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, cati on-sensing receptor (CaR). We used microphysiometry to determine wheth er an extracellular cation-sensing mechanism exists in Madin-Darby can ine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellula r activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PC R) using nested CaR-specific primers, identification of an appropriate ly located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP phot oaffinity label [alpha-P-32]GTP azidoanalide (AA-GTP). After stimulati on with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for G(q/11)alpha) and G(i) a lpha family members. Gd3+ increased incorporation of AA-GTP into G(q/1 1)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 G d3+ on the G proteins were ruled out using partially purified mammalia n G proteins expressed in Escherichia coli or Sig cells. We conclude t hat 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).