A key step in cell migration is the dynamic formation and disassembly of adhesions at the front and the concomitant movement and release of adhesions in the rear of the cell. Fibroblasts maintained in the absence of serum have stable adhesions within the rear of the cell and exhibit reduced trailing-edge retraction resulting in an elongated cell phenotype. Addition of lysophosphatidic acid (LPA) induced the movement of adhesions and retraction of the trailing edge, thus mimicking tail retraction in a migrating cell. Focal adhesion kinase (FAK), guanine nucleotide exchange factors (GEF) for Rho and the Rho effector Rho kinase II (ROCKII) are crucial for the regulation of adhesion movement and trailing-edge retraction. Downregulation of FAK by small interfering RNAs or small hairpin RNAs blocked LPA-induced adhesion movement and restoration of cell shape. This phenotype was rescued by the ectopic expression of PDZ-RhoGEF or a RhoA-effector-domain mutant that activates ROCK. Knockdown of PDZ-RhoGEF or ROCKII inhibited LPA-induced trailing-edge retraction and adhesion movement. Moreover, overexpressed PDZ-RhoGEF co-immunoprecipitated with FAK and localized to FAK-containing adhesions. These studies support a model in which FAK and PDZ-RhoGEF cooperate to induce Rho/ROCKII-dependent focal adhesion movement and trailing-edge retraction in response to LPA.

Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.

Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGSRhoGEFs) directly link activated forms of the G12 family of heterotrimeric G protein α subunits to the small GTPase Rho. Stimulation of G 12/13 -coupled GPCRs or expression of constitutively activated forms of α 12 and α 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 α 13 -induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent α 12 -induced PM translocation of p115-RhoGEF. Additional differences between α 13 and α 12 in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated α 12 effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas α 13 only weakly does. On the other hand, α 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, α 12 is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of α 12/13 -mediated PM recruitment of p115-RhoGEF.

PDZ-RhoGEF is a member of the regulator of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein α subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561-585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as necessary for binding to actin and for co-localization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the Nterminus of the RhoGEF frabin, and, as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate a motif of LIxxFE, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure independent of its ability to activate RhoA. KeywordsG protein; Rho; guanine-nucleotide exchange factor; actin; localization Rho family GTPases are members of the Ras super family of monomeric G-proteins (1). The Rho family comprises six small GTPase subfamilies including Rho, Rac, Cdc42, Rnd, RhoBTB and RhoT/Miro (2). Out of these, the most well studied members include Rho (A, B and C), Rac (1 and 2) and Cdc42 proteins. Their roles in cell regulation include modulation of cytoskeletal structure, motility, cell division, gene transcription, vesicular transport and various enzymatic activities. As key regulators of the actin cytoskeleton, in fibroblasts, RhoA induces the formation of actin stress fibers and focal adhesions, Rac1 stimulates the protrusion of lamellipodia and membrane ruffles and Cdc42 promotes extension of filopodia and actin microspikes (3)(4)(5). Rho GTPases are found in all eukaryotic cells and so far twenty-two mammalian genes encoding Rho GTPases have been described (6).+ This work was supported by NIH grant GM62884 (P.B.W.). *Corresponding Author: Philip B. Wedegaertner, Ph.D., Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 S. 10 th St., 839 BLSB, Philadelphia, PA 19107, tel: 215-503-3137, fax: 215-923-2117, P_Wedegaertner@mail.jci.tju.edu. Supporting information available: A figure showing an actin polymerization assay is available as Supplemental Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 ...