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Monoclonal Antibodies Against Synthetic Peptides Corresponding to the Extracellular Domain of the Human Ca 2+ Receptor: Characterization and Use in Studying Concanavalin A Inhibition
Author(s) -
Goldsmith Paul K.,
Fan Gaofeng,
Miller Jeffery L.,
Rogers Kimberly V.,
Spiegel Allen M.
Publication year - 1997
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.1997.12.11.1780
Subject(s) - monoclonal antibody , epitope , internalization , concanavalin a , receptor , microbiology and biotechnology , flow cytometry , extracellular , antibody , chemistry , transfection , biology , biochemistry , in vitro , immunology , gene
We generated monoclonal antibodies against two synthetic peptides corresponding to residues 214–235 (ADD) and 374–391 (LRG) of the human Ca 2+ 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 Ca 2+ . Our studies show that Con A inhibition is partially accounted for by receptor internalization but, additionally, Con A may prevent Ca 2+ stimulation directly by binding to carbohydrate residues in the receptor ECD.