We propose to continue our studies of the biochemical and biophysical mechanisms used by G protein signaling modules to organize, integrate and transmit information. The diversity, number and ubiquity of G protein-coupled receptors (GPCRs) accounts both for their involvement in a huge number of diseases and for their being efficacious targets of numerous drugs. While all G protein modules contain the same basic components ?GPCR, heterotrimeric G protein, effector protein, GTPase-activating protein (GAP) ?and follow the same cycle of activation and deactivation, their detailed behaviors vary enormously. We propose to discover in quantitative terms how the underlying protein-protein interactions within a G protein module determine the amplitude, timing and specificity of its output. The dynamics of G protein signaling reflects multiple regulatory interactions among Go.and Gpy subunits, GPCR, GAP and effector. We will use kinetic and equilibrium methods to monitor the GTPase cycle reactions, conformational states of the proteins and protein-protein binding events. Our three specific aims are inter-related by this focus. 1. We will study the independent effects of receptors, GAPs and Gpy on the rates of G protein activation and deactivation, and the relation of these rates to the steady-state activation of G protein and effector. Experiments will utilize a reconstituted system of purified proteins at known concentrations, including fluorescent sensors of protein conformation and protein-protein binding. We will use a quantitative computational model of these events to guide experimental design and analysis, and to help interpret the complex behaviors of this system. Key findings will be qualitatively tested in cultured cells. We will focus on the mechanism(s) whereby GAPs modulate the speed of response to receptor input without significantly inhibiting signal output. 2. Receptor conformation is regulated by both agonist and G proteins, but the relationship between agonist binding, receptor conformation and G protein regulation remains unclear. We will use fluorescent reporters within ml muscarinic receptors to study their conformational changes in response to agonists, Ga and Cfty subunits. This study should identify the equilibrium poise and reaction rates among the receptor's conformational states and their energetic coupling to ligands and G proteins. 3. Gpy subunits are required for normal stimulation of G protein activation by receptors and regulate many effector proteins. G0Yalso inhibits GAP activity and, at high concentrations, inhibits receptor-mediated G protein activation. We will determine the mechanisms that coordinate these effects, focusing on the kinetics of the cooperative and competitive binding interactions among the proteins involved.
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