Abstract
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.
Highlights
The calcitonins (CT) are a group of evolutionary conserved 32 amino acid peptide hormones, which have been isolated from many different species over the last 50 years and divided into several distinct groups based on structure and sequence [1]
The calcitonins bind to the calcitonin receptor (CT(a)R), which belongs to the class B subfamily of G-protein coupled receptors (GPCRs) [2,3]
The teleost/avian calcitonins are a unique group among the calcitonins as they bind with high affinity and potency to the CT(a)R [1] as well as to the amylin receptor (AMY-R) with salmon calcitonin (sCT) being a more potent agonist compared to amylin [11]. sCT binds almost irreversibly, with a very slow off rate from the CT(a)R [12] and induces a sustained cAMP accumulation [3,13,14]
Summary
The calcitonins (CT) are a group of evolutionary conserved 32 amino acid peptide hormones, which have been isolated from many different species over the last 50 years and divided into several distinct groups based on structure and sequence [1]. The teleost/avian calcitonins are a unique group among the calcitonins as they bind with high affinity and potency to the CT(a)R [1] as well as to the amylin receptor (AMY-R) with sCT being a more potent agonist compared to amylin [11]. SCT is much more potent in vivo [1], and this has assumptively been associated with longer plasma half-life. This clear cut difference is somewhat blurred as this is not always reflected in comparable in vitro studies [3,12,13,14,18,19,20]. It is apparent that sCT is different from hCT, as they only share 50% sequence homology, see Table 1, but whether this difference is purely a consequence of activation potency or whether other mechanisms are involved remains to be elucidated
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