The Rho family of G proteins regulate diverse cellular processes involving coordinate alterations in cytoskeletal rearrangements and transcriptional control, and ultimately are necessary for processes as varied as chemotaxis, phagocytosis, and cellular growth and differentiation. Conversely the aberrant functioning of Rho-family G proteins promotes various malignancies from irregular development to cellular transformation and oncogenesis. A large and diverse class of guanine nucleotide exchange factors related to Db1 control the regulated activation of Rho-family G proteins by facilitating the release of GDP bound to inactive G proteins and catalyzing the concomitant loading of GTP to produce G proteins active in downstream signaling. Consequently, dysfunctional constitutive activation of Dbl-related GEFs abnormally elevates intracellular concentrations of active, GTP-bound G proteins contributing to tumorigenesis and developmental disorders. Although GEFs specific for Rho-family G proteins vary greatly in size and domain architecture, all contain a Db1-homology domain (DH) invariantly associated with a juxtaposed, C-terminal pleckstrin homology domain (PH). DH domains catalyze guanine nucleotide exchange, while functions for the associated PH domains are less well defined and more variable. This proposal describes experiments designed to illuminate, at atomic resolution, the conserved mechanism of guanine nucleotide exchange catalyzed by Dbl-related GEFs. We have recently solved the crystal structures of the DH/PH fragments of Tiam1 and Dbs in complex with their cognate G proteins, Rac1 and Cdc42, respectively. These structures form the basis for understanding guanine nucleotide exchange catalyzed by Dbl-related GEFs and guide many of the biophysical, biochemical, and in vivo experiments described. Complementary aspects of this proposal are designed to elucidate mechanistic details required for efficient guanine nucleotide exchange catalyzed by Dbl-related GEFs, including assigning specific functions to PH domains invariantly associated with DH domains. It is anticipated that this information will be invaluable for understanding and controlling the activation of Rho-family G proteins culminating in the amelioration of pathologies associated with constitutively active G proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062299-04
Application #
6747897
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Anderson, Richard A
Project Start
2001-06-01
Project End
2006-06-30
Budget Start
2004-06-01
Budget End
2006-06-30
Support Year
4
Fiscal Year
2004
Total Cost
$251,996
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Cherkis, Karen A; Temple, Brenda R S; Chung, Eui-Hwan et al. (2012) AvrRpm1 missense mutations weakly activate RPS2-mediated immune response in Arabidopsis thaliana. PLoS One 7:e42633
Klein, Ryan R; Bourdon, David M; Costales, Chester L et al. (2011) Direct activation of human phospholipase C by its well known inhibitor u73122. J Biol Chem 286:12407-16
Hamel, Brant; Monaghan-Benson, Elizabeth; Rojas, Rafael J et al. (2011) SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC. J Biol Chem 286:12141-8
Huang, Weigang; Hicks, Stephanie N; Sondek, John et al. (2011) A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes. ACS Chem Biol 6:223-8
Gresset, Aurelie; Hicks, Stephanie N; Harden, T Kendall et al. (2010) Mechanism of phosphorylation-induced activation of phospholipase C-gamma isozymes. J Biol Chem 285:35836-47
Waldo, Gary L; Ricks, Tiffany K; Hicks, Stephanie N et al. (2010) Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex. Science 330:974-80
Yohe, Marielle E; Rossman, Kent; Sondek, John (2008) Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins. Biochemistry 47:6827-39
Mitin, Natalia; Betts, Laurie; Yohe, Marielle E et al. (2007) Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nat Struct Mol Biol 14:814-23
Rojas, Rafael J; Yohe, Marielle E; Gershburg, Svetlana et al. (2007) Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain. J Biol Chem 282:29201-10
Chhatriwala, Mariya K; Betts, Laurie; Worthylake, David K et al. (2007) The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. J Mol Biol 368:1307-20

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