Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-P-32]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for G(q/11)alpha) and G(i) alpha family members. Gd3+ increased incorporation of AA-GTP into G(q/11)alpha precipitates by 146 +/- 48% and into G alpha(i-2) and G alpha(i-3) to a lesser extent but not into G alpha(i-1). Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sig cells. We conclude that MDCK cells possess a cell-surface CaR that activates G(q/11)alpha, G alpha(i-2), and G alpha(i-3) but not G alpha(i-1).