G-Protein Mediated Signalling

A major focus of research concerns signalling pathways that utilizecell surface receptors and membrane associated GTP-dependent proteins(G proteins). Thus, these G proteins mediate hormonal regulationof intracellular enzymes which produce various second messengers.G proteins have been implicated in the regulation of adenylylcyclase,phosphatidylinositol metabolism, arachidonic acid release, anda variety of ion channels. Two major goals have dominated thisarea in recent years. The first is to identify all of the potentialparticipants in these pathways; the second is to identify thecircuitry which defines interactions among all of these components.

A major approach we use is to identify and purify various componentsof these systems and test their mechanisms of interaction by reconstitutionin well defined milieus. Recent work has concentrated on the pathwayswhich regulate phospholipases. We have identified a new subfamilyof G protein a subunits, the aq family, which is capable of mediatingregulation of phospholipase C (PLC) and thus the generation oftwo second messengers, IP3 and diacylglycerol. We are currentlyexpressing native and mutated PLCb isozymes for structure/functionanalysis.

A second major focus has developed from attempts to examine regulationof phospholipase D (PLD) activity by G proteins. We have identifieda monomeric G protein, Arf, as a stimulator of this enzymaticactivity. The role of the Arf protein in vesicle mediated proteintraffic suggests that PLD and the subsequent formation of negativelycharged phosphatidic acid could play a key role in these processes.Current goals include purification of the Arf-sensitive PLD enzyme,identification of other cytosolic factors that regulate this activity,and attempts to demonstrate the functional relevance of this activityand its sequelae in membrane traffic and in the process of membranefusion in general.