Receptor-stimulated, phospholipase C-catalyzed hydrolysis of phosphoinoistides represents a major signalling mechanism for hormones and neurotransmitters. While knowledge of the biochemical sequelae resulting from inositol-1,4,5 P3 and diacylglycerol formation has advanced markedly in recent years, the receptor-initiated trans-plasma membrane steps involved in the formation of these second messengers are not well understood. The long-term goal of this work is to identify the components of this membrane hormone signalling mechanism, and to describe in molecular terms the steps involved in their purposeful interaction. As with hormonal regulation of adenylate cyclase, a guanine nucleotide regulatory protein (G-protein) apparently subserves a necessary role in receptor-stimulated activation of phospholipase C. A major aim of this proposal is to identify and characterize this protein. Turkey erythrocyte membranes, which we recently have shown express a phospholipase C that is remarkably responsive to activators of G- proteins, will be used as a model membrane system. The properties of activation of phospholipase C by guanine nucleotides and by a putative P2-purinergic receptor will be examined, as will the potential regulation of the turkey erythrocyte enzyme by a pertussis or cholera toxin substrate. Activation of phospholipase C by reconstitution of GTP gamma S-pre-activated proteins will be used to identify the protein in turkey erythrocytes that activates phospholipase C and to compare the relative activities of G-proteins purified from turkey erythrocytes, and G alpha- 25,000, Gi, Go, and Gs purified from other sources. To compare the G-protein-mediated regulation of phospholipase C to other G- protein-regulated responses, the effects of purified beta gamma subunits on inositol phosphate formation will be examined as will the effects of the resolved alpha-subunit of any protein active in stimulation of phospholipase C. The observation that epidermal growth factor (EGF) receptors activate phospholipase C has added support to the idea that multiple mechanisms of regulation of phospholipase C exist. Experiments will be carried out to confirm preliminary data suggesting that the presence of guanine nucleotides is necessary for observation of EGF receptor stimulation of phospholipase C in membranes from (3H) inositol- labelled WB cells. Moreover, the guanine nucleotide selectively, substrate specificity and sensitivity to bacterial toxins of the phospholipase activity(s) stimulated by EGF and that stimulated by receptors for hormones, e.g. angiotensin II, that have been more closely associated with the phosphoinositide/Ca++ signalling response will be examined. The completion of the experiments should provide an identification and the initial characterization of the G-protein responsible for regulation of phospholipase C in at least one model membrane system and should provide considerable advances in delineating possible multiple mechanisms of hormmone-stimulated phospholipase C.
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