Identification of Potential Mechanisms for Regulation of p115 RhoGEF through Analysis of Endogenous and Mutant Forms of the Exchange Factor

Clark Wells, Stephen Gutowski, Gideon Bollag, Paul C. Sternweis

Research output: Contribution to journalArticle

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Abstract

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p 115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by Gα13. Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p115 Rho-GEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252-288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p115 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sphingosine 1-phosphate was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.

Original languageEnglish (US)
Pages (from-to)28897-28905
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number31
DOIs
StatePublished - Aug 3 2001
Externally publishedYes

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Rho Guanine Nucleotide Exchange Factors
G-Protein-Coupled Receptors
Membranes
Cytosol
Amino Acids
rho GTP-Binding Proteins
Ion exchange
Thermodynamic properties
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of Potential Mechanisms for Regulation of p115 RhoGEF through Analysis of Endogenous and Mutant Forms of the Exchange Factor. / Wells, Clark; Gutowski, Stephen; Bollag, Gideon; Sternweis, Paul C.

In: Journal of Biological Chemistry, Vol. 276, No. 31, 03.08.2001, p. 28897-28905.

Research output: Contribution to journalArticle

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