Mechanisms for reversible regulation between G13 and Rho exchange factors.
The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.
Wells
Clark D
CD
Department of Pharmacology, University of Texas, Southwestern Medical Center at Dallas, Texas 75390-9041, USA.
Liu
Mu-Ya
MY
Jackson
Mandy
M
Gutowski
Stephen
S
Sternweis
Pamela M
PM
Rothstein
Jeffrey D
JD
Kozasa
Tohru
T
Sternweis
Paul C
PC
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017470:Receptors, Glutamate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors
C576600:ARHGEF1 protein, human; C577234:ARHGEF11 protein, human; D020662:Guanine Nucleotide Exchange Factors; D017470:Receptors, Glutamate; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D020962:Heterotrimeric GTP-Binding Proteins