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).
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.
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.
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.
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