THYROTROPIN-RELEASING-HORMONE AND GONADOTROPIN-RELEASING-HORMONE RECEPTORS ACTIVATE PHOSPHOLIPASE-C BY COUPLING TO THE GUANOSINE TRIPHOSPHATE-BINDING PROTEIN-GQ AND PROTEIN-G11

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alphabetagamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (G(p)) was suggested to mediate receptor regulation. The required G(p) protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the G(q) subfamily (alpha(q) and alpha11) which regulate the activity of the beta1 isoenzyme of PLC. G(q) and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common G(q/11) alpha-subunit carboxy-terminal sequence was generated and shown to react with unique 42-kilodalton G(qalpha) and 43-kilodalton G11alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alphaT3-1 pituitary cells. The G(q/11alpha) peptide antibody was shown to immunodeplete the G(p) activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta1. In addition, the immunoglobulin G fraction of G(q/11alpha) peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alphaT3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a G(p) protein(s) which corresponds to G(q), G11 or both.