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

Chunfa Huang, Kristine M. Hujer, Zhenzhen Wu, R. Tyler Miller

Research output: Contribution to journalArticle

61 Scopus citations

Abstract

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gαi to inhibit the activity of adenylyl cyclase and activate ERK, Gαq to 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 Gα 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 [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 CaRWT but not the CaRR796W. 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 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 Gαi or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gαi and Gαq, and a p115RhoGEF construct containing the RGS domain for Gα12/13). 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 Gα12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Gαi and Gαq. This suggests that the CaR may regulate cytoskeleton via Gα12/13, Rho, and PLD.

Original languageEnglish (US)
Pages (from-to)C22-C30
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number1 55-1
StatePublished - Jan 1 2004

Keywords

  • Calcium-sensing receptor
  • G proteins
  • RGS proteins

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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