Abstract
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.
Highlights
Heterotrimeric G proteins mediate signals from seven transmembrane receptors to a wide array of effectors, including adenylyl cyclase, ion channels, phospholipases, and the exchange factor p115 RhoGEF [1]
A chimeric protein, which contained the regulators of G protein signaling (RGS) region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by G␣13. These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of G␣13 to both its RGS and Dbl homology (DH) domains
Further deletion up to aa 17 or 21 resulted in fragments that did not express intact protein. This N-terminal region, which apparently interferes with protein stability, ends at or before amino acid 25, because a fragment of p115 RhoGEF encoded by aa 25–252 can be expressed at 100 –200 g/gram of E. coli
Summary
Heterotrimeric G proteins mediate signals from seven transmembrane receptors to a wide array of effectors, including adenylyl cyclase, ion channels, phospholipases, and the exchange factor p115 RhoGEF [1]. The smallest region capable of accelerating GTPase activity is called the RGS box This domain is characterized by strong primary sequence identity and structural similarity among the four RGS family members for which structures are known: RGS4 [18], GAIP [19], axin/conductin [20], and RGS9 [21]. LARG [22], PDZ RhoGEF [23], p115 RhoGEF [24], and GTRAP48 [25] are guanine nucleotide exchange factors for Rho; all share a highly conserved region that interacts with G␣12 and/or G␣13 [22, 23, 25, 26] This region invariantly lies N-terminal to the tandem Dbl homology (DH) [27] and Pleckstrin homology (PH) [28] domains found in all four proteins. The rgRGS domain within the N terminus of p115 RhoGEF was subsequently established as a GAP for G␣12 and
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