Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors

This study investigates the poor reversibility of salmon calcitonin (sCT) b inding to rat and human calcitonin receptors. Efficacy of CT and analogue p eptides in I-125- sCT binding competition and cAMP assays was compared with the dissociation kinetics of I-125-labelled peptides. Assessment was perfo rmed on cells stably expressing either rat or human calcitonin receptors. D issociation kinetics of the antagonists, sCT(8-32) and AC512, revealed that binding was rapidly and completely reversible at the receptors, despite hi gh affinity binding, suggesting that poor reversibility required the active conformation of the receptor. G protein coupling was not essential as the dissociation kinetics of I-125-sCT binding to cell membranes did not signif icantly alter in the presence of GTP gamma S. Time course experiments estab lished that the transition to irreversibility was slow, while the reversibl e component of binding appeared to involve a single population of either re ceptor states or binding sites. Pre-bound I-125-human CT dissociated rapidl y from the receptors, indicating that not all agonists bound irreversibly. To identify structural features of sCT that contribute to its poor reversib ility, dissociation kinetics of sCT analogues with various structural modif ications were examined. Increasing truncation of N-terminal residues of sCT analogues led to a corresponding increase in the rate of peptide dissociat ion. Salmon CT peptides which had been substituted at the N-terminus by 13- 21 residues of human CT (hCT) were equipotent with sCT in binding competiti on and cAMP accumulation assays but exhibited a dissociation rate similar t o hCT. In contrast, despite lower affinity and efficacy at the receptors, t he chimeric analogue sCT(1-16)-hCT(17-32) displayed poorly reversible bindi ng, similar to sCT. Analysis of the dissociation kinetics of sCT analogues with differing alpha-helix forming potential indicated that the ability to form alpha-helical secondary structure was an important factor in the rate of ligand dissociation. We hypothesise that poor reversibility results from a conformational change in the receptor and/or ligand and that this is dep endent, at least in part, on interaction with residues constrained within t he alpha-helix of the peptide.