Human Calcitonin Receptor Research Articles

Development of the novel amylin and calcitonin receptor activators by peptide mutagenesis

The amylin peptide hormone receptor is the complex of the calcitonin peptide hormone receptor and an accessory protein. The calcitonin receptor activation controls calcium homeostasis, while it also functions as the main component of the amylin receptor. Amylin receptor activation in brains controls blood glucose and appetite. Currently, non-selective amylin and calcitonin receptor activators have been tested for body weight reduction to treat obesity. Here, multiple peptide activators for human amylin and calcitonin receptors were developed by introducing comprehensive mutagenesis to rat amylin peptide. The rat amylin peptide C-terminal fragment that interacts with amylin receptor extracellular domain was used to screen for affinity-enhancing mutations. Up to twelve mutational combinations were found to significantly increase peptide affinity both for amylin and calcitonin receptor extracellular domains by over 100-fold. Using these affinity-enhancing mutations, three representative rat amylin analogs with thirty-seven amino acids were made to test the potency increase for amylin and calcitonin receptor activation. All three mutated rat amylin analogs showed significant potency increases by 5- to 10-fold compared to endogenous rat amylin. These mutated peptide activators also showed higher potency for human amylin and calcitonin receptor activation than a clinically available amylin receptor activator pramlintide. These amylin and calcitonin receptor activators developed in this study may be useful as the valuable pharmacological tools that activate amylin receptors in cell-based systems.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

GC- MS SCREENING AND IN-SILICO PREDICTION OF PILA GLOBOSA EXTRACT AGAINST THE BONE DISEASES IN CALCITONIN RECEPTOR

India’s flora and fauna has diverse medicinal properties and constitutes the major portion of our traditional medicine system. One among them is snails. Snails have a long history of being used for treating diseases in traditional medicinal systems around the world including India and China. Pila globosa is one of the largest land snails, mainly used as food. Here in this study, the gas chromatography and mass spectrometry discovered the presence of thirteen bio active constituents with various medicinal applications. The constituent Stigmasta-4, 22-dien-3-beta-ol had the highest retention time and exhibited a efficient binding with the human protein calcitonin receptor ectodomain. The calcitonin receptor types is expressed widely in various tissues and cells but most importantly in the osteoclates and hence a very important receptor in the treatment for bone disorders. The binding of the Pila globosa snail constituent with the human calcitonin receptor proved that a novel drug can be developed from the snail for the treatment of bone disorders such as osteoporosis, hypercalcemia of malignancy and paget’s disease. In addition to that, the snail extract exhibited no cytotoxicity and an efficient antioxidant property against DPPH and H2O2 free radicals. The FTIR analysis revealed the snail contained biologically significant functional groups with properties suitable for a drug and prevalent in most of the pharmaceutical drugs. Thus, these results imply the safety and efficacy of the snail Pila globosa for a novel drug development.

Characterization of Ubrogepant: A Potent and Selective Antagonist of the Human Calcitonin Gene‒Related Peptide Receptor.

A growing body of evidence has implicated the calcitonin gene-related peptide (CGRP) receptors in migraine pathophysiology. With the recent approval of monoclonal antibodies targeting CGRP or the CGRP receptor, the inhibition of CGRP-mediated signaling has emerged as a promising approach for preventive treatments of migraine in adults. However, there are no small-molecule anti-CGRP treatments available for treating migraine. The current studies aimed to characterize the pharmacologic properties of ubrogepant, an orally bioavailable, CGRP receptor antagonist for the acute treatment of migraine. In a series of ligand binding assays, ubrogepant exhibited a high binding affinity for native (K i=0.067 nM) and cloned human (K i=0.070 nM) and rhesus CGRP receptors (K i=0.079 nM), with relatively lower affinities for CGRP receptors from rat, mouse, rabbit and dog. In functional assays, ubrogepant potently blocked human α-CGRP stimulated cAMP response (IC50 of 0.08 nM) and exhibited highly selective antagonist activity for the CGRP receptor compared with other members of the human calcitonin receptor family. Furthermore, the in vivo CGRP receptor antagonist activity of ubrogepant was evaluated in a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans. Results demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV with a mean EC50 of 3.2 and 2.6 nM in rhesus monkeys and humans, respectively. Brain penetration studies with ubrogepant in monkeys showed a CSF/plasma ratio of 0.03 and low CGRP receptor occupancy. In summary, ubrogepant is a competitive antagonist with high affinity, potency, and selectivity for the human CGRP receptor. SIGNIFICANCE STATEMENT: Ubrogepant is a potent, selective, orally delivered, small-molecule competitive antagonist of the human calcitonin generelated peptide receptor. In vivo studies using a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV, indicating a predictable pharmacokinetic-pharmacodynamic relationship.

Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy

Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hotspot for distinct ligand- and pathway-specific effects, and this has implications for the future design of biased agonists of class B GPCRs.

Characterization of signalling and regulation of common calcitonin receptor splice variants and polymorphisms

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is a therapeutic target for the treatment of hypercalcaemia of malignancy, Paget’s disease and osteoporosis. In primates, the CTR is subject to alternative splicing, with a unique, primate-specific splice variant being preferentially expressed in reproductive organs, lung and kidney. In addition, humans possess a common non-synonymous single-nucleotide polymorphism (SNP) encoding a proline/leucine substitution in the C-terminal tail. In low power studies, the leucine polymorphism has been associated with increased risk of osteoporosis in East Asian populations and, independently, with increased risk of kidney stone disease in a central Asian population. The CTR is pleiotropically coupled, though the relative physiological importance of these pathways is poorly understood. Using both COS-7 and HEK293 cells recombinantly expressing human CTR, we have characterized both splice variant and polymorphism dependent response to CTs from several species in key signalling pathways and competition binding assays. These data indicate that the naturally occurring changes to the intracellular face of CTR alter ligand affinity and signalling, in a pathway and agonist dependent manner. These results further support the potential for these primate-specific CTR variants to engender different physiological responses. In addition, we report that the CTR exhibits constitutive internalization, independent of splice variant and polymorphism and this profile is unaltered by peptide binding.

Frozen in action: cryo-EM structure of a GPCR-G-protein complex.

Interaction with heterotrimeric G proteins is a hallmark of G-protein-coupled receptor (GPCR) family members, and it is the key step for a diverse range of cell-signaling cascades. A recent cryo-EM structure of the human calcitonin receptor (CTR) in complex with a G-protein heterotrimer reveals novel insights into receptor–G-protein coupling.

Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR). The crystal structure of the human calcitonin receptor ectodomain (CTR ECD) in complex with a truncated analogue of salmon calcitonin ([BrPhe(22)]sCT(8-32)) has been determined to 2.1-Å resolution. Parallel analysis of a series of peptide ligands showed that the rank order of binding of the CTR ECD is identical to the rank order of binding of the full-length CTR, confirming the structural integrity and relevance of the isolated CTR ECD. The structure of the CTR ECD is similar to other Class B GPCRs and the ligand binding site is similar to the binding site of the homologous receptors for the calcitonin gene-related peptide (CGRP) and adrenomedulin (AM) recently published (Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Mol. Cell 58, 1040-1052). Interestingly the receptor-bound structure of the ligand [BrPhe(22)]sCT(8-32) differs from the receptor-bound structure of the homologous ligands CGRP and AM. They all adopt an extended conformation followed by a C-terminal β turn, however, [BrPhe(22)]sCT(8-32) adopts a type II turn (Gly(28)-Thr(31)), whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.

Prolonged Calcitonin Receptor Signaling by Salmon, but Not Human Calcitonin, Reveals Ligand Bias

Salmon calcitonin (sCT) and human calcitonin (hCT) are pharmacologically distinct. However, the reason for the differences is unclear. Here we analyze the differences between sCT and hCT on the human calcitonin receptor (CT(a)R) with respect to activation of cAMP signaling, β-arrestin recruitment, ligand binding kinetics and internalization. The study was conducted using mammalian cell lines heterologously expressing the human CT(a) receptor. CT(a)R downstream signaling was investigated with dose response profiles for cAMP production and β-arrestin recruitment for sCT and hCT during short term (<2 hours) and prolonged (up to 72 hours) stimulation. CT(a)R kinetics and internalization was investigated with radio-labeled sCT and hCT ligands on cultured cells and isolated membrane preparations from the same cell line. We found that sCT and hCT are equipotent during short-term stimulations with differences manifesting themselves only during long-term stimulation with sCT inducing a prolonged activation up to 72 hours, while hCT loses activity markedly earlier. The prolonged sCT stimulation of both cAMP accumulation and β-arrestin recruitment was attenuated, but not abrogated by acid wash, suggesting a role for sCT activated internalized receptors. We have demonstrated a novel phenomenon, namely that two distinct CT(a)R downstream signaling activation patterns are activated by two related ligands, thereby highlighting qualitatively different signaling responses in vitro that could have implications for sCT use in vivo.

Receptor activity‐modifying protein‐dependent impairment of calcitonin receptor splice variant Δ(1–47)hCT(a) function

Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [Δ(1-47)hCT((a)) ]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. Δ(1-47)hCT((a)) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. Despite lacking 47 residues at the N terminus, Δ(1-47)hCT((a)) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. Δ(1-47)hCT((a)) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. Δ(1-47)hCT((a)) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ and amylin may express their effects through the amylin receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the amylin receptor family, amylin receptor-3 (AMY3). Aβ1-42 and human amylin (hAmylin) increase cytosolic cAMP and Ca(2+), trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1-42 and hAmylin also induce cell death during exposure for 24-48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1-42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin receptor antagonist AC253 blocks increases in intracellular Ca(2+), activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1-42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

Bioportide: an emergent concept of bioactive cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: Possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.

Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization

Dimerization of the prototypic family B G protein-coupled secretin receptor is determined by the lipid-exposed face of transmembrane segment four (TM4), and has substantial functional importance, facilitating G protein coupling. Recently, we demonstrated that the human secretin receptor elicits an inter-receptor bioluminescence resonance energy transfer (BRET) signal with most other human family B peptide receptors, except for the calcitonin receptor. In this study we have explored the occurrence and importance of calcitonin receptor oligomerization. Static and saturation receptor BRET were utilized to demonstrate that, unlike the human calcitonin receptor that does not yield a significant homomeric BRET signal, the rabbit calcitonin receptor exhibits strong resonance energy transfer. Within the lipid-exposed face of TM4, rabbit and human calcitonin receptors differ by a single amino acid (Arg236 in human; His in rabbit), while Thr253 that occurs in human and rabbit calcitonin receptors is unique across family B receptors. Mutating Arg236 or Thr253 of the human calcitonin receptor to residues found in the rabbit calcitonin receptor or the human secretin receptor (R236H, R236Y and T253A) resulted in generation of significant BRET signals. Similarly, mutation of Val250 of the human calcitonin receptor to another key lipid-facing residue found in the secretin receptor (V250I) also increased the receptor BRET signal. These data support the consistent theme of lipid-exposed residues of TM4 being important for the dimerization of the calcitonin receptor. However, rabbit and human calcitonin receptor constructs bound calcitonin and stimulated cAMP similarly, suggesting that differences in BRET could reflect differences in orientation or in the stability of homo-dimeric receptor complexes, which were nevertheless similarly effective in eliciting the functions attributed to that complex. The likelihood of human calcitonin receptor dimerization, even in the absence of a significant BRET signal, was further supported by data demonstrating that the peptide representing TM4 of this receptor that disrupts the rabbit receptor BRET signal, produced a right shift in the cAMP concentration–response curves for both rabbit and human receptors.

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.

Calcitonin in a protochordate, Ciona intestinalis – the prototype of the vertebrate calcitonin/calcitonin gene‐related peptide superfamily

The calcitonin (CT)/CT gene-related peptides (CGRPs) constitute a large peptide family in vertebrates. However, no CT/CGRP superfamily members have so far been identified in invertebrates, and the evolutionary process leading to the diverse vertebrate CT/CGRP superfamily members remains unclear. In this study, we have identified an authentic invertebrate CT, Ci-CT, in the ascidian Ciona intestinalis, which is the phylogenetically closest invertebrate chordate to vertebrates. The amino acid sequence of Ci-CT was shown to display high similarity to those of vertebrate CTs and to share CT consensus motifs, including the N-terminal circular region and C-terminal amidated proline. Furthermore, the Ci-CT gene was found to be the only Ciona CT/CGRP superfamily gene. Ci-CT also exhibited less potent, but significant, activation of the human CT receptor, as compared with salmon CT. Physiological analysis revealed that Ci-CT reduced the osteoclastic activity that is specific to vertebrate CTs. CD analysis demonstrated that Ci-CT weakly forms an alpha-helix structure. These results provide evidence that the CT/CGRP superfamily is essentially conserved in ascidians as well as in vertebrates, and indicate that Ci-CT is a prototype of vertebrate CT/CGRP superfamily members. Moreover, expression analysis demonstrated that Ci-CT is expressed in more organs than vertebrate CTs in the cognate organs, suggesting that an original CT/CGRP superfamily member gene was also expressed in multiple organs, and each CT/CGRP superfamily member acquired its current specific tissue distribution and physiological role concomitantly with diversification of the CT/CGRP superfamily during the evolution of chordates. This is the first report on a CT/CGRP superfamily member in invertebrates.

A novel promoter regulates calcitonin receptor gene expression in human osteoclasts

The calcitonin receptor (CTR) is expressed in a wide variety of tissues and cell types. In bone, its expression is restricted to osteoclasts, the cells that mediate bone resorption. The human CTR ( hCTR) gene has a complex structural organization that exhibits similarity to the porcine ( pCTR) and mouse ( mCTR) CTR genes. In these species, alternative splicing of a single gene generates multiple CTR isoforms that are distributed in both tissue-specific and species-specific patterns. However, the structural organization of the 5′ putative regulatory region and transcriptional mechanisms responsible for tissue-specific expression of the different CTR isoforms are not fully defined. The present studies were undertaken to characterize the structural organization of the 5′-region of the hCTR and identify the regulatory regions involved in osteoclast-specific transcriptional activation. Analysis of mRNA prepared from human osteoclasts using reverse transcription-polymerase chain reaction (RT-PCR) and transient transfection of hCTR promoter-luciferase reporter constructs identified two regions in the 5′-flanking sequence of the hCTR gene that regulated CTR gene expression in osteoclasts. Both of these putative promoters were responsive to the osteoclast-inducing cytokine, receptor activator of NF-κB ligand (RANKL) and demonstrated trans-activation by the RANKL-induced transcription factor nuclear factor of activated T cells (NFATc1), consistent with a role in regulating CTR gene expression in osteoclasts.

Agonist‐dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor

Transmembrane proline (P) residues in family A G protein-coupled receptors (GPCRs) form functionally important kinks in their helices. These residues are little studied in family B GPCRs but experiments with the VPAC1 receptor and calcitonin receptor-like receptor (CL) show parallels with family A receptors. We sought to determine the function of these residues in the insert negative form of the human calcitonin receptor, a close relative of CL. Proline residues within the transmembrane domains of the calcitonin receptor (P246, P249, P280, P326, P336) were individually mutated to alanine (A) using site-directed mutagenesis. Receptors were transiently transfected into Cos-7 cells using polyethylenimine and salmon and human calcitonin-induced cAMP responses measured. Salmon and human calcitonin competition binding experiments were also performed and receptor cell-surface expression assessed by whole cell ELISA. P246A, P249A and P280A were wild-type in terms of human calcitonin-induced cAMP activation. P326A and P336A had reduced function (165 and 12-fold, respectively). In membranes, human calcitonin binding was not detectable for any mutant receptor but in whole cells, binding was detected for all mutants apart from P326A. Salmon calcitonin activated mutant and wild-type receptors equally, although B(max) values were reduced for all mutants apart from P326A. P326 and P336 are important for the function of human calcitonin receptors and are likely to be involved in generating receptor conformations appropriate for agonist binding and receptor activation. However, agonist-specific effects were observed , implying distinct conformations of the human calcitonin receptor.

Fish calcitonin receptor has novel features

Calcitonin (CT), a 32-amino acid peptide, was initially isolated from fish. Fish CT has higher affinity to mammalian CT receptor (CTR), and has activity on calcium homeostasis. Therefore, fish CT has been used as a drug for the treatment of human bone diseases. However, the physiological roles of CT in fish as well as the characteristics of the fish CTR have not been clarified. Here, we cloned and characterized CTR from mefugu ( Takifugu obscurus). Full-length cDNA sequencing revealed that mfCTR (mf, mefugu) consists of N-terminal four tandem putative hormone-binding domains (HBDs). Database mining showed that the multiple HBD-containing CTR is a common feature for some other fishes. Detailed pharmacological studies revealed that mfCTR generated cAMP in response to (1) fish CT, (2) calcitonin gene-related peptide (CGRP) in combinations with receptor activity-modifying proteins (mfRAMPs) 1 and 4, and (3) amylin in combinations with mfRAMPs 1−5. Unlike mammalian CTR, mfCTR showed dual affinity sites. Corresponding EC 50 values of those are in close proximity of the in vivo concentration of CT in fish. Analyses of the deletion mutants of mfCTR demonstrated that only the nearmost HBD to the first transmembrane region is functional to the ligands. Although, fish CT has higher affinity to the human CTR, human CT did not bind to the mfCTR. This is the first report that demonstrates the structure and property of fish receptor for CT, CGRP, and amylin. Fish CTR is the first example that has multiple HBD-like sequences.