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

J Biol Chem. 2015 Jun 12;290(24):15197-209. doi: 10.1074/jbc.M114.628164. Epub 2015 Apr 28.

Abstract

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 Gα13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα13, 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 Gα13. Rgnef co-immunoprecipitated with activated Gα13Q226L but not Gα12Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα13. Point mutations of Rgnef-CT residues disrupt association with active Gα13 but not Gαq. These results show that Rgnef functions as an effector of Gα13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.

Keywords: G protein-coupled receptor (GPCR); Gastrin; cell signaling; guanine nucleotide exchange factor (GEF); heterotrimeric G protein; regulator of G protein signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Colonic Neoplasms / metabolism*
  • Colonic Neoplasms / pathology
  • Focal Adhesion Protein-Tyrosine Kinases / metabolism
  • GTP-Binding Protein alpha Subunits, G12-G13 / physiology*
  • Gastrins / physiology*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HEK293 Cells
  • Humans
  • Paxillin / chemistry
  • Paxillin / metabolism
  • Phosphorylation
  • Receptor, Cholecystokinin B / metabolism
  • Rho Guanine Nucleotide Exchange Factors / chemistry
  • Rho Guanine Nucleotide Exchange Factors / metabolism
  • Tyrosine / metabolism

Substances

  • Gastrins
  • Guanine Nucleotide Exchange Factors
  • Paxillin
  • Receptor, Cholecystokinin B
  • Rho Guanine Nucleotide Exchange Factors
  • p190RhoGEF protein, human
  • Tyrosine
  • Focal Adhesion Protein-Tyrosine Kinases
  • GTP-Binding Protein alpha Subunits, G12-G13

Associated data

  • PDB/3AB3
  • PDB/3CX8