Intracellular retention and rapid degradation of human calcitonin receptors overexpressed in COS cells.
Overexpression of native and epitope-tagged human calcitonin (CT) receptors (hCTR-2) in COS-1 cells was performed to permit identification of the receptor protein and begin studies of receptor turnover. Data obtained with immunological techniques and cross-linking of radiolabeled salmon CT ([125I]sCT) revealed two forms of hCTR-2 in transfected cells: a larger, mature cell surface receptor (apparent size, 81 kDa) and a smaller, intracellular form (apparent size, 66 kDa). These conclusions are based on the following observations. 1) Only the larger hCTR-2 was visualized by cell surface [125I]sCT binding, whereas both species were identified by [125I]sCT binding to cell lysates. 2) Immunofluorescence studies with antibodies directed against the epitope confirmed the presence of cell surface and intracellular hCTR-2s; there were apparently many more receptors intracellularly than on the cell surface. 3) Both hCTR-2 forms were changed to a similar size of approximately 57-60 kDa by deglycosylation with endoglycosidase F; this size is consistent with that predicted by the amino acid sequence. Metabolic studies 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 cells leads to their intracellular retention and rapid degradation.
Dermer
S J
SJ
Cohen
D P
DP
Thaw
C N
CN
Nussenzveig
D R
DR
Gershengorn
M C
MC
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018003:Receptors, Calcitonin / Q000276:immunology / Q000378:metabolism*; D016324:Sequence Tagged Sites; D014162:Transfection
D000939:Epitopes; D018003:Receptors, Calcitonin