Interaction kinetics between p115-RhoGEF and Gα13 are determined by unique molecular interactions affecting agonist sensitivity

Abstract

The three RH-RhoGEFs (Guanine nucleotide exchange factors) p115-RhoGEF, LARG (leukemia-associated RhoGEF) and PDZ-RhoGEF link G-protein coupled receptors (GPCRs) with RhoA signaling through activation of Gα12/13. In order to find functional differences in signaling between the different RH-RhoGEFs we examined their interaction with Gα13 in high spatial and temporal resolution, utilizing a FRET-based single cell assay. We found that p115-RhoGEF interacts significantly shorter with Gα13 than LARG and PDZ-RhoGEF, while narrowing the structural basis for these differences down to a single amino acid in the rgRGS domain of p115-RhoGEF. The mutation of this amino acid led to an increased interaction time with Gα13 and an enhanced agonist sensitivity, comparable to LARG, while mutating the corresponding amino acid in Gα13 the same effect could be achieved. While the rgRGS domains of RH-RhoGEFs showed GAP (GTPase-activating protein) activity towards Gα13 in vitro, our approach suggests higher GAP activity of p115-RhoGEF in intact cells.

Allosteric Activation of RhoA Complexed with p115-RhoGEF Deciphered by Conformational Dynamics

“…31 The heterotrimeric G protein Gα 13 stimulates RH-RhoGEFs, leading to the activation of RhoA. 32,33 RhoGEFs fall into two different classes: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (DOCK) family of proteins. 34 The Dbl family of the GEF is a direct activator of the Rho family proteins.…”

“…26,47 One of the RH-RhoGEFs is the 115 kDa guanine nucleotide exchange factor (p115-RhoGEF, encoded by ARHGEF1). 33 Among its known roles are regulation of epithelial plasticity, 48 regulation of extracellular Ca 2+ -induced choline kinase activation and prostate cancer cell proliferation, 49 activating RhoA to support tight junction maintenance and remodeling by repairing localized leaks, 50 and recently platelet exocytosis. 51 In this work, we use MD simulations, allosteric communication, and essential dynamics analysis to study the structure and dynamics of the p115 RhoGEF when bound to RhoA− GDP and RhoA−GTP, with the aim of elucidating the activation mechanism of this key small GTPase by its GEF, how RhoA−GDP is initially recruited by the DH domain, and how RhoA−GTP is released from the DH domain after the catalytic exchange event.…”

“…The heterotrimeric G protein Gα 13 stimulates RH-RhoGEFs, leading to the activation of RhoA. , RhoGEFs fall into two different classes: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (DOCK) family of proteins . The Dbl family of the GEF is a direct activator of the Rho family proteins. − RH-RhoGEFs are a family of GEFs that contain the N-terminal RGS homology (RH) domain and the C-terminal Dbl homology (DH) and Pleckstrin homology (PH) domains (Figure c).…”

“…One of the RH-RhoGEFs is the 115 kDa guanine nucleotide exchange factor (p115-RhoGEF, encoded by ARHGEF1 ) . Among its known roles are regulation of epithelial plasticity, regulation of extracellular Ca 2+ -induced choline kinase activation and prostate cancer cell proliferation, activating RhoA to support tight junction maintenance and remodeling by repairing localized leaks, and recently platelet exocytosis …”