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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030355-25
Application #
6930983
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Dunsmore, Sarah
Project Start
1981-08-01
Project End
2006-12-31
Budget Start
2005-08-01
Budget End
2006-12-31
Support Year
25
Fiscal Year
2005
Total Cost
$390,000
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Navaratnarajah, Punya; Gershenson, Anne; Ross, Elliott M (2017) The binding of activated G?q to phospholipase C-? exhibits anomalous affinity. J Biol Chem 292:16787-16801
Kadamur, Ganesh; Ross, Elliott M (2016) Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-? Exposes a G?? Protein Binding Site. J Biol Chem 291:11394-406
Dyachok, Julia; Earnest, Svetlana; Iturraran, Erica N et al. (2016) Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism. J Biol Chem 291:22414-22426
Wauson, Eric M; Guerra, Marcy L; Dyachok, Julia et al. (2015) Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells. Mol Endocrinol 29:1114-22
Ross, Elliott M (2014) G Protein-coupled receptors: Multi-turnover GDP/GTP exchange catalysis on heterotrimeric G proteins. Cell Logist 4:e29391
Kadamur, Ganesh; Ross, Elliott M (2013) Mammalian phospholipase C. Annu Rev Physiol 75:127-54
Chang, Seungwoo; Ross, Elliott M (2012) Activation biosensor for G protein-coupled receptors: a FRET-based m1 muscarinic activation sensor that regulates G(q). PLoS One 7:e45651
An, Sung-Wan; Cha, Seung-Kuy; Yoon, Joonho et al. (2011) WNK1 promotes PIP? synthesis to coordinate growth factor and GPCR-Gq signaling. Curr Biol 21:1979-87
Rebres, Robert A; Roach, Tamara I A; Fraser, Iain D C et al. (2011) Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q. J Biol Chem 286:942-51
Philip, Finly; Kadamur, Ganesh; Silos, Rosa Gonzalez et al. (2010) Synergistic activation of phospholipase C-beta3 by Galpha(q) and Gbetagamma describes a simple two-state coincidence detector. Curr Biol 20:1327-35

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