Salmon Calcitonin Binding Research Articles

Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors.

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.

Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity.

A series of mutant porcine calcitonin receptors with progressively truncated carboxy termini have been expressed in COS and HEK 293 cells. All forms of the receptor, including those totally lacking the cytoplasmic tail, were able to bind 125I-labeled salmon calcitonin. However, removal of C-terminal domains resulted in multiple functional changes in the receptor. First, compared with the wild type receptor, affinity of binding of salmon calcitonin was increased for truncated receptors, whether determined in intact transfected cells or in cell membranes. Second, internalization of the ligand-receptor complex was greatly attenuated for mutants truncated by 44 or 83 amino acids but not for an intermediate form truncated by 63 amino acids. Third, truncation affected signal transduction, which for the porcine calcitonin receptor occurs by generation of intracellular cAMP and Ca2+. The magnitude of adenylate cyclase responses was much reduced for the same mutants defective in internalization. Under conditions where expression of each receptor form was approximately equal, the magnitude of intracellular Ca2+ responses was decreased by C-terminal truncation. These results draw attention to the functional significance of the cytoplasmic tail of the porcine calcitonin receptor and suggest intramolecular interactions between the carboxy terminus and other receptor domains and/or cellular regulatory elements.

Islet Amyloid Polypeptide Stimulates Cyclic AMP Accumulation via the Porcine Calcitonin Receptor

CHO-cells stably transfected with an expression vector for the porcine calcitonin receptor were exposed to various concentrations of IAPP, CGRP or calcitonin from different species. In these, but not in untransfected cells, rat IAPP mediated cAMP accumulation at concentrations above 25 nM. This potency was three orders of magnitude lower than that of porcine calcitonin and two and four orders of magnitude lower than those of human and salmon calcitonin, respectively. Human β-CGRP had an effect similar to rat IAPP whereas human α-CGRP was at least one order of magnitude less potent than rat IAPP. COS cells expressing recombinant porcine calcitonin receptors transiently or stably showed a different pattern of responses to calcitonin, IAPP and the CGRPs. In these cells, rat IAPP was as potent an inducer of cAMP as was salmon or porcine calcitonin and more potent than human calcitonin or the CGRPs. The dissociation constants for salmon calcitonin binding to the porcine calcitonin receptors on CHO and COS cells were 0.2 nM and 2.0 nM and the corresponding number of binding sites per cell were approximately 7 × 10 4 and 2 × 10 6, respectively. These results demonstrate that IAPP and CGRP can mediate signal transduction via the porcine calcitonin receptor and provide a possible explanation for the calcitonin-like effects of pharmacological levels of IAPP and CGRP administrated in vivo. The difference between CHO and COS cells in their relative response to the various ligands may relate to the difference in receptor number, post-transcriptional processing, or to dissimilarities in the signal transduction pathways between the two cell types.

Modulation of Calcitonin Binding by Calcium: Differential Effects of Divalent Cations

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.

Effects of cerebral lesions on binding sites for calcitonin and calcitonin gene-related peptide in the rat nucleus accumbens and ventral tegmental area

Binding site densities of [ 125I]-labelled salmon calcitonin and human calcitonin gene-related peptide were investigated in the rat nucleus accumbens and ventral tegmental area by means of quantitative autoradiography following selective brain lesions. [ 125I]salmon calcitonin and [ 125I]human calcitonin gene-related peptide binding sites were highly concentrated in the accumbens, whereas the ventral tegmental area only contained [ 125I]salmon calcitonin binding sites. Unilateral injection of 6-hydroxydopamine into the ventral tegmental area did not alter [ 125I]salmon calcitonin and [ 125I]human calcitonin gene-related peptide binding site densities in the ipsilateral accumbens, while it produced a significant decrease in [ 125I]salmon calcitonin binding sites in the lesioned ventral tegmental area (−50%). In contrast, following unilateral injection of quinolinic acid into the accumbens, the densities of [ 125I]salmon calcitonin and [ 125I]human calcitonin gene-related peptide binding sites were significantly decreased in the lesioned accumbens (−57% and −56%, respectively), while [ 125I]salmon calcitonin binding site densities were not modified in the ipsilateral ventral tegmental area. The present study clearly suggests that [ 125I]salmon calcitonin and [ 125I]human calcitonin gene-related peptide binding sites are located on intrinsic neurons but not on the dopaminergic nerve terminals in the accumbens. Moreover, a certain proportion of [ 125I]salmon calcitonin binding sites could be present on dopaminergic cell bodies in the ventral tegmental area.

Analogue separates biological effects of salmon calcitonin on brain and renal cortical membranes

The conformation-activity relationship of salmon calcitonin in kidney and brain was investigated with regard to effects on membrane binding and adenylate cyclase activity. Since an amphipathic α-helical conformation on the calcitonin molecule is associated with high potency in lowering serum calcium, the activity of the parent peptide was compared to that of [Gly 8, D-Arg 24]des-Leu 16-salmon calcitonin, a calcitonin analogue (CTA) with less helix forming potential. The results indicate that while salmon calcitonin possesses similar potency in brain and kidney, CTA is effective only in brain. Furthermore, CTA did not inhibit the binding of 125I-labeled human calcitonin gene-related peptide (HCGRP) to brain membranes. Our findings suggest that the specific binding and effects of salmon calcitonin on adenylate cyclase activity in brain do not depend on conformational features in the middle region of the molecule, although the α-helical structure in this region does appear to be an important property for salmon calcitonin binding to renal cortical membranes.

Identification of calcitonin receptors in human spermatozoa.

Calcitonin (CT) is a powerful hypocalcemic hormone which regulates calcium balance in cells. The presence of CT and CT receptors has been demonstrated in many extrathyroidal tissues, including the male genital tract. CT immunoreactivity has also been found in human seminal fluid, and an inhibitory effect of salmon CT on human sperm motility in vitro was recently reported. In this study the presence of specific binding sites for synthetic salmon CT in intact human spermatozoa was investigated using [125I]salmon CT. Binding experiments demonstrated a CT-sperm interaction involving a receptor-mediated mechanism. The binding was very rapid and minimally reversible, with the maximal site saturation occurring at approximately 2 nM labeled peptide. The dissociation of the CT-receptor complex was only slightly influenced by the addition of unlabeled hormone. Increasing concentrations of unlabeled salmon, eel, and human CT produced a dose-dependent inhibition of [125I]salmon CT binding. These data fulfill the major criteria for demonstration of specific receptors for salmon CT in human spermatozoa. Owing to the key role of calcium ions in regulating sperm motility and the onset of the acrosomal reaction, CT receptors could be important in male gamete physiology.

Conformational requirements for activity of salmon calcitonin.

A series of deletion-substitution analogs of salmon calcitonin (SCT) have been prepared containing combinations of a glycine substitution in position 8 and deletions of serine-2 and tyrosine-22. Biological activity of the analogs with respect to native SCT were determined in the rat hypocalcemic assay and by studying stimulation of cAMP formation and competition for binding of 125I-labeled SCT in T47 D human breast cancer cells. It was found that each of the analogs retained full potency, irrespective of the means of assessment. It is suggested that conservation of the alpha-helical region of SCT, along with the overall tertiary structure, are more important for peptide potency than chain length per se.

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK1 cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.

Characterization of an osteoblast-like clonal cell line which responds to both parathyroid hormone and calcitonin.

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.

Amphipathic helix and its relationship to the interaction of calcitonin with phospholipids.

Salmon, porcine, and human calcitonins interact with phosphatidylglycerol to form water-soluble complexes, but these peptides do not interact with the zwitterionic lipids phosphatidylcholine or sphingomyelin. The calcitonins are more helical in the presence of dimyristoylphosphatidylglycerol than in its absence, but human calcitonin is considerably less helical than the other two, particularly in the presence of the lipid. This may explain the previously reported faster rate of degradation of human compared with salmon calcitonin in vivo. The ability of human calcitonin to solubilize dimyristoylphosphatidylglycerol and to alter the phase transition properties of this phospholipid while maintaining a low content of helix indicates that the presence of an amphipathic helix is not a requirement for these effects. The binding of salmon calcitonin to dimyristoylphosphatidylglycerol has been studied by determining the dependence of the circular dichroism properties of the peptide on the concentration of lipid. At 25 degrees C, salmon calcitonin binds to five molecules of dimyristoylphosphatidylglycerol with an affinity constant of 1 X 10(5) M-1. Little change in these parameters is observed at 38 degrees C, and the complex is stable over a wide range of temperatures both above and below the phase transition temperature. The rate of reaction of salmon calcitonin with dimyristoylphosphatidylglycerol is rapid at or above the phase transition temperature of the lipid but not at low temperatures. Salmon calcitonin also interacts with egg phosphatidylglycerol. These results demonstrate that salmon calcitonin can react with phosphatidylglycerol at or above its phase transition temperature to form complexes which are at least kinetically stable both above and below the phase transition temperature. Salmon calcitonin can solubilize mixtures of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine containing 25% or more of the former phospholipid. The helical content of the peptide in the presence of these lipid mixtures is dependent on the fraction of the lipid which is phosphatidylglycerol, with larger fractions of this lipid leading to the formation of a higher helical content. At 25% phosphatidylglycerol, salmon calcitonin can solubilize the lipid mixture without much increase in the helix content of the peptide, again demonstrating that an amphipathic helical structure is not required for the solubilization of phospholipids. Ionic bonding appears to be an important component in the binding of the cationic calcitonins to phospholipids. Salmon calcitonin binds to the acidic phospholipids phosphatidylinositol and phosphatidic acid, but not to zwitterionic phospholipids. In addition, high concentrations of NaCl cause the dissociation of the complex between salmon calcitonin and dimyristoylphosphatidylglycerol.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of salmon calcitonin binding sites in rat brain by autoradiography

The regional distribution of [ 125I]salmon calcitonin (sCT) binding sites was examined in rat brain by receptor autoradiography. Dense specific labeling was recognized over the hypothalamus, preoptic and accumbent nuclei, amygdala, zona incerta, interpeduncular nucleus, periventricular gray and the reticular formation. Intermediate binding was seen over the arcuate and supramamillary nuclei and substantia nigra, and no binding in the neocortex, cerebellum, thalamus, basal ganglia and mamillary bodies. The well defined topographical distribution strongly indicates physiological roles for sCT in the mammalian brain.

Salmon calcitonin binding sites in rat pituitary

The distribution of calcitonin binding sites in the pituitary of the rat was examined by membrane binding studies and in vitro autoradiography. Anterior and particularly the intermediate, but not the posterior lobe have binding sites for [ 125I]salmon calcitonin. These results are congruent with the existence of immunoreactive calcitonin in the pituitary.

Characterization of specific receptors for calcitonin in porcine lung.

The binding of salmon calcitonin was investigated in subcellular fractions obtained from normal porcine lung. Only the membrane fraction (density, 1.14 g/cm3) showed specific binding for calcitonin. Specific binding of 125I-labeled salmon calcitonin was competitively inhibited by concentrations of unlabeled homologous hormone in the range 0.01-1 nM. Half-maximal inhibition of binding was observed with 0.12 nM salmon calcitonin. Scatchard analysis of the data suggested the presence of one class of binding sites with a mean affinity constant of 0.9 X 10(10) M-1 and a mean receptor number of 40 X 10(8)/mg of protein. The binding of salmon calcitonin was highly specific; half-maximal inhibition of binding was observed with 63.8 nM bovine calcitonin, the hormone corticotropin having no effect in this system.

Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D).

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.

Demonstration of specific receptors for calcitonin in isolated trout gill cells

1. 1. Salmon calcitonin binding by isolated gill cells from rainbow trout, Salmo gairdneri has been investigated. 2. 2. The calcitonin receptor interaction is time- and temperature-dependent. 3. 3. 50% of inhibition of the 125I labeled calcitonin binding is observed in presence of 1.5 ng/ml unlabeled salmon calcitonin. 4. 4. Two types of receptors are described: a high affinity-low capacity site and a low affinity-large capacity site. 5. 5. These studies strongly support the role of calcitonin as a hormone regulating the gill function in physiological conditions.

Calcitonin-responsive adenylate cyclase in a calcitonin-producing human cancer cell line

A calcitonin-responsive adenylate cyclase has been found in a cell line of a poorly differentiated bronchial carcinoma (BEN cells). The cells have previously been shown to secrete an immunoreactive form of calcitonin in culture. Salmon calcitonin (SCT), porcine calcitonin (PCT) and human calcitonin (CT-M) all stimulated adenylate cyclase activity in particulate preparations. CT-M sulphoxide had little effect. The concentrations of the calcitonins required for half the maximum activation of adenylate cyclase were 6-8, 18 and 90 nm respectively. SCT (30pm) and CT-M (60 pm) increased the intracellular concentration of cyclic AMP from 11-2+/-0-2 (s.e.) to 18-2+/-0-2 and 16-7+/-0-2 respectively over a 2-5-min period. SCT (labelled with 125I) bound to particulate preparations of Ben cells, and competition for binding occurred with unlabelled SCT and CT-M. The concentration of SCT required for half the maximum inhibition of [125I]SCT binding was 11 nm. CT-M sulphoxide inhibited only at high concentration (3 micron). The characteristics of the adenylate cyclase response to SCT did not change over the period between cell adhesion (after subculture) and confluence. However, pre-incubation of cells for 4 h with SCT (150 nm) abolished the subsequent adenylate cyclase response of particulate preparations to further hormone. The practical difficulties encountered in purifying and quantifying the large-mol.-wt. form of CT-M secreted by BEN cells has precluded direct investigation of the potential relationship between hormone secretion and the occurrence of the calcitonin receptor. This relationship is discussed in terms of its possible biological significance.