Characterization of G alpha 13-dependent plasma membrane recruitment of p115RhoGEF.
The Ras homology (Rho) guanine nucleotide exchange factor (GEF), p115RhoGEF, provides a direct link between the G-protein alpha subunit, alpha(13), and the small GTPase Rho. In the present study, we demonstrate that activated mutants of alpha(13) or alpha(12), but not alpha(q), 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 A(2) 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 alpha(13)-promoted PM recruitment, even though it strongly interacts with activated alpha(13). We also identify the pleckstrin homology domain as essential for alpha(13)-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 alpha(13)-mediated PM translocation of p115RhoGEF. The results suggest a mechanism whereby multiple signals contribute to regulated PM localization of p115RhoGEF.
Bhattacharyya
Raja
R
Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., 839 BLSB, Philadelphia, PA 19107, USA.
Wedegaertner
Philip B
PB
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction
D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D044365:GTP-Binding Protein alpha Subunits, G12-G13