Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Calcitonin Receptor

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101586

Synonyms

Historical Background

A hypocalcemic factor that lowers blood calcium in response to hypercalcemia in dogs was first discovered and named by Copp et al. as calcitonin (CT) (Copp et al. 1962). The parafollicular cells of the thyroid in mammals were indicated as the source of calcitonin production (Fig. 1). Calcitonin extracted from thyroid in mammals inhibited the release of calcium from bones (Pondel 2000), and subsequently the amino acid sequence of calcitonin from multiple species was revealed. Salmon calcitonin (sCT) showed the highest potency among the various calcitonins, which resulted in the prevalent use of sCT to understand calcitonin physiology. In 1986, 125I-labeled-sCT binding to isolated rat osteoclasts was visualized by Nicholson et al. indicating that the CT receptor (CTR) was a cell surface receptor highly expressed in osteoclasts (Sexton et al. 1999). In addition, large numbers of 125I-sCT binding sites were demonstrated in the brain in the 1980s (Sexton 1991). A porcine CTR was first cloned in 1991 (Lin et al. 1991), and subsequently human CTR cDNA was reported. Overexpression of porcine and human CTR showed adenylyl cyclase activation confirming CTR as a G protein-coupled receptor (GPCR) (Sexton et al. 1999). In 1986, the peptide amylin (Amy), which is similar in sequence to CT, was identified as the major component of islet amyloid deposits. Amy was subsequently shown to be a hormone released from the pancreas that acts at receptors in the brain to regulate food intake and energy expenditure and contribute to blood glucose control (Hay et al. 2015) (Fig. 1). Consistent with noted similarities between CT and Amy receptors and in part explaining the prominent expression of CTR in the brain, in 1999, it was demonstrated that coexpression of CTR with accessary proteins called receptor activity-modifying proteins (RAMPs) gives rise to Amy receptors (Christopoulos et al. 1999). Based on the antiresorptive effect of CT on osteoclasts, the potent CTR agonist sCT was developed as a drug to treat osteoporosis, Paget’s disease, and hypercalcemia. An Amy analog was developed for the treatment of diabetes based on the ability of Amy to regulate blood glucose.
Calcitonin Receptor, Fig. 1

Overview of the calcitonin receptor function and its peptide hormone ligands calcitonin and amylin. CT calcitonin, CTR calcitonin receptor, RAMP receptor activity-modifying protein

Expression, Splice Variants, and Regulation

The human Calcr gene encoding the ~500 amino acid CTR is located on chromosome 7q21.3 and contains 14 exons spread over ~70 kb and at least three promoters from which transcription can be initiated. CTR expression is widespread in many tissues. In addition to its prominent expression in bone osteoclasts and in many areas of the brain, CTR is also present in kidney, pituitary, spinal cord, placenta, testis, spermatozoa, lung, stomach, skeletal muscle, lymphocytes, and many cancer cell lines (Pondel 2000; Sexton et al. 1999). At least five human CTR isoforms resulting from alternative splicing are known, with two of these that differ by the presence or absence of a 16 amino acid insert in intracellular loop 1 being most common (Sexton et al. 1999). Gorn et al. showed that hCTR gene cloned from ovarian carcinoma cells contained the 16 amino acid insert as compared to porcine CTR (Gorn et al. 1992). The insert negative form of human CTR was cloned from a breast cancer cell line and it is more abundant in most tissues than the insert positive variant for which expression is limited to lung, ovary, bone marrow, and placenta (Purdue et al. 2002). Notably, the insert positive hCTR variant has altered signaling properties as compared to the insert negative form despite their identical sCT binding affinity (Purdue et al. 2002). The insert positive variant exhibited decreased internalization and decreased downstream signaling. The International Union of Pharmacology guidelines recommends the nomenclature of hCT receptor(a) for the insert negative variant and hCT receptor(b) for the insert positive variant.

CTR is regulated by internalization and its gene expression is subject to regulation at the level of transcription (Sexton et al. 1999). The receptor is downregulated by exposure to CT. As for many GPCRs, agonist-induced phosphorylation of the CTR C-terminal tail via G protein-coupled receptor kinases and second messenger kinases leads to β-arrestin recruitment, desensitization, and internalization with receptor degradation. At the level of transcription, CT causes a decrease in CTR mRNA production, whereas glucocorticoids such as dexamethasone increase CTR gene transcription.

Physiological and Pharmacological Functions and Signaling Mechanisms

The most well-characterized function of CTR is its role in mediating the inhibitory actions of CT on bone osteoclasts. CT activation of CTR decreases osteoclast motility and results in loss of the ruffled border and cell retraction. CT was originally thought to be a counter-regulatory hormone to parathyroid hormone (PTH) in calcium homeostasis. Whereas PTH causes the release of calcium from bone in response to low-serum calcium, CT lowers serum calcium by inhibiting osteoclast-mediated bone resorption and increasing renal calcium excretion, at least in the hypercalcemic state (Sexton et al. 1999). Although the pharmacological use of CT to inhibit bone resorption is well established, the relevance of CT function for calcium homeostasis in normal adults has been debated for many years in part because overexpression or loss of CT in humans seems to have no physiological effect. Recent work with genetically engineered mouse models revealed an unexpected role for CT-CTR signaling in inhibiting bone formation (Davey and Findlay 2013; Keller et al. 2014).

Overexpression of hCTR in vitro showed cAMP production by CT indicating that the adenylyl cyclase/cAMP/protein kinase A (PKA) pathway is an important CTR signaling mechanism (Gorn et al. 1992). Gαs protein is known to mediate adenylyl cyclase activation by CTR (Findlay et al. 2015). In addition, cAMP analogs were shown to inhibit rodent osteoclast activity. CTR also couples with Gαq to activate phospholipase C (PLC), which produces signaling molecules (diacylglycerol and inositol trisphosphate) for protein kinase C (PKC) activation and intracellular calcium increase (Findlay et al. 2015). This pathway is also important for CT inhibition of osteoclast activity. In addition, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was observed with CTR activation and these effects appear to be dependent on both Gi and PKC (Findlay et al. 2015). Together, there are at least three important downstream signaling mediators of CTR: adenylyl cyclase, PLC, and ERK1/2 activation (Fig. 2). CT-CTR signaling can also regulate adhesion complexes via effects on focal adhesion kinase and other adhesion-related proteins, which is likely relevant for osteoclast attachment to bone (Zhang et al. 2002).
Calcitonin Receptor, Fig. 2

Calcitonin receptor signaling pathways. CTR triggers two main signaling pathways mediated by adenylyl cyclase and phospholipase C for the inhibition of osteoclast activity. ERK activation by CTR has been demonstrated to regulate cell growth in various cell lines. Recently, CTR was shown to inhibit bone formation by downregulating an S1P transporter Spns2 and consequently decreasing extracellular S1P and S1P3 activation in osteoblasts. CT calcitonin, CTR calcitonin receptor, ECD extracellular domain, TMD transmembrane domain, AC adenylyl cyclase, PLC phospholipase C, ERK extracellular signal-regulated kinase, cAMP cyclic adenosine monophosphate, PKA protein kinase A, IP3 inositol trisphosphate, DAG diacylglycerol, PKC protein kinase C, Spns2 spinster homolog 2, S1P sphingosine 1-phosphate, S1P 3 sphingosine 1-phosphate receptor 3

Some species- and tissue-dependent differences have been reported with regard to which pathway mediates osteoclast inhibition. In human osteoclast-like cells, PKC activators reproduced the antiresorptive effects of sCT, but the activators for PKA and calcium/calmodulin-dependent kinase (downstream signaling of intracellular calcium increase) failed to reproduce sCT effects (Findlay et al. 2015). In human teeth odontoclasts, PKA activators, but not the PKC activator, exhibited antiresorptive activity comparable with hCT (Findlay et al. 2015). In contrast to human, PKA activation was more critical for antiresorptive activity of CT than PKC activation in mouse osteoclast-like cells (Findlay et al. 2015). The intracellular calcium increase was also reported to inhibit osteoclast activity (Zaidi et al. 1990). Although the mechanisms of how ERK1/2 activation mediates CT effects are not entirely clear, ERK1/2 activation by CT was reported to regulate cell growth in various cell lines (Purdue et al. 2002).

Genetically engineered mice lacking CT or CTR surprisingly displayed a phenotype of increased bone mass suggesting an unexpected role for CT-CTR signaling in inhibiting bone formation (Davey and Findlay 2013). A recent report indicated that the increase in bone formation resulting from CTR knockout (globally or in osteoclasts) was mediated by sphingosine 1-phosphate (S1P) release from osteoclasts and its activation of the S1P3 receptor on osteoblasts. CTR activation in osteoclasts was shown to reduce the expression of an S1P transporter Spns2, which consequently decreases the extracellular S1P and the S1P3 receptor activation in osteoblasts (Fig. 2) (Keller et al. 2014). This work appears to indicate that the function of CT-CTR signaling in physiology is different than its function in a pharmacological setting.

Structure and Mechanism of Calcitonin Recognition

CTR is a member of the class B GPCR subfamily. In humans, there are 15 class B receptors that mediate the actions of a diverse collection of peptide ligands including CT, Amy, PTH, glucagon, and several other endocrine hormones, paracrine factors, and neuropeptides. Class B GPCRs have an N-terminal extracellular domain of ~150 residues followed by a plasma membrane-embedded seven-transmembrane domain (7TMD) and a cytoplasmic C-terminal tail. Binding of peptide ligands to class B GPCRs is described by a “two-domain” model in which the C-terminal half of the peptide binds the ECD and the N-terminal half of the peptide binds the juxtamembrane portion of the 7TMD. The former interaction contributes to affinity and selectivity and the latter interaction activates the receptor to act as a guanine nucleotide exchange factor for the associated heterotrimeric G-protein. A useful consequence of this binding mechanism is that N-terminally truncated peptides act as antagonists.

Studies of CT-CTR interactions and chimeric receptors and peptides support the two-domain model (Purdue et al. 2002). Understanding the details of peptide binding and receptor activation ultimately requires high-resolution structures, but no structure of a full-length class B GPCR is available. Fortunately, progress has been made on structural studies of the free peptides and individual receptor domains (Fig. 3). NMR studies of sCT and analogs indicated the three regions of the free peptide: an N-terminal ring structure comprising the C1–C7 disulfide, an amphipathic α-helix extending to approximately residue 21, and an unstructured C-terminal 22–32 segment ending in the proline-amide that is required for bioactivity. The N-terminal ring structure is involved in receptor activation. Truncated sCT (8–32) lacking the ring is widely used as a CTR antagonist. The crystal structure of the CTR ECD in complex with a C-terminal sCT fragment revealed that it maintains a largely unstructured conformation when bound to the ECD, but a crucial β-turn structure is adopted near the C-terminus that enables the proline-amide to occupy a small ECD pocket (Johansson et al. 2016). Other sCT residues important for ECD-binding include G28, which provides flexibility for β-turn formation, and T25 and T27 that make hydrophobic and polar contacts with the ECD. Binding of the sCT N-terminal region to the 7TMD is poorly understood. Only two class B GPCR 7TMD structures are available, those of the glucagon receptor (GCGR) and the corticotropin-releasing factor receptor, and these lack peptide ligands. Some information constraining the orientation of full-length CT with respect to the domains in the full-length receptor is available from cross-linking experiments (Dong et al. 2014). These studies indicated that CT residues 16 and 19 in the helical region are in proximity to the CTR segment connecting the ECD and the first TM helix and that CT residue 8 is in proximity to extracellular loop 3 of the 7TMD. The spatial relationship of the ECD and 7TMD in the peptide-free and peptide-bound receptor states is unclear.
Calcitonin Receptor, Fig. 3

Calcitonin receptor and RAMP structure and hormone binding. As no structures of the intact receptors are available, CTR and RAMP1 are represented by structures of isolated domains including a crystal structure of the CTR ECD with a bound sCT fragment (residues 21–32) (PDB ID: 5II0), a crystal structure of the peptide-free 7TM domain of the glucagon receptor (PDB ID: 5EE7), and a crystal structure of the RAMP1 ECD (PDB ID: 4RWG). The RAMP TM segment is represented by a generic alpha helix. The relative orientations of the ECDs and their cognate membrane spanning domains are arbitrary. NMR structures of free full-length sCT (PDB ID: 2GLH) and hAmy (PDB ID: 2KB8) are shown on the far left and right, respectively

Association with Receptor Activity-Modifying Proteins (RAMPs)

As noted, CTR plays an important role in mediating the actions of the hormone Amy, which regulates food intake and energy expenditure and contributes to blood glucose control (Hay et al. 2015). Amy receptors arise from association of CTR with each of three receptor activity-modifying proteins (RAMP1, -2, and -3). The receptor complexes are designated the AMY1, AMY2, and AMY3 receptors for CTR in complex with RAMP1, -2, and -3, respectively (Purdue et al. 2002). RAMPs are single transmembrane-spanning proteins with a short cytoplasmic C-terminal tail and an N-terminal ECD that forms a disulfide-linked three-helix bundle (Fig. 3). RAMP association with CTR enhances binding of Amy and decreases binding of hCT, although sCT retains high affinity for CTR:RAMP complexes. Evidence suggests that Amy, which has a structure similar to that of CT, binds its receptors in a manner similar to CT. Chimeric receptor studies indicated that the RAMP ECDs dictate receptor phenotype. By analogy to RAMP modulation of calcitonin receptor-like receptor (CLR) selectivity for the CT family peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (Booe et al. 2015), CTR:RAMP complexes are likely heterodimers and the RAMP subunits probably make minimal contact with Amy and may allosterically modulate CTR to alter its phenotype. Notably, RAMP1 association with CTR also significantly enhances its affinity for CGRP, although the physiological relevance of this for CGRP actions is unclear.

Amy is an anorectic hormone that reduces food intake by inducing satiation (Hay et al. 2015). Amy also increases energy expenditure and inhibits gastric emptying. Amy is cosecreted with insulin from pancreatic β-cells and it contributes to blood glucose regulation by inhibiting glucagon release from pancreatic α-cells. The ability of Amy to suppress the abnormal postprandial rise in glucagon that is common in both type I and type II diabetes provides the basis for the use of AMY receptor agonists as diabetes drugs. These Amy effects are mediated by activation of central AMY receptors. The area postrema (AP) of the brain is known to be an important site mediating Amy actions, and AP neurons express both CTR and RAMPs. In heterologous in vitro expression systems, the AMY receptors activate many of the same pathways as CTR alone and the cAMP pathway is most commonly monitored for pharmacological studies. In AP neurons signaling downstream of Amy involves cyclic guanosine monophosphate (cGMP) and phosphorylation of ERK1/2 (Hay et al. 2015), but in general the downstream signaling pathways mediating Amy actions in physiologically relevant cell types remain poorly understood. Similarly, little is known regarding which of the AMY receptor subtypes is most relevant for various Amy functions.

CTR and CTR:RAMP Complexes as Therapeutic Targets

sCT as an injectable drug has been used for decades to treat Paget’s disease, hypercalcemia, and osteoporosis, although bisphosphonates are now the preferred treatment. Unlike the bisphosphonates, sCT also has analgesic effects for bone pain (Bandeira et al. 2016). Other routes of administration for sCT (nasal spray and an oral formulation) have been developed to improve drug compliance. Administration of sCT via nasal spray has shown a significant increase in bone mineral density in small- and mid-scale clinical studies with postmenopausal osteoporotic women. The nasal spray of sCT was approved as a drug by US Food and Drug Administration (FDA) for postmenopausal osteoporosis in 1995. A Phase III clinical trial (PROOF study) demonstrated that sCT nasal spray significantly decreased the risk of new vertebral fractures by 33% compared to placebo, although this effect was not dose-dependent (Bandeira et al. 2016). The Phase III clinical trial with oral sCT formulated with an acid-resistant enteric coat (ORACAL study) showed a mild but significant increase in lumbar bone mineral density accompanied by greater reductions in bone resorption markers than placebo and even nasal spray groups (Bandeira et al. 2016). However, new vertebral fractures of postmenopausal osteoporotic women were not prevented by oral sCT in a separate Phase III clinical trial, although it is unclear if this result was related to much lower plasma sCT concentration than that observed with other clinical trials using oral sCT (Henriksen et al. 2016).

Despite decades of apparently safe use of sCT as a drug, a concern regarding possible cancer risk increased by sCT use was recently raised. Although meta-analysis with currently available clinical data was unable to show a causal relationship, there was a statistical weak association of sCT use with the increased cancer risk (Wells et al. 2016). US FDA performed its own analysis for risks and benefits of sCT in 2013 and retained the use of nasal sCT despite the potential for a cancer risk and the lack of strong evidence for antifracture efficacy of sCT.

An amylin analog pramlintide has been approved for type I and type II diabetes as adjunct therapy with insulin. Based on benefits for blood glucose control mediated by amylin receptor activation, several independent research groups have actively pursued next generation amylin analogs and tested in preclinical studies for advanced diabetes and obesity therapeutics. There are other potential therapeutic areas where amylin receptor activation may provide clinical benefits (Hay et al. 2015). The potential of the amylin receptor for other diseases remains to be explored in future studies.

Summary

CTR is a cell surface class B GPCR that mediates the actions of the peptide hormones CT and Amy. CTR is highly expressed in bone osteoclasts where it mediates the action of CT to inhibit osteoclastic bone resorption. The association of CTR with RAMPs enables it to mediate Amy actions in the brain that result in the reduction of food intake, gastric empyting, and glucagon secretion. CTR couples to several G proteins including Gs, Gq, and Gi that enable downstream signaling through the adenylyl cyclase/cAMP/PKA, PLC/Ca2+/PKC, and ERK1/2 pathways. Utilization of these pathways can vary depending on cell type. The PKA and PKC pathways are important for CT inhibition of osteoclastic bone resorption. Paradoxically, CT-CTR signaling in osteoclasts also leads to inhibition of bone formation. This indirect effect involves inhibition of the release of the lipid signaling molecule S1P, which acts on osteoblasts to promote bone formation (Keller et al. 2014). CTR is a proven therapeutic target for bone disorders and diabetes. The potent CTR agonist sCT has been used for decades as a drug for osteoporosis, Paget’s disease, and hypercalcemia taking advantage of the pharmacologic action of CT to inhibit osteoclastic bone resorption. The Amy analog pramlintide is used as insulin adjunct therapy to provide better glycemic control in diabetes based on its ability to reduce postprandial glucagon secretion. Structural studies of CTR and other class B GPCRs revealed the folds adopted by the ECD and 7TMD. The binding of CT to CTR follows a “two-domain” model in which the hormone C-terminal region contacts the ECD and the N-terminal region binds and activates the 7TMD. The crystal structure of sCT-bound CTR ECD revealed the peptide β-turn structure that enables the crucial C-terminal proline-amide to occupy a pocket on CTR (Johansson et al. 2016). How CT binds the 7TMD of CTR and activates the receptor and how RAMPs interact with CTR to enhance its affinity for Amy remain unclear. Future studies seeking to elucidate the structure and dynamics of hormone-bound CTR and CTR:RAMP complexes and define the functions of the receptors in a variety of cell types will undoubtedly advance our understanding of CTR biology and facilitate the development of next generation drugs targeting CTR.

References

  1. Bandeira L, Lewiecki EM, Bilezikian JP. Pharmacodynamics and pharmacokinetics of oral salmon calcitonin in the treatment of osteoporosis. Expert Opin Drug Metab Toxicol. 2016;12(6):681–9.CrossRefPubMedGoogle Scholar
  2. Booe JM, Walker CS, Barwell J, Kuteyi G, Simms J, Jamaluddin MA, et al. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor. Mol Cell. 2015;58(6):1040–52.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, et al. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol. 1999;56(1):235–42.PubMedCrossRefGoogle Scholar
  4. Copp DH, Cameron EC, Cheney BA, Davidson AG, Henze KG. Evidence for calcitonin – a new hormone from the parathyroid that lowers blood calcium. Endocrinology. 1962;70:638–49.CrossRefPubMedGoogle Scholar
  5. Davey RA, Findlay DM. Calcitonin: physiology or fantasy? J Bone Miner Res Off J Am Soc Bone Miner Res. 2013;28(5):973–9.CrossRefGoogle Scholar
  6. Dong M, Koole C, Wootten D, Sexton PM, Miller LJ. Structural and functional insights into the juxtamembranous amino-terminal tail and extracellular loop regions of class B GPCRs. Br J Pharmacol. 2014;171(5):1085–101.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Findlay DM, Sexton PM, Martin TJ. Calcitonin. Endocrinology: adult and pediatric I. 7th ed: Elsevier; Philadelphia, PA. 2015. p. 1004–17.Google Scholar
  8. Gorn AH, Lin HY, Yamin M, Auron PE, Flannery MR, Tapp DR, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J Clin Invest. 1992;90(5):1726–35.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hay DL, Chen S, Lutz TA, Parkes DG, Roth JD. Amylin: pharmacology, physiology, and clinical potential. Pharmacol Rev. 2015;67(3):564–600.CrossRefPubMedGoogle Scholar
  10. Henriksen K, Byrjalsen I, Andersen JR, Bihlet AR, Russo LA, Alexandersen P, et al. A randomized, double-blind, multicenter, placebo-controlled study to evaluate the efficacy and safety of oral salmon calcitonin in the treatment of osteoporosis in postmenopausal women taking calcium and vitamin D. Bone. 2016;91:122–9.Google Scholar
  11. Johansson E, Hansen JL, Hansen AM, Shaw AC, Becker P, Schaffer L, et al. Type II turn of receptor-bound salmon calcitonin revealed by X-ray crystallography. J Biol Chem. 2016;291(26):13689–98.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Keller J, Catala-Lehnen P, Huebner AK, Jeschke A, Heckt T, Lueth A, et al. Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts. Nat Commun. 2014;5:5215.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Lin HY, Harris TL, Flannery MS, Aruffo A, Kaji EH, Gorn A, et al. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science. 1991;254(5034):1022–4.CrossRefPubMedGoogle Scholar
  14. Pondel M. Calcitonin and calcitonin receptors: bone and beyond. Int J Exp Pathol. 2000;81(6):405–22.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Purdue BW, Tilakaratne N, Sexton PM. Molecular pharmacology of the calcitonin receptor. Recept Channels. 2002;8(3–4):243–55.CrossRefPubMedGoogle Scholar
  16. Sexton PM. Central nervous system binding sites for calcitonin and calcitonin gene-related peptide. Mol Neurobiol. 1991;5(2–4):251–73.CrossRefPubMedGoogle Scholar
  17. Sexton PM, Findlay DM, Martin TJ. Calcitonin. Curr Med Chem. 1999;6(11):1067–93.PubMedGoogle Scholar
  18. Wells G, Chernoff J, Gilligan JP, Krause DS. Does salmon calcitonin cause cancer? A review and meta-analysis. Osteoporos Int. 2016;27(1):13–9.CrossRefPubMedGoogle Scholar
  19. Zaidi M, Datta HK, Moonga BS, MacIntyre I. Evidence that the action of calcitonin on rat osteoclasts is mediated by two G proteins acting via separate post-receptor pathways. J Endocrinol. 1990;126(3):473–81.CrossRefPubMedGoogle Scholar
  20. Zhang Z, Neff L, Bothwell AL, Baron R, Horne WC. Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts. Bone. 2002;31(3):359–65.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA