G protein signaling is determined by a balance of the rates of GTP binding and hydrolysis, the first catalyzed by receptors and the second accelerated by GTPase-activating proteins (GAPs). These rates also determine the rates of signal onset and termination. GAPs can thus sharpen signal termination upon removal of agonist, attenuate signal amplitude or both. It is uncertain, though, how G protein systems can generate either high or low amplitude signaling with either fast or slow response times. We propose that GAPs can sharpen signaling kinetics without inhibiting output by promoting receptor-G protein binding throughout the GTPase cycle, a process of """"""""kinetic scaffolding."""""""" Thus, if bound GTP is hydrolyzed fast, receptor will not have time to dissociate from 0-GTP and will, without delay, catalyze GDP/GTP exchange to reactivate the system. The goal of this project is to determine how the kinetic balance of the GTPase cycle determines the intensity, speed and specificity of signaling over multiple time scales and in response to multiple simultaneous inputs.1. We will determine the mechanism of integration of receptor-catalyzed activation and GAP-promoted deactivation that allows independent control of response times and signal outputs. We will relate the rates of intermediary GTPase cycle reactions, determined by quench flow and stopped flow assays, to the accumulation and turnover of receptor-G protein-GAP complexes measured by fluorescence resonance energy transfer between attached fluorophores. We will probe the apparently related mechanism by which GAPs enhance receptor-G protein selectivity. We will also determine how kinetic scaffolding modulates signaling by GBetagamma and, reciprocally, how Gbetagamma alters GAP function. 2. We will study the mechanism, regulatory significance and structural basis of the Gq GAP activity of phospholipase C-beta, the major Gq-regulated effector protein. Based on this work we will prepare mutants of Galphaq and or PLC-1 that do not GAP, and use these mutants to evaluate the importance of having GAP activity intrinsic to an effector protein in cellular signaling.
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