Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

Abstract

The calcitonin receptor is a member of the class B family of G protein-coupled receptors, which contains numerous potentially important drug targets. Delineation of themes for agonist binding and activation of these receptors will facilitate the rational design of receptor-active drugs. We reported previously that a photolabile residue within the carboxyl-terminal half (resi- Calcitonin (CT), 1 secreted by the thyroid gland in response to elevations in blood calcium levels, is a peptide hormone that regulates calcium by inhibition of osteoclast-mediated bone resorption (1, 2). CT acts on bone and kidney to maintain calcium homeostasis and is also present in the central nervous system, where it has anorectic and analgesic effects (3). It has been used therapeutically for the treatment of Paget's disease, the hypercalcemia associated with certain types of tumors, and osteoporosis (1, 2).dueCT is a relatively large peptide that contains 32 amino acids and has a diffuse pharmacophoric domain. Although residues throughout the entire length have been demonstrated to be critical for its biological activity, the amino-terminal residues of CT contain key determinants for its receptor agonist selectivity (1, 2). Truncation of the first seven amino-terminal residues that includes a disulfide bond between residues 1 and 7 results in antagonist action (4, 5). Residues 8 through 22 tend to form an amphiphilic ␣-helical structure that is important for high affinity binding (1).CT exhibits its agonist activities through binding to the CT receptor, a member of the class B family of guanine nucleotidebinding protein (G protein)-coupled receptors that have the seven-transmembrane-domain structure. Although they have topology similar to that of class A receptors, members of class B family share less than 12% amino acid identity with the more extensively studied receptors in the class A family. Class B receptors have distinct signature sequences, including a long complex amino-terminal domain with six conserved cysteine residues that are believed to be involved in intradomain disulfide bonds critical for establishing functional receptor conformation (6 -10). Members included in this family are receptors for moderately large peptides having diffuse pharmacophoric domains, such as secretin, calcitonin, glucagon, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, and parathyroid hormone, sharing 30 to 50% homology with each other.The unique amino-terminal domain of the CT receptor has been shown to be critical for agonist binding and receptor activation using chimeric receptor studies (11)(12)(13). This represents a consistent theme for other class B family members (14 -19). Photoaffinity labeling is a more direct approach for exploration of spatial approximations between residues within a ligand and within its receptor. Using this approach, we have recently demonstrated that probes incorporated a photolabile p-benzoyl-L-phenylalanine (Bpa) residue in the carboxyl-terminal half and mid-region of the ...

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

“…However, another two carboxyl-terminal position 23 and 28 probes were shown to label the distal amino terminus (49,50). The labeling of the calcitonin receptor is quite consistent with the data from the secretin and PTH receptors (44,51), but the labeling of the VPAC1 receptor suggests a different mechanism. All the vasoactive intestinal polypeptide (VIP) probes tested so far label the amino-terminal domain adjacent to the transmembrane domain.…”

“…Based on these studies, a "two-step" or "tethering" mechanism has been proposed, where the carboxyl-terminal region of the peptide ligand initially interacts with the amino-terminal domain of the receptor, and in the second step, the amino-terminal region of the ligand interacts with the core domain of the receptor, which leads to receptor activation (27). This mechanism has also been proposed for other members of Family B GPCRs (42)(43)(44). Our current data show that both the position 24 and 35 residues of GLP1 are in proximity with the residues in the amino-terminal domain of its receptor, which is consistent with this mechanism.…”

Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Has a Critical Role in GLP-1 Peptide Binding and Receptor Activation

“…Each receptor possesses a characteristically large and sequence-divergent extracellular N-terminal domain; however, there is conservation of key residues, including three disulfide bonds within this domain, that aids in stability and confers similarities in secondary structure (3,4). The widely accepted peptide-receptor binding model for family B GPCRs is the two domain model, whereby the ␣-helical C terminus of the endogenous ligand interacts with the N-terminal domain of the receptor, and the N terminus of the peptide interacts with the core domain of the receptor, which includes both the extracellular loops and TM bundle (5)(6)(7). Generically, the N-terminal domain of the receptor is primarily responsible for ligand recognition and specificity, whereas the core of the receptor has a major influence in signaling specificity and transmission (8).…”

Insights into Interactions between the α-Helical Region of the Salmon Calcitonin Antagonists and the Human Calcitonin Receptor using Photoaffinity Labeling

“…In human CT (hCT), amino acid 16, located one helical turn apart from residue 19, cross-linked to Phe 137 , consistent with orientation of the ␣-helix of agonist peptides with the membrane-proximal region of the receptor amino terminus (46). We have also demonstrated that hCT amino acid 26 interacts with Thr 30 in the distal amino terminus (46), whereas hCT amino acid 8 interacts with Leu 368 in the third extracellular loop (47).…”