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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.

Stable transfectants expressing a recombinant human calcitonin receptor respond to calcitonin via increased cyclic adenosine 3',5' monophosphate (cAMP, EC50 = 0.06 nM salmon calcitonin [sCT]) and a transient mobilization of intracellular calcium ([Ca2+]i) coincident with turnover of inositol phosphate (IP; EC50 = 6 nM sCT). Millimolar increases in extracellular calcium ([Ca2+]o, EC50 = 8 mM) cause a rapid elevation in [Ca2+]i after a calcitonin dose-dependent pretreatment of cells (pretreatment EC50 = 0.2 nM sCT). Cells exhibit persistent sensitivity to increased [Ca2+]o up to 3 h after hormone exposure and even after multiple cycles of increased [Ca2+]o followed by wash. Calcitonin pretreatment of cells also allows apparent influx of elevated extracellular strontium and manganese, but little or no effect is observed on addition of barium, cadmium, or lanthanum. Human amylin (100 nM) causes a rapid and transient increase in [Ca2+]i comparable to that of calcitonin; however, no significant response to increased [Ca2+]o is observed after amylin addition. Human calcitonin gene-related product (hCGRP) (300 nM) and forskolin do not increase [Ca2+]i or activate a sensitivity to increased [Ca2+]o. Nevertheless, human amylin and human calcitonin gene-related product (hCGRP) activate adenylate cyclase with EC50s of 0.7 nM and 8 nM, respectively. The calcium-channel drugs verapamil, BAY K 8644, diltiazem, and nifedipine have little effect on [Ca2+]i increases. The calcitonin-induced transient mobilization of calcium is inhibited by treatment of cells with cholera toxin or 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8); whereas, the response to subsequent increased [Ca2+]o is inhibited by lanthanum chloride (200 microM) and lower pH (6.0).(ABSTRACT TRUNCATED AT 250 WORDS)