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

Abstract

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gα_i to inhibit the activity of adenylyl cyclase and activate ERK, Gα_q to stimulate phospholipase C and phospholipase A₂, and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as 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 CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Gα_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 Gα_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gα_i and Gα_q, and a p115RhoGEF construct containing the RGS domain for Gα_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT 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 Gα_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Gα_i and Gα_q. This suggests that the CaR may regulate cytoskeleton via Gα_12/13, Rho, and PLD.