EVIDENCE FOR TIGHT COUPLING OF GONADOTROPIN-RELEASING-HORMONE RECEPTORS TO PHOSPHATIDYLINOSITOL KINASE IN PLASMA-MEMBRANE FROM OVARIAN CARCINOMAS

Gonadotropin-releasing hormone (Gn-RH) analogs inhibit ovarian cancer cell proliferation in vivo and in vitro. To examine whether Gn-RH rece ptor (Gn-RHR) mediates direct antiproliferative effects, we attempted to determine inhibitory regulation by Gn-RH of phosphatidylinositol (P tdIns) kinase activity, known to stimulate mitogenic response, in plas ma membranes isolated from ovarian carcinoma samples. Ovarian carcinom as surgically removed and cloned cell line SK-OV3 had been screened fo r Gn-RHR expression prior to plasma membrane isolation. PtdIns kinase activity was measured as phosphorylation of exogenous substrate PtdIns by the purified plasma membranes. Incubation of the plasma membranes isolated from Gn-RHR-positive specimens with [gamma-P-32]ATP and PtdIn s caused [P-32]phosphate incorporation into PtdIns phosphate (PtdInsP) in a time-dependent manner. Concomitant exposure of the membrane prep arations to Gn-RH analog buserelin (1 mu M) led to a 70% inhibition of the PtdInsP production, when compared to control. After 10 or 15 min of an initial incubation, the addition of analog resulted in similar s uppression of PtdIns phosphorylation. This inhibition was dependent on the buserelin dose, and a half-maximal effect occurred at a concentra tion 0.1 to 1 nM of buserelin. Degradation of the produced PtdInsP in the plasma membranes was not affected by the Gn-RH analog. Similar inh ibition of PtdIns kinase activities was observed in membranes prepared from cells that had been pretreated with buserelin (1 mu M) for 48 hr prior to assay. These findings demonstrate that PtdIns kinase activit y is suppressed by Gn-RH analog in plasma membrane isolated from GnRHR -expressing ovarian carcinomas, suggesting a tight coupling of Gn-RHR to PtdIns. The inhibition of membrane-associated PtdIns kinase by Gn-R HR occupancy may mediate the antimitogenic action of the hormone on hu man ovarian carcinomas. (C) 1995 Academic Press, Inc.