INTRACELLULAR RETENTION AND RAPID DEGRADATION OF HUMAN CALCITONIN RECEPTORS OVEREXPRESSED IN COS CELLS

Overexpression of native and epitope-tagged human calcitonin (CT) rece ptors (hCTR-2) in COS-1 cells was performed to permit identification o f the receptor protein and begin studies of receptor turnover. Data ob tained with immunological techniques and crosslinking of radiolabeled salmon CT ([I-125]sCT) revealed two forms of hCTR-2 in transfected cel ls: a larger, mature cell surface receptor (apparent size, 81 kDa) and a smaller, intracellular form (apparent size, 66 kDa). These conclusi ons are based on the following observations. 1) Only the larger hCTR-2 was visualized by cell surface [I-125]sCT binding, whereas both speci es were identified by [I-125]sCT binding to cell lysates. 2) Immunoflu orescence studies with antibodies directed against the epitope confirm ed the presence of cell surface and intracellular hCTR-2s; there were apparently many more receptors intracellularly than on the cell surfac e. 3) Both hCTR-2 forms were changed to a similar size of approximatel y 57-60 kDa by deglycosylation with endoglycosidase F; this size is co nsistent with that predicted by the amino acid sequence. Metabolic stu dies with radioactive amino acids labeled only the intracellular form. This immature form exhibited a rapid half-life of 30 min. We conclude that overexpression of native and epitope-tagged hCTR-2s in COS-1 cel ls leads to their intracellular retention and rapid degradation.