The study of the structure and the function of GTP-binding protein which involved in the mechanism of TRH-sensitive phospholipase C activation

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The hypothalamic tripetide TRH (thyrotropin-releasing hormone) binds to specific high affinity receptors on target cells of the pituitary within seconds, stimulates the degradation of polyphosphoinositides by phospholipase C and the concomitant formation of inositol phosphates and diacylglycerol. Recently, guanine nucleotide-binding protein, G protein has been proposed to coupled cell-surface receptors to phospholipase C-mediated polyphosphoinositide breakdown. In order to determine the structure and the function of G protein coupled TRH-receptor, we have characterized the relationship between TRH-receptor and G protein in GH3 clonal pituitary cells and membranes which are frequently used to study for the mechanism of TRH action.We found that TRH stimultes the activity of phospholipase C using exogenous substrate [^3H]phosphatidylinositol-4,5 bisphosphate with the dependency of guanine nucleotide in these cell membranes. And also we found that the cholera toxin-treated membranes demonstrated a partial reduction in the activity of TRH-induced low Km GTPase activity and a 10-fold increase in the concentration of guanine nuclerotide required for a half-maxmimal effect in regulating the TRH-receptor affinity for TRH-ligand. These data suggested that cholera toxin may act directly on a G rotein that is associated with TRH-receptors. There have been one report that cholera toxin inhibits secretin-mediated increase in the hydrolysis of polyphosphoinositide by a cAMP-independent mechanism. Recently it was found that Gs, the stimulatory G protein for adenylate cyclase, can stimulete the activity of ca channel. Therefore, cholera toxin-sensitive G protein will be considered about a new role of the signal transduction system and our results will help to study these purpose.