Structure/Function Relationships of Calcitonin Analogues as Agonists, Antagonists, or Inverse Agonists in a Constitutively Activated Receptor Cell System

Abstract

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

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

“…7B). These data are consistent with photocross-linking to the amino terminus of the mutant receptor with cleavage at Met 49 and confirm Met 49 as the site of cross-linking for [Bpa 8 ]sCT (8 -32) (Fig. 7C) 19 ]sCT (8 -32) efficiently labeled the HA-hCTR a receptor transiently expressed in COS-7 cells with a single radioactive band of M r ϳ97,000, which shifted to M r ϳ52,000 after Nglycosidase F deglycosylation (Fig.…”

“…11, A, C, and E), Leu 368 and Met 49 , the Bpa 8 cross-linking site of agonist and antagonist peptides, respectively, are located in close proximity to each other. In one model (Fig.…”

“…Amino-terminal truncation of the disulfide-bridged loop (amino acids 1-7) of sCT leads to the generation of potent antagonists (48,49) and allows for comparative analysis of the site of cross-linking of equivalent amino acids of agonist and antagonist peptides. In this study we have generated antagonist peptides substituted with the photolabile amino acid pbenzoyl-L-phenylalanine (Bpa) at residue 8 ( …”

Involvement of Microphthalmia in the Inhibition of Melanocyte Lineage Differentiation and of Melanogenesis by Agouti Signal Protein

“…The effects of ASP on basal and forskolin-induced differentiation fit better with the hypothesis that ASP would be an inverse agonist of the MC1 receptor. Inverse agonists, ligands that suppress spontaneous receptor signaling activity, have been described for a growing number of G-protein coupled receptors: the ␤ 2 -adrenoreceptor of myocardial cells (41), the 5-hydroxytryptamine receptors in NIH-3T3 fibroblasts (42), and the calcitonin receptors (43). Hence, ASP appears likely to act both as an antagonist of ␣MSH binding on MC1R and an inverse agonist to block both the effects induced by cAMP elevating agents and basal effects.…”

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

“…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).…”