The Ca2+-sensing receptor couples to Gα12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Galpha(i) to inhibit the activity of adenylyl cyclase and activate ERK, Galpha(q) to stimulate phospholipase C and phospholipase A(2), and Gbetagamma to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Galpha(12/13), we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Galpha(12/13) target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W as a negative control, prelabeled these cells with [H-3] palmitic acid, and measured CaR-stimulated PLD activity as the formation of [H-3] phosphatidylethanol (PEt). The formation of [H-3] PEt increased in a time-dependent manner in the cells that overexpress the CaRWT but not the CaRR796W. Treatment of the cells with C-3 exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Galpha(12/13). To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Galpha(i) or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Galpha(i) and Galpha(q), and a p115RhoGEF construct containing the RGS domain for Galpha(12/13)). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to Galpha(12/13) to regulate PLD via a Rho-dependent mechanism and does so independently of Galpha(i) and Galpha(q). This suggests that the CaR may regulate cytoskeleton via Galpha(12/13), Rho, and PLD.