The Ca²⁺-sensing receptor couples to Gα_12/13to activate phospholipase D in Madin-Darby canine kidney cells

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gαito inhibit the activity of adenylyl cyclase and activate ERK, Gαqto stimulate phospholipase C and phospholipase A2, and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα12/13target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796Was a negative control, prelabeled these cells with [3H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that overexpress the CaRWTbut not the CaRR796W. Treatment of the cells with C3exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Gα12/13. 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 Gαior coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gαiand Gαq, and a p115RhoGEF construct containing the RGS domain for Gα12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWTinhibited 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 Gα12/13to regulate PLD via a Rho-dependent mechanism and does so independently of Gαiand Gαq. This suggests that the CaR may regulate cytoskeleton via Gα12/13, Rho, and PLD.