intra-cellularly by binding to its receptor, the calcitonin receptor (CTR or calcr), a G protein-coupled cell surface receptor with complex intracellular signalling that includes activation of adenylate cyclase and phospholipase C ( Martin et al. 2010
Rachel A Davey, Michele V Clarke, Suzanne B Golub, Patricia K Russell, and Jeffrey D Zajac
Jonathan H Gooi, Ling Yeong Chia, Nicole C Walsh, Morten A Karsdal, Julian M W Quinn, T John Martin, and Natalie A Sims
osteocytes from calvarial bone it was not possible to detect Calcr mRNA in the isolated osteocytes from the long bones of these older mice ( Fig. 4 B). Figure 4 Semi-quantitative PCR analysis of calcitonin receptor ( Calcr ) mRNA levels in UMR106.01 cells
JM Hilton, M Dowton, S Houssami, and PM Sexton
This study investigates the poor reversibility of salmon calcitonin (sCT) binding to rat and human calcitonin receptors. Efficacy of CT and analogue peptides in (125)I-sCT binding competition and cAMP assays was compared with the dissociation kinetics of (125)I-labelled peptides. Assessment was performed on cells stably expressing either rat or human calcitonin receptors. Dissociation kinetics of the antagonists, sCT(8-32) and AC512, revealed that binding was rapidly and completely reversible at the receptors, despite high affinity binding, suggesting that poor reversibility required the active conformation of the receptor. G protein coupling was not essential as the dissociation kinetics of (125)I-sCT binding to cell membranes did not significantly alter in the presence of GTP gamma S. Time course experiments established that the transition to irreversibility was slow, while the reversible component of binding appeared to involve a single population of either receptor states or binding sites. Pre-bound (125)I-human CT dissociated rapidly from the receptors, indicating that not all agonists bound irreversibly. To identify structural features of sCT that contribute to its poor reversibility, dissociation kinetics of sCT analogues with various structural modifications were examined. Increasing truncation of N-terminal residues of sCT analogues led to a corresponding increase in the rate of peptide dissociation. Salmon CT peptides which had been substituted at the N-terminus by 13-21 residues of human CT (hCT) were equipotent with sCT in binding competition and cAMP accumulation assays but exhibited a dissociation rate similar to hCT. In contrast, despite lower affinity and efficacy at the receptors, the chimeric analogue sCT(1-16)-hCT(17-32) displayed poorly reversible binding, similar to sCT. Analysis of the dissociation kinetics of sCT analogues with differing alpha-helix forming potential indicated that the ability to form alpha-helical secondary structure was an important factor in the rate of ligand dissociation. We hypothesise that poor reversibility results from a conformational change in the receptor and/or ligand and that this is dependent, at least in part, on interaction with residues constrained within the alpha-helix of the peptide.
M. Kurokawa, V. P. Michelangeli, and D. M. Findlay
When T47D cells were maintained long term in medium containing 0·1 μmol cortisol/l, calcitonin receptor (CTR) expression was stimulated compared with the very low levels of binding in untreated cells grown from frozen stocks. The time-course of the appearance of CTR following treatment with cortisol was slow, requiring up to 3 weeks of continuous exposure of the cells to the steroid. Binding capacity of control cells also increased slowly with time in culture, but after 3 months was only 20–30% of that in cells continuously treated with cortisol. Removal of cortisol resulted in rapid loss of CTR so that binding was reduced to ∼ 50% of treated cell levels within 1 week of removal. Scatchard analysis of the binding data showed that the increased binding capacity induced by cortisol was due solely to a change in average receptor number per cell, with no change in receptor affinity. That this induction of CTR was due to a glucocorticoid effect was shown by the more rapid (< 96 h) and more potent (< 1 nmol/l) action of dexamethasone than of cortisol. In addition, induction was inhibited by the glucocorticoid inhibitor RU486. The induced receptors were shown to be functional, since salmon calcitonin-stimulated adenylate cyclase was induced in parallel with CTR. These results indicate that glucocorticoids are potential regulators of the CTR.
Journal of Endocrinology (1991) 130, 321–326
M. C. Eliam, M. Baslé, Z. Bouizar, J. Bielakoff, M. Moukhtar, and M. C. de Vernejoul
Isolated osteoclasts obtained from young chickens fed a normal (+ Ca) or deficient ( − Ca) calcium and vitamin D diet for 3 weeks, were studied for their ability to bind salmon calcitonin (sCT). Osteoclasts obtained from −Ca chickens, when incubated with 0·1 μmol sCT/l, doubled cyclic (c)AMP production and retracted from a glass support, as observed by scanning electron microscopy. The presence of receptors was also demonstrated by autoradiography and competition analysis of 125I-labelled sCT binding. The number of receptors per cell was 0·9 ± 0·1 × 104. In contrast, osteoclasts obtained from + Ca chickens did not increase cAMP production and did not retract in the presence of 0·1 μmol sCT/l. No specific binding of 125I-labelled sCT could be demonstrated on these osteoclasts.
Plasma levels of calcium and calcitonin were measured in +Ca and − Ca chickens. The plasma concentration of calcium was markedly lower at 3 weeks in −Ca than in +Ca chickens. The plasma concentration of calcitonin was decreased in − Ca chickens compared with +Ca chickens at the first week and kept decreasing during the 3 weeks.
These results strongly support the hypothesis that calcium and vitamin D intake regulate plasma calcitonin levels in chickens, and that calcitonin receptors can be detected on chicken osteoclasts only when blood calcium is decreased by a diet deficient in calcium and vitamin D.
J. Endocr. (1988) 119, 243–248
D. M. FINDLAY, M. DE LUISE, V. P. MICHELANGELI, and T. J. MARTIN
A human lung cancer cell line (BEN cells) with a calcitonin receptor and calcitonin-responsive adenylate cyclase also possesses an insulin receptor. This has been characterized and found to have properties similar to those of other mammalian cell insulin receptors. A receptor number of 58 000 per cell was calculated from curvilinear Scatchard plots, and dissociation of bound labelled insulin by dilution was facilitated by the addition of unlabelled insulin, consistent with negatively co-operative interactions among binding sites.
Preincubation of cells with either calcitonin or insulin led to loss of hormone binding in washed cells. In the case of calcitonin this was associated with loss of adenylate cyclase response. For each hormone the state of down-regulation was characterized by a decrease in receptor number, and for calcitonin there was also a loss in sensitivity of adenylate cyclase. Down-regulation to calcitonin was more rapid than that to insulin and in each case recovery had occurred by 16 h after removal of the hormone. Induction of down-regulation was specific, in that preincubation with one hormone did not influence the subsequent binding or response of the other. Such data are consistent with independent modulation of peptide receptors in the same cell.
Christopher J Charles, Takeshi Katafuchi, Timothy G Yandle, and Naoto Minamino
Hamano K Kangawa K Matsuo H Minamino N 2003a Calcitonin receptor-stimulating peptide, a new member of the calcitonin gene-related peptide family . Journal of Biological Chemistry 278 12046 – 12054 . Katafuchi T Hamano K Kikumoto K
U Zimmermann, B Fluehmann, W Born, JA Fischer, and R Muff
Amylin, calcitonin (CT) and calcitonin gene-related peptide (CGRP) share limited structural homology including amino-terminal ring structures linked by a disulfide bridge and amidated carboxy-termini. Here, we have compared [125I]Bolton-Hunter-[Lys1] rat amylin ([125I]amylin) binding and the stimulation of cyclic AMP accumulation by human (h) amylin, hCT and hCGRP-I in the human breast carcinoma cell lines MCF-7 and T47D, which predominantly express hCT1a and hCT1b receptor isoforms (hCTR1a, hCTR1b) at a similar total number of hCT-binding sites. In MCF-7 cells, half-maximal inhibition (IC50) of [125I]amylin binding by human amylin was observed at 3.6 +/- 0.8 nM (n = 6). hCT and hCGRP-I displaced [125I]amylin binding with 22 and 66 times higher IC50. [125I]hCT binding was inhibited by hCT with an IC50 of 8.1 +/- 1.9 nM (n = 5), and human amylin and hCGRP-I were over 100 times less potent. In T47D cells, on the other hand, specific binding of [125I]amylin was not observed, but hCT inhibited [125I]hCT binding with an IC50 of 3.2 +/- 0.4 nM (n = 3), and human amylin and hCGRP-I had over 200 times higher IC50. In MCF-7 cells, half-maximal stimulation (EC50) of cyclic AMP accumulation by human amylin, hCT and hCGRP-I occurred at 1.4 +/- 0.2, 1.7 +/- 0.4 and 6.3 +/- 1.3 nM respectively. In T47D cells, the EC50 of hCT was 0.32 +/- 0.02 nM (n = 3), and 30- and 1900-fold higher with human amylin and hCGRP-I. In conclusion, the expression of hCTR1a and hCTR1b and [125I]hCT binding were indistinguishable in MCF-7 and T47D cells. Yet, [125I]amylin binding was only recognized in MCF-7 cells, consistent with a distinct amylin receptor.
LJ Raggatt, A Evdokiou, and DM Findlay
Recently we reported that calcitonin (CT) induces growth arrest at the G2 stage of the cell cycle in HEK-293 cell lines expressing the most abundant, insert-negative, isoform of the human CT receptor (insert -ve hCTR). The present study investigates the involvement of the MAPK signalling pathway in the anti-proliferative actions of CT and compares the activity of an isoform of the hCTR that contains a 16 amino acid insert in the first putative intracellular loop (insert +ve hCTR). Comparison of HEK-293 cells stably transfected with the insert -ve or the insert +ve hCTR, showed that accumulation of cAMP and intracellular free calcium in response to CT were specific for the insert -ve receptor isoform. However, a novel acidification of the extracellular medium was mediated by both isoforms. Treatment with CT of cells expressing the insert -ve hCTR, caused a decrease in cell growth associated with an induction of p21(WAF1/CIP1). Analysis by fluorescence-activated cell scanning showed that growth inhibition was associated with an accumulation of cells in G2. CT treatment of cells expressing the insert -ve, but not insert +ve hCTR, induced the phosphorylation of Erk1/2 MAPK, which persisted for at least 72 h. Treatment of cells expressing the insert -ve hCTR with the MAPK kinase (MEK) inhibitor, PD-98059, inhibited the phosphorylation of Erk1/2 and abrogated the growth inhibitory effects of salmon CT, the accumulation of cells in G2, and the associated induction of p21(WAF1/CIP1). These data suggest that activation of Erk1/2 are downstream effectors of the insert -ve hCTR in modulating cell cycle progression.
Y Takei, H Hashimoto, K Inoue, T Osaki, K Yoshizawa-Kumagaye, M Tsunemi, T X Watanabe, M Ogoshi, N Minamino, and Y Ueta
intermedin ( Roh et al . 2004 , Takei 2006 ). In addition, another member of the CGRP family, calcitonin receptor-stimulating peptide (CRSP), was identified in the pig ( Katafuchi et al . 2003 ), which was generated by tandem duplication of the CGRP gene