Strategies are proposed for the elucidation of mechanisms by which a large family of membrane-associated proteins, G protein, mediate regulation of intracellular processes by extracellular signals (hormones, neurotransmitters, light, etc.). This family includes the Gs and Gt proteins which stimulate adenylyl cyclase and the visual cGMP- phosphodiesterase, respectively. Other G proteins are implicated in the regulation of phospholipase activities and ion channels. They mediate hormonal regulation of inositol polyphosphates, diacylglycerol, arachidonic acid, membrane potential, and Ca2+ influx. Three major questions are addressed. First, how are more that 100 receptors, 15 or more, and several regulated activities linked together? Second, what is the specificity of subunit interaction within the G protein family? Third, how extensive is the spectrum of regulation of the G proteins? The first question is addressed in part by attempts to reconstitute regulation of signalling pathways with purified G proteins. A specific focus will be on the characterization of a new G protein, Gq, and other similar proteins that are not substrates for bacterial toxins. Receptor interaction will be examined in membranes as well as by reconstitution with purified receptors. Functional studies will focus on the regulation of phospholipases. More general techniques will use immobilized alpha and betagamma subunit matrices in attempts to isolate, and thus identify regulated proteins. Interaction among unique G protein subunits will be examined in detail. This will be supported by the preparation of specific complexes of the beta and gamma subunits through expression in a baculovirus system. The interaction of these complexes with individual alpha subunits, their role in receptor interaction, and their potential regulation of effector molecules will be examined. Post-translational modification, which effects the functional interaction of the alpha and betagamma subunits with each other and effectors, will be examined further. This is facilitated by isolation of labeled subunits with affinity matrices. The potential spectrum of regulation by G proteins will be examined be characterizing other proteins with which they interact. Strategies for exploiting subunit affinity columns in this endeavor are proposed. One focus is on an 80 kDa protein that binds to the betagamma subunits in the presence of Ca2+. In summary, this proposal examines several aspects of G protein regulation. It will help define the specificity and action of these common signalling pathways for hormones.
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