I propose to study the mechanism of regulation of G proteins by receptors; the structural determinants of receptor function, specificity and regulation; and the organization of G protein-mediated signaling pathways. (1) We will investigate the mechanism of action of a cytoplasmic C-terminal domain that alters several aspects of receptors' activity and regulation, probably by anchoring receptor to a fixed structure at or on the plasma membrane. We will evaluate similar domains on other receptors and attempt to purify the protein(s) to which this domain binds. (2) We recently found that the m1 muscarinic receptor regulates phospholipase C activity through Gq and that PLC-beta1 promotes the deactivation of Gq by stimulating the GTPase activity of Gq. We will characterize this feedback regulation in receptor-Gq-PLC vesicles and relate it kinetically to the overall activation of the Gq pathway. We will also determine if other G protein-regulated effectors have GTPase- stimulating activity. (3) We will continue to study the mechanisms by which receptors selectively regulate specific G proteins. We will define the important structural features of the selectivity-determining domains that we have identified in the receptors' second and third cytoplasmic loops. We will use defined, recombinant betagamma subunit dimers purified from Baculovirus-infected Sf9 cells to analyze the contribution of individual beta and gamma subunits to receptor-G protein selectivity. (4) We will evaluate the extent of Gs-independent signaling by the beta- adrenergic receptor in Gs-deficient cyc- cells and explore its mechanism. We will determine the pathway by which this receptor stimulates the Na+/H+ exchanger, a widespread beta-adrenergic response that does not require Gs or cyclic AMP. We will focus on whether stimulation is direct or second messenger-mediated. We will evaluate the ability of the receptor to regulate Gq and Gi both in cyc- cells and in vitro. Receptor-Gq coupling will be evaluated as a possible mechanism for beta-adrenergic regulation of the exchanger. (5) We will continue studying mutations of the beta-adrenergic receptor that improves its expression. We will collaborate with Richard Henderson on the receptor's large scale purification, stabilization and crystallization.
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