The Ras homology (Rho) guanine nucleotide exchange factor (GEF), p115RhoGEF, provides a direct link between the G-protein α subunit, α13, and the small GTPase Rho. In the present study, we demonstrate that activated mutants of α13 or α12, but not αq, promote the redistribution of p115RhoGEF from the cytoplasm to the plasma membrane (PM). We also show that the PM translocation of p115RhoGEF is promoted by stimulation of thromboxane A2 receptors. Furthermore, we define domains of p115RhoGEF required for its regulated PM recruitment. The RhoGEF RGS (regulators of G-protein signalling) domain of p115RhoGEF is required for PM recruitment, but it is not sufficient for strong α13-promoted PM recruitment, even though it strongly interacts with activated α13. We also identify the pleckstrin homology domain as essential for α13-mediated PM recruitment. An amino acid substitution of lysine to proline at position 677 in the pleckstrin homology domain of p115RhoGEF inhibits Rho-mediated gene transcription, but this mutation does not affect α13-mediated PM translocation of p115RhoGEF. The results suggest a mechanism whereby multiple signals contribute to regulated PM localization of p115RhoGEF.
- heterotrimeric G-protein
- plasma membrane translocation
- regulators of G-protein signalling
- signal transduction
Abbreviations used: α13QL (etc.), constitutively active α13 (etc.); COS, cellular origin of SV-40; DH, Dbl homology; DMEM, Dulbecco's modified Eagle's medium; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HA, haemagglutinin; LARG, leukaemia-associated RhoGEF; P fraction, particulate fraction; PDZ, post synaptic density protein, discs large protein, zonula occludens; PH, pleckstrin homology; PI 3-kinase, phosphoinositide 3-kinase; p115GFP, GFP-tagged p115RhoGEF; PM, plasma membrane; RGS, regulators of G-protein signalling; S fraction, soluble fraction; SRF, serum response factor; TP, thromboxane A2 receptor; wt, wild-type.
- The Biochemical Society, London ©2003