Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of the human Ca²⁺ receptor: Characterization and use in studying concanavalin a inhibition

We generated monoclonal antibodies against two synthetic peptides corresponding to residues 214-235 (ADD) and 374-391 (LRG) of the human Ca²⁺ receptor (hCaR) extracellular domain (ECD). Although both antibodies reacted well with their respective immunizing peptides on peptide-based enzyme linked immunosorbent assay, ADD was much more strongly reactive with the hCaR than LRG in assays such as immunoblots done under denaturing conditions. The opposite pattern was seen in flow cytometry analysis of the native receptor stably expressed in transfected 293 cells. We speculate that the ADD epitope is unexposed in the native receptor while the reverse is true for the LRG epitope. The ability to measure cell surface expression of the hCaR under native conditions using flow cytometry with the LRG monoclonal allowed us to study the basis for Concanavalin A (Con A) inhibition of CaR activation by Ca²⁺. Our studies show that Con A inhibition is partially accounted for by receptor internalization but, additionally, Con A may prevent Ca²⁺ stimulation directly by binding to carbohydrate residues in the receptor ECD.