals the presence of 3 hGnRH-receptor mRNAs and Southern blot analysis of human genomic DNA suggests the existence of more than one GnRH receptor gene. Reverse transcriptase/PCR analysis of RNA revealed that the receptor mRNA is expressed in pituitary, ovary, testis, adrenal, breast, breast tumor, prostate, prostate tumor, and breast tumor cell line (MCF-7). The specific aims of the proposal are to: clone, sequence, express and characterize GnRH receptor cDNAs for all three mRNAs; to clone, sequence, and characterize all the genes encoding the GnRH- receptors; to determine the presence and amount of each receptor mRNA in normal and malignant tissues; to prepare monoclonal antibodies to the receptor(s) for detection and measurement of the amount of receptor protein in breast and prostate cancers; to study the effects of monoclonal antibodies on growth of breast and prostate tumor cells in culture and; to localize the gene for each receptor(s) on the chromosome. These studies should provide extensive new information about the potential role of GnRH- receptors in hormonally-dependent cancers.