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

Abstract

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 calcitoninsecretin 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 wildtype 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 (CT), 3 a 32-amino acid peptide secreted by the thyroid gland in response to elevations in blood calcium levels, acts on bone and kidney to maintain calcium homeostasis (1, 2). CT is widely used therapeutically for the treatment of bonerelated disorders such as osteoporosis, hypercalcemia of malignancy, and Paget disease (1, 2). This peptide must be administered parenterally, whereas small-molecule agonist ligands that can be administered orally have substantial clinical advantage, particularly for long-term treatment.CT acts on the CT receptor, a Family B G protein-coupled receptor (GPCR). Members of this family include receptors for secretin, vasoactive intestinal polypeptide, parathyroid hormone, corticotrophin-releasing factor, glucagon, glucagon-like peptide 1, CT, and CT gene-related peptide (3), with each having a long extracellular amino-terminal domain containing six conserved cysteine residues that form three intradomain disulfide bonds. This region has been shown to play a critical role in natural peptide ligand binding and receptor activation (4 -11).Development of small-molecule ligands for Family B GPCRs represents an area of great interest. For the CT receptor, several such ligands have been developed (12)(13)(14). However, the mechanisms of their actions at their receptors remai...

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…Class B peptide hormone GPCRs have been particularly difficult to target therapeutically because of the diffuse pharmacophore associated with the peptide orthosteric site, which is extracellular and involves multiple points of interaction with the N terminus and top of the transmembrane bundle. However, a number of small molecules have recently emerged that act allosterically, examples of which are found with molecules targeting the corticotrophin releasing factor-1 receptor (Hoare et al, 2008), the calcitonin receptor (Dong et al, 2009), and the glucagon-like peptide-1 receptor (Knudsen et al, 2007;Wootten et al, 2011). These findings suggest that the allosteric approach represents a viable path forward for discovering small molecules directed against these peptide-hormone receptors.…”

Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

“…Mutagenesis studies have also focused interest on this region (47,(51)(52)(53). Of note, the same region has also recently been shown to play a critical role in small molecule agonist action at the calcitonin receptor (54).…”

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor

“…This is also the analogous region of the VPAC 1 receptor labeled by amino-terminal positions 0 and 6, and carboxyl-terminal positions 22, 24, and 28 vasoactive intestinal polypeptide probes (47)(48)(49). Interestingly, this juxtamembranous region of the amino terminus of the calcitonin receptor has been recently shown to play a critical role in small-molecule agonist action (50).…”