Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for G alpha(13) downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of G alpha(13), gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active G alpha(13). Rgnef co-immunoprecipitated with activated G alpha(13)Q226L but not G alpha(12)Q229L. The Rgnef C-terminal (CT, 1279 - 1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented G alpha(13)-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to G alpha(13). Point mutations of Rgnef-CT residues disrupt association with active G alpha(13) but not G alpha(q). These results show that Rgnef functions as an effector of G alpha(13) signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.