Mapping the Galpha13 binding interface of the rgRGS domain of p115RhoGEF.
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).
Chen
Zhe
Z
Department of Biochemistry, the University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Singer
William D
WD
Wells
Clark D
CD
Sprang
Stephen R
SR
Sternweis
Paul C
PC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D005822:Genetic Vectors; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006801:Humans; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D064067:Rho Guanine Nucleotide Exchange Factors; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D020710:RGS Proteins; D011994:Recombinant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13