DIMERIZATION OF THE EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CAR) ON THE CELL-SURFACE OF CAR-TRANSFECTED HEK293 CELLS

The extracellular calcium (Ca-o(2+))-sensing receptor (CaR) is a G pro tein-coupled receptor that plays important roles in calcium homeostasi s, In this study, we employed epitope tagging, cell-surface biotinylat ion, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surfa ce proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa, Most of the se high molecular mass forms of the receptor could be converted to a s ingle monomeric species at 160 kDa under reducing conditions. This res ult suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitiv e to reducing agents. Consistent with this hypothesis, use of a cell-s urface cross-linking agent substantially increases the proportion of t he putative dimeric CaR at 280 kDa relative to the monomeric form of t he receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected an d co-immunoprecipitated the wild type, full-length receptor and a trun cated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the ce ll surface of transfected HEK293 cells in the form of a dimer.