COUPLING OF GONADOTROPIN-RELEASING-HORMONE RECEPTOR TO GI PROTEIN IN HUMAN REPRODUCTIVE-TRACT TUMORS

The signaling pathway by which GnRH acts in peripheral tumors is disti nct from that in the anterior pituitary. We attempted to identify the guanosine triphosphate (GTP)-binding protein (G protein) subtypes link ed to GnRH receptor in the genital tract tumor membranes. Surgically r emoved ovarian carcinomas and uterine leiomyosarcomas were screened fo r GnRH receptor expression before plasma membrane isolation. The G alp ha i was detected by immunoblotting of membrane extracts with specific antibody and pertussis toxin-catalyzed ADP-ribosylation from nicotina mide adenine dinucleotide. Membrane phosphotyrosine phosphatase activi ty was determined as a GnRH-sensitive membrane event using synthetic s ubstrate p-nitrophenyl in a spectrophotometric assay. Pertussis toxin, but not cholera toxin, brought about ADP-ribosylation of an immunodet ected G alpha i of 41 kDa in the GnRH receptor-positive tumor membrane . incubation with a GnRH analog and GTP decreased the ADP-ribosylation activity in a dose-dependent manner; a half-maximal effect occurred w ith 30 nmol/L buserelin (P < 0.01). The apparent inhibition by GnRH of the ADP-ribosylation demonstrated that GnRH resolved the alpha-subuni t of the Gi to GTP-bound form in the membranes. The action of GnRH was neutralized by a competitive antagonist, antide. Pretreatment of the membrane with the pertussis toxin completely inhibited GnRH-sensitive phosphotyrosine phosphatase activity (P < 0.01). These data demonstrat e the coupling of GnRH receptor to Gi protein subfamily. The Gi which couples GnRH receptor to the effector may define the difference of res ponses by peripheral tumor and the anterior pituitary.