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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.

High affinity binding was characterized for a number of salmon calcitonin (sCT) analogs to a chimeric receptor (NtCTr) constructed by splicing the N-terminal domain of the human CT receptor onto the C-terminal, transmembrane loop region of the receptor for glucagon. Another chimeric receptor (NtGGr) with the N-terminal domain of the glucagon receptor spliced onto the C-terminal regions of the CT receptor shows no high affinity binding of sCT. Nevertheless, sCT and a number of analogs of the hormone are able to elevate cAMP levels in cells transfected with NtGGr. The least helical analog, des-1-amino-[Ala1,7,Gly8]des-Leu19-sCT, is one of the most active in this regard. Two hormone analogs with modifications in the amino-terminal region, des-Ser2-sCT and [Gly2,3,4,5,6]sCT, show reduced or no activity, respectively, for elevating cAMP in cells expressing the NtGGr. In addition, a 15-fold excess of the peptide sCT-(8-32) antagonizes sCT activation of this receptor. In contrast, these calcitonin analogs exhibited a different rank order for binding to the NtCTr receptor. In fact, des-Ser2-sCT and [Gly8]-des-Leu19-sCT along with the native hormone had the highest helical content as well as the highest binding affinities to the NtCTr receptor. These studies suggest that the helical portion of the hormone within residues 8-22 of sCT is the principal determinant for binding to the receptor N-terminus. Residues 2-6 of sCT interact with the receptor transmembrane loop region and are critical for activation of adenylate cyclase; however, residues 8-32, including Leu16, are responsible for most of the hormone interaction with the transmembrane loop region. Thus, unique requirements exist for CT interaction at the receptor N-terminus relative to the receptor transmembrane loop region, yet there is significant overlap in the hormone determinants that facilitate these interactions.

Two chimeric receptors were constructed by transposing the coding regions for the putative N-terminal domains of the human calcitonin (hCTR) and glucagon (hGGR) receptors. These receptors were stably expressed as glycosylated proteins with molecular masses of 80 kDa for the calcitonin receptor N-terminus chimera (NtCTr) and 65 kDa for the glucagon receptor N-terminus chimera (NtGGr). The NtCTr chimera binds salmon calcitonin (sCT) with an apparent Kd of 12 nM relative to 0.3 nM for the native hCTR. However, this chimera does not mediate a cAMP response even with a transfectant expressing 1.8 x 10(6) cell surface receptors. Stable transfectants expressing the NtGGr chimera show no detectable binding of 125I-sCT or 125I-human glucagon. Surprisingly, adenylate cyclase is activated through the NtGGr chimera by sCT, pCT, and hCT with half-maximal activation at 2.2 +/- 0.6, 5.8 +/- 2.1, and 810 +/- 151 nM, respectively, and the maximum response is similar to that induced by 25 microM forskolin. The rank-order of competition for sCT binding to the NtCTr chimera is similar to the hCTR (sCT > pCT > hCT), but the concentrations required for half-maximal competition are 100- to > 2000-fold higher. In addition, salmon calcitonin binds with a much more rapid on-rate and off-rate to the NtCTr chimera relative to the hCTR which binds hormone irreversibly. Cross-linking of 125I-sCT to the NtCTr chimera with bis(sulfosuccinimidyl) suberate is much greater than to the hCTR, suggesting unique conformations for the two receptor-hormone complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin, a peptide hormone active in calcium homeostasis, is used in the treatment of bone loss disorders because it inhibits osteoclast function. A human calcitonin receptor was cloned and expressed in baby hamster kidney cells. Three independent stable transfectants respond to calcitonin via increased intracellular calcium ([Ca2+]i) and cyclic adenosine 3',5'-monophosphate (cAMP). We made the interesting observation that these cells also respond to millimolar increases in extracellular calcium via a rapid and sustained elevation in [Ca2+]i, whereas three calcitonin receptor-negative baby hamster kidney cell lines, two of which express recombinant receptors related to the calcitonin receptor, show no sensitivity to changes in extracellular calcium. The increase of [Ca2+]i in response to both calcitonin and extracellular calcium is a function of the average number of calcitonin receptors per cell. These studies suggest a dual role for the calcitonin receptor as a hormone receptor and an extracellular calcium sensor.

Binding of salmon calcitonin to bovine hypothalamic membranes is enhanced about 25% by calcium with a half-maximal effect at 15 mM calcium. In contrast, membranes prepared from a cell line expressing a recombinant human calcitonin receptor show no effect of calcium under similar conditions. The hypothalamic calcitonin receptor solubilized with CHAPS detergent retains an apparent Kd of 0.3 nM for salmon calcitonin; however, binding of calcitonin to the detergent-solubilized receptor complex can be inhibited by divalent cations in order of potency Mn > Ca approximately Sr approximately Mg >> NaCl with Mn and Ca having apparent Ki's of 5 mM and 20 mM respectively. Dixon and Scatchard plots of Mn and Ca inhibition of binding to the soluble receptor complex suggest a noncompetitive mechanism of inhibition. Calcium also inhibits calcitonin binding to a detergent-solubilized recombinant human calcitonin receptor. Inhibition of calcitonin binding is observed using two independent methods for determining soluble receptor-hormone complex and inhibition is reversed by EDTA.