Phosphorylation of the human calcitonin receptor by multiple kinases is localized to the C-terminus.
The calcitonin receptor is a seven-transmembrane G-protein coupled receptor which is located on osteoclasts, in kidney, and in brain. The receptor signals through multiple pathways, including activation of adenylate cyclase, leading to inhibition of bone resorption. In the present study, we used antibodies raised against the C-terminus of the human calcitonin (CT) receptor to study receptor phosphorylation. In baby hamster kidney cells transfected with the human CT receptor, phosphorylation of the receptor increased approximately 2.5-fold after cells were treated with calcitonin, phorbol ester, forskolin, or calcitonin plus phorbol ester. Phosphorylation reached a maximum 20 minutes after treatment with sCT and half-maximal phosphorylation was observed at 0.1 nM sCT, a hormone concentration related to receptor occupancy. Digestion of the immunoprecipitated receptor with cyanogen bromide (CNBr) yielded a single 32P-labeled fragment which migrates at Mr 14 kD on gel electrophoresis. This corresponds to the predicted size of the CNBr fragment containing the C-terminal domain of the receptor. No 32P-labeled bands were observed for CNBr fragments predicted to contain the first, second, or third intracellular loops. An identical labeling pattern was seen with cells expressing an alternatively spliced isoform of the human receptor (insert-positive isoform). Phosphorylation of the receptor by phorbol ester and forskolin was further localized to a Mr 6 kD proteolytic fragment within the C-terminus. The protein kinase A and C inhibitors staurosporine, chelerythrine, and H-89 had little effect on CT-induced phosphorylation, suggesting that nonsecond messenger-activated kinases are involved in hormone-dependent CT receptor phosphorylation.
Nygaard
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SC
ZymoGenetics, Inc., Seattle, Washington 98102, U.S.A.
Kuestner
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Moore
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Stroop
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D005576:Colforsin; D006224:Cricetinae; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D018003:Receptors, Calcitonin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection
D010761:Phosphorus Radioisotopes; D018003:Receptors, Calcitonin; D005576:Colforsin; D002116:Calcitonin; D013755:Tetradecanoylphorbol Acetate; D003488:Cyanogen Bromide