Abstract
Structural requirements for function of the Rho GEF (guanine nucleotide exchange factor) regulator of G protein signaling (rgRGS) domains of p115RhoGEF and homologous exchange factors differ from those of the classical RGS domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS domain was undertaken to determine its functional interface with the Galpha(13) subunit. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Galpha(13) and the interactions of RGS4 and RGS9 with their Galpha substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Lack of sequence conservation within the N terminus may also explain the lack of GTPase-activating protein (GAP) activity in a subset of the rgRGS domains. For all mutations, loss of functional GAP activity is paralleled by decreases in binding to Galpha(13). The same mutations, when placed in the context of the p115RhoGEF molecule, produce deficiencies in GAP activity as observed with the rgRGS domain alone but show no attenuation of the regulation of Rho exchange activity by Galpha(13). This suggests that the rgRGS domain may serve a structural or allosteric role in the regulation of the nucleotide exchange activity of p115RhoGEF on Rho by Galpha(13).
Highlights
The intrinsic guanosine triphosphatase (GTPase) activity of the ␣ subunits of certain heterotrimeric guanine nucleotidebinding proteins (G proteins) can be stimulated by members of the regulators of G protein signaling (RGS)1 family [1,2,3]
The N terminus of p115RhoGEF is not present in the crystal structure, it is required for GTPase-activating protein (GAP) activity [15], and structural modeling of the rgRGS1⁄7G␣13 complex suggests that N-terminal residues of the regulator of G protein signaling (rgRGS) domain might interact with the helical domain and switch regions of G␣13 [26]
The mutagenesis experiments that we have conducted are consistent with the hypothesis that the G␣13 interaction surface of the RGS box region in p115RhoGEF is roughly similar to that observed in the structures of RGS4 and RGS9 bound to their G␣ substrates [5, 7]
Summary
The intrinsic guanosine triphosphatase (GTPase) activity of the ␣ subunits of certain heterotrimeric guanine nucleotidebinding proteins (G proteins) can be stimulated by members of the regulators of G protein signaling (RGS) family [1,2,3]. Two members of the p115RhoGEF family, GTRAP48 [13, 15] and PDZRhoGEF, bind to G␣13 but have little or no GAP activity; these form a distinct sequence subset with respect to both the RGS box domain and the N-terminal segment. Structural [5, 7] and mutagenesis (16 –19) studies have defined three distinct regions of the RGS domain which interact with G␣ and convey GAP activity These correspond to two surface polypeptide turns that join helical segments, together with the surface of the C-terminal ␣ helix of the RGS domain. The N terminus of p115RhoGEF is not present in the crystal structure, it is required for GAP activity [15], and structural modeling of the rgRGS1⁄7G␣13 complex suggests that N-terminal residues of the rgRGS domain might interact with the helical domain and switch regions of G␣13 [26]. Placement of the same mutations in the context of the p115RhoGEF molecule produces the same deficiencies in GAP activity as observed with the rgRGS domain alone but shows no attenuation of the regulation of Rho exchange activity by G␣13
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
