Prolonged Calcitonin Receptor Signaling by Salmon, but Not Human Calcitonin, Reveals Ligand Bias

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.

Can residence time offer a useful strategy to target agonist drugs for sustained GPCR responses?

“…Long residence time could drive sustained receptor activation through a single preformed stable binding event that persists after removal of unbound agonist (Fig. 1a), as has been implicated for a number of GPCR agonists [18,20,23]. In the simplest model, ligand binds to receptor to form ligand-receptor complexes and, in binding kinetic experiments, residence time is calculated at the reciprocal of the rate of dissociation (K off ) of these complexes (Eqn 1).…”

“…Using in vitro experiments this can be exhibited by 'wash-resistant' receptor activation, where signaling continues even after excess agonist is removed from the receptor by infinite dilution of the drug-containing buffer [8,9,[16][17][18][19][20][21][22]. It is anticipated that this could Thyroid-stimulating hormone SSIR >30 min a [9] Sphingosine-1 phosphate-1 SSIR; residence time and rebinding 300 min a Q6 [16,42] b2-adrenergic SSIR; exosite; membrane deposition >180 min a or >10 min b [25,27,43,44] Vasopressin-2 SSIR >40 min a [17] Glucagon-like peptide SSIR n.d. [37] Angiotensin-II SSIR >30 min b [45] Calcitonin a SSIR; residence time > 4320 min b [18] Serotonin 5-HT2B SSIR; residence time and rebinding >240 min a [19] GPR119…”

“…Anecdotal evidence suggests that agonist dissociation kinetics can regulate whether a ligand can evoke SSIR. For PTH receptor, calcitonin and serotonin 5-HT2B receptors the observation of SSIR appears to be characteristic of agonists with slow dissociation from these receptors, while being absent with rapidly dissociating ligands [8,18,19]. It is therefore tempting to speculate that long residence time is a general feature of SSIR agonists, although this remains to be characterized for other receptors.…”

“…We speculate that the residence time of an agonist relative to receptor internalization rate will determine the proportion of agonist co-internalized with the receptor. Based on measurements made at 378C for multiple receptor systems, GPCR ligands have been observed to have residence times ranging from less than a minute to a number of hours [8,18,23,32,33]. In this model we describe ligands that fall within this range.…”

Amylin: Pharmacology, Physiology, and Clinical Potential

“…Activation of ERK1/2 is clearly a pathway that amylin can use, but more work is needed to determine the precise circumstances under which this occurs and the mechanism involved. There has been no specific investigation of whether amylin-induced ERK activation is G protein dependent or arrestin mediated; however, the calcitonin receptor can recruit arrestin in response to human and salmon calcitonin (Andreassen et al, 2014). Amylin was recently reported to either increase or decrease ERK and Akt phosphorylation in dispersed islets, depending on the amylin and glucose concentration (Visa et al, 2015).…”