We propose to study the biochemical mechanisms that underlie the timing and integration of G protein-mediated signals in cells. G protein signaling modules control diverse cellular functions in response to equally diverse inputs. Consequently, G protein signaling is involved in many disease processes, and is the target of a huge number of drugs. G protein modules consist of a conserved group of interacting proteins: receptors, G protein Ga and Gbg subunits, GTPase activating proteins (GAP) and effector proteins. A mammalian cell typically expresses about 30 G protein-coupled receptors, half-dozen G proteins and GAPs and a dozen effectors. Mechanism of action is conserved, but varies quantitatively to allow different cells to respond to extracellular signals with a wide variety of kinetic patterns, intracellular outputs and modes of signal integration. Output from a G protein module quantitatively reflects a balance of receptor-catalyzed G protein activation and GAP-promoted deactivation. The rates of signal initiation and termination thus seem linked to the level of output, but cells can control response kinetics and response levels independently. We developed a quantitative framework for analyzing how receptors and GAPs interact to solve this problem. We will test mechanisms of receptor-GAP interaction and evaluate how and when each contributes to the temporal control of signaling. They include the ability of GAPs to stabilize the association of receptors and G proteins and to directly potentiate receptor function. We recently discovered that Gaq and Gbg, each of which stimulates phospholipase C-bs (PLC- bs) in response to different receptors, together stimulate the PLC-b3 isoform with strong synergism, 10 times the sum of the activities evoked by each subunit individually. Gaq-Gbg synergism is observed in diverse animal cells. We showed that Gaq-Gbg synergism can be explained by a classical two-state allosteric model, and we propose to test the physical basis of that interaction. Further, this model predicts that any two-state enzyme that is stimulated by two different ligands will display significant synergism only if its basal activity is very low, <0.1% of maximum. We will test this prediction by evaluating constitutively active PLC-b3 mutants for loss of synergism and constitutively deactivated PLC- b2 mutants, which should acquire synergistic regulation. We will also use this prediction to search for novel regulatory enzymes that can act as coincidence detectors for two or more ligands.

Public Health Relevance

The proposed research aims to expand our understanding of how cells use simple chemical reactions to amplify and integrate information. The relevance, or applied value, of studying G protein signaling mechanisms lies in the ubiquity of G protein modules among all animals, plants and fungi;the variety and importance of the physiology that they control;the numerous diseases in which they are involved;and the huge number of drugs that act on them. The goal of the proposed research is to develop quantitative understanding of the mechanisms that control cellular information processing by G protein pathways. Such understanding will both help us analyze their malfunction in disease and inform their therapeutic manipulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM030355-30A1
Application #
8236486
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
1981-08-01
Project End
2015-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
30
Fiscal Year
2012
Total Cost
$396,666
Indirect Cost
$146,666
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
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
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
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
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
Ross, Elliott M (2008) Coordinating speed and amplitude in G-protein signaling. Curr Biol 18:R777-R783
Turcotte, Marc; Tang, Wei; Ross, Elliott M (2008) Coordinate regulation of G protein signaling via dynamic interactions of receptor and GAP. PLoS Comput Biol 4:e1000148
Ross, Elliott M; Mateu, Dania; Gomes, Aldrin V et al. (2006) Structural determinants for phosphatidic acid regulation of phospholipase C-beta1. J Biol Chem 281:33087-94
Tang, Wei; Tu, Yaping; Nayak, Surendra K et al. (2006) Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor. J Biol Chem 281:4746-53
Carrasco, G A; Barker, S A; Zhang, Y et al. (2004) Estrogen treatment increases the levels of regulator of G protein signaling-Z1 in the hypothalamic paraventricular nucleus: possible role in desensitization of 5-hydroxytryptamine1A receptors. Neuroscience 127:261-7

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