HORMONE REGULATION OF PHOSPHOINOSITIDE HYDROLYSIS

A major mechanism of transmembrane signal transduction utilizes regulatory GTP-binding proteins to mediate events from receptor activation to the effector. Adenylyl cyclase, cyclic GMP phosphodiesterase and a number of ion channels are regulated by heterotrimeric G proteins. Increasing evidence indicates that another class of important enzymes, phospholipases, are also regulated through a receptor-G protein dependent mechanism. The identity and properties of the GTP-binding proteins which regulate phospholipase activity has eluded identification suggesting that regulation of this class of enzymes occurs through a novel G protein or novel G protein regulated mechanism. The phosphoinositide specific phospholipase C is linked to receptor mechanisms involved in elevation of cytosolic Ca2+ levels and increase in protein kinase C activity. The identity of phospholipase C and its regulatory GTP-binding protein (Gp) however has yet to be established. Guanine nucleotide-dependent inhibition of phospholipase C activity has been demonstrated in membranes, suggesting that phospholipase C activity may be regulated by both stimulatory and inhibitory G proteins. A major goal of the proposed studies is to purify the Gp-regulated PLC and to characterize its regulation by Gp. To accomplish these goals, conditions which solubilize a Gp-regulated phospholipase C activity from bovine brain membranes have been established. Purification of the solubilized activity results in the resolution of two distinct activities, a Gp-insensitive and Gp-sensitive phospholipase C. GTP-gamma-S promotes a marked increase in the Ca2+ sensitivity of the Gp regulated activity. This suggests that the Gp regulated phospholipase C is associated with its regulatory component and constitutes a source of the regulated phospholipase C. The properties of Gp will be characterized. The nature of the interaction between Gp and phospholipase C will be ascertained by hydrodynamic studies. The Gp regulated PLC will be used to purify or identify Gp. The interaction between these components will be studied in a reconstituted system. These studies will extend our knowledge concerning the mechanism of regulation of phospholipase C by GTP-binding proteins and provide insight into the regulation of this important class of enzymes.