CCK activates RhoA and Rac1 differentially through Gα₁₃ and Gα_q in mouse pancreatic acini

Abstract

Cholecystokinin (CCK) has been shown to activate RhoA and Rac1, as well as reorganize the actin cytoskeleton and, thereby, modify acinar morphology and amylase secretion in mouse pancreatic acini. The aim of the present study was to determine which heterotrimeric G proteins activate RhoA and Rac1 upon CCK stimulation. Gα13, but not Gα12, was identified in mouse pancreatic acini by RT-PCR and Western blotting. Using specific assays for RhoA and Rac1 activation, we showed that only active Gα13 activated RhoA. By contrast, active Gα13 and Gαq, but not Gαs, slightly increased GTP-bound Rac1 levels. A greater increase in Rac1 activation was observed when active Gα13 and active Gαq were coexpressed. Gαi was not required for CCK-induced RhoA or Rac1 activation. The regulator of G protein signaling (RGS) domain of p115-Rho guanine nucleotide exchange factor (p115-RGS), a specific inhibitor of Gα12/13-mediated signaling, abolished CCK-stimulated RhoA activation. By contrast, both RGS-2, an inhibitor of Gαq, and p115-RGS abolished CCK-induced Rac1 activation, which was PLC pathway-independent. Active Gαq and Gα13, but not Gαs, induced morphological changes and actin redistribution similar to 1 nM CCK. CCK-induced actin cytoskeletal reorganization was inhibited by RGS-2, but not by p115-RGS, whereas CCK-induced amylase secretion was blocked by both inhibitors. Together, these findings indicate that, in mouse pancreatic acini, Gα13 links CCK stimulation to the activation of RhoA, whereas both Gα13 and Gαq link CCK stimulation to the activation of Rac1. CCK-induced actin cytoskeletal reorganization is mainly mediated by Gαq. By contrast, Gα13 and Gαq signaling are required for CCK-induced amylase secretion.

Balanced Tiam1-Rac1 and RhoA Drives Proliferation and Invasion of Pancreatic Cancer Cells

“…In most situations, rac1 and RhoA exhibit an antagonistic relationship at multiple levels and there is balance between the activities of rac1 and RhoA (22)(23)(24)(25). This opposition can be reciprocal and unidirectional, as observed in NIH3T3 cells, in which rac1 activation induces epithelial cell morphology, including cadherin-based junctions, accompanied by decreased RhoA activity.…”

Galpha 12/13

“…In this study CCK was shown to rapidly increase the expression of both α12 and α13 as well as increasing the association of RhoA and Vav2 with Gα 13 but not Gα 12 . In mouse pancreas and pancreatic acini both PCR and Western blotting revealed the presence of Gα 13 but not Gα 12 (29). In accord with previous studies (1), Gα 13 was associated with a membrane fraction in both control and stimulated acini.…”

“…Similar results had been reported earlier in intestinal smooth muscle cells expressing Gα13 (23) and in NIH 3T3 cells stabely transfected with CCKA receptors (19). A mutant form of p115-RGS (E29K) failed to modify CCK-induced RhoA activation (29). The effect of p115-RGS expression was shown to be specific for G 12/13 signaling as it had no effect on Ca 2+ mobilization or cAMP formation.…”

“…Constitutively active Gα 13 (Q226L) delivered by adenoviral vector was shown to activate RhoA similar to CCK in mouse acini and to alter the actin cytoskeleton leading to bleb formation (29). In this study, expression of a p115 RhoGEF RGS like domain (p115-RGS) abolished RhoA activation in response to CCK suggesting that the action of CCK receptors to activate RhoA was mediated by Gα 12/13 .…”

“…We have used antibodies from Santa Cruz to Gα 12 (sc-409) and Gα 13 (sc-410) and a rabbit polyclonal to Gα 12 from Abcam (ab35016) for Western blotting in our studies of mouse pancreas (29).…”