CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells.
Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G13- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active alpha-subunits; Galpha13 and Galpha12 but not Galphaq induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G12/13 inhibited active alpha12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit Gq, had no effect. Cotransfection with plasmids coding for the G protein alpha-subunit carboxy-terminal peptide from alpha13 and, to a lesser extent alpha12, also inhibited the effect of CCK, whereas the peptide from alphaq did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G13, leading to rearrangement of the actin cytoskeleton.
Le Page
Sophie L
SL
Department. of Molecular & Integrative Physiology, University of Michigan, 7744 Medical Science II, Ann Arbor, MI 48109-0622, USA.
Bi
Yan
Y
Williams
John A
JA
D000818:Animals; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D051381:Rats; D043886:Receptor, Cholecystokinin A / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D043886:Receptor, Cholecystokinin A; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein