Calcitonin, human – Agonist of the calcitonin receptor CTR
Human Calcitonin (hCT) is a 32-amino acid hormone peptide belonging to the Calcitonin/Calcitonin-gene related peptide (CGRP) family, that also comprises amylin, adrenomedullin (AM) and adrenomedullin2/intermedin (AM2/IMD). Calcitonin is characterized by a N-terminal disulfide bond that forms a 7 amino acid ring structure, a region with a α-helix tendency and an amidated C-terminus1.
Calcitonin peptide is produced by C cells of the thyroid gland and binds preferentially to the G-protein coupled receptor, calcitonin receptor (CTR)1. Calcitonin receptor signals through activated Gs protein and the production of cAMP second messenger molecules by adenylyl cyclase1. The dissociation constant (Kd) of calcitonin receptors expressed by a human ovarian small cell carcinoma line is approximately 4.6 nM for human Calcitonin2.
Calcitonin is involved in the homeostasis of calcium and phosphorus, and the regulation of bone dynamics. At the basal state, calcitonin secretion reduces plasma calcium and phosphorus levels, and promote bone formation. In Calca -/- mice, a murin particle-induced osteolysis model, Calcitonin peptide has been used as a test compound for studying the effects of calcitonin deficiency. It has been shown that artificial calcitonin subsitution inhibits bone resorption by reducing the formation of osteoclasts and thereby the resulting osteolytic reaction3. The Hypocalcemic Human calcitonin was also used as a positive control to monitor changes in serum calcium levels in HHD transgenic mice vaccinated with Parathyroid hormone-related protein (PTH-rP)-derived peptides in the context of anti-cancer immunotherapy study4. Moreover, it has been reported that calcitonin peptide is a potent stimulator of uncapacitated mouse spermatozoa by regulating a specific isoform of adenylyl cyclase and the production of cAMP, which plays a pivotal role in mammalian sperm function5.
AA sequence: CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH2
Disulfide bonds: Cys1-Cys7
Length (aa): 32
Molecular Weight: 3417.85 g/mol
CAS number: 21215-62-3
Purity rate: >95%
Preparation and storage : Lyophilized powder, trifluoroacetate (TFA) salt format. Shipped at ambient temperature. Upon receipt, store lyophilized peptide at -20°C or lower. Reconstituted peptide can be aliquoted and stored at -20°C or lower. Avoid repeated freeze/thaw cycles.
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1-Hay, D. L. et al., J. Pharmacol. 175, 3–17 (2018)
The calcitonin/CGRP family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b) ). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clinical trials, and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues.
2-Gorn, A. H. et al., J. Clin. Invest. 90, 1726–1735 (1992)
Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line
A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.
3-Kauther, M. D. et al., BMC Musculoskelet. Disord. 12, 186 (2011)
Background Periprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice.
Methods We used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells.
Results Bone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution.
Conclusions As anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.
4-Francini, G. et al., J. Immunol. Baltim. Md 1950 169, 4840–4849 (2002)
High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects
Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.
5-Adeoya-Osiguwa, S. A. & Fraser, L. R., Mol. Reprod. Dev. 65, 228–236 (2003)
Calcitonin stimulates capacitation in uncapacitated mouse spermatozoa and then inhibits spontaneous acrosome loss in capacitated cells, responses similar to those elicited by fertilization promoting peptide (FPP), a peptide known to regulate the adenylyl cyclase/cAMP pathway. This study investigated the hypothesis that calcitonin also modulates this pathway. Calcitonin significantly stimulated cAMP production in uncapacitated spermatozoa and then inhibited it in capacitated cells; the magnitude of both stimulatory and inhibitory changes was similar to that obtained with FPP but the inhibitory responses to FPP preceded those of calcitonin. This possibly reflects the involvement of two different adenosine receptors in response to FPP compared with one calcitonin receptor. Calcitonin receptors were located on the acrosomal cap and the flagellum, the midpiece having a greater abundance than the principal piece. Although both calcitonin and adenosine receptors are found in the head and flagellum, there was no evidence for cross-talk between them. Chlortetracycline investigations to determine the minimum extracellular Ca(2+) requirement for responses to calcitonin revealed that calcitonin significantly stimulated capacitation in Ca(2+)-deficient medium but FPP did not. Calcitonin also significantly stimulated cAMP production under these conditions, and similarly preincubated suspensions, when diluted into +Ca(2+) medium, were significantly more fertile in vitro than untreated controls. These results indicate that calcitonin, like FPP, acts as a first messenger to regulate the production of cAMP and mammalian sperm function, but the differences in Ca(2+) requirements suggest that calcitonin and FPP may regulate different isoforms of adenylyl cyclase.