title: Differences in Galpha12- and Galpha13-mediated plasma membrane recruitment of p115-RhoGEF.
abstract: Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein alpha subunits to the small GTPase Rho. Stimulation of G(12/13)-coupled GPCRs or expression of constitutively activated forms of alpha(12) and alpha(13) has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for alpha(13)-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent alpha(12)-induced PM translocation of p115-RhoGEF. Additional differences between alpha(13) and alpha(12) in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated alpha(12) effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas alpha(13) only weakly does. On the other hand, alpha(13) strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, alpha(12) is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of alpha(12/13)-mediated PM recruitment of p115-RhoGEF.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Raja
authors.initials: R
authors.lastname: Bhattacharyya
authors.forename: Jayashree
authors.initials: J
authors.lastname: Banerjee
authors.forename: Kamel
authors.initials: K
authors.lastname: Khalili
authors.forename: Philip B
authors.initials: PB
authors.lastname: Wedegaertner
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C576600:ARHGEF1 protein, human; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020741:rho GTP-Binding Proteins