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.

Figure 1. Functional output of cAMP, β-arrestin, ligand binding on membranes and live cells in response to calcitonins.

Head-to-head comparison of dose dependent cAMP accumulation (A) or β-arrestin recruitment (B), mediated by sCT and hCT after one (cAMP assay) and two (β-arrestin assay) hours of stimulation, respectively. The cAMP experiment was conducted with 100 μM IBMX in the medium to assess total cAMP production. Homologous competitive binding (C) and Ligand association (D) profiles of sCT and hCT using U2OS CALCR membranes preparations. Homologous competitive binding (E) and association (F) profiles of sCT and hCT using U2OS CALCR cells in culture. Data are shown as mean ± SEM and representative of three individual conducted experiments with six replicates (A,B) or three replicates (C–F).

Figure 2. Difference in CT_(a)R -mediated cAMP response by prolonged continuous hCT and sCT stimulation.

A) cAMP production as a function of ligand concentration as U2OS CALCR cells were stimulated with sCT or hCT at a dose range of 10 pM to 10 nM for a prolonged period of time, ranging from 1, 4, 24, 48 to 72 hours. Ligands and medium were only added during the initiation of the experiment. Assays were conducted without IBMX in the medium to assess time dependent cAMP production. B) Single dose of 10 nM sCT, 10 nM hCT and Vehicle shown in (A) plotted as a function of time. Asterisk (*) indicate significant difference between AUC sCT and AUC hCT, p<0.05 was considered to be significant. *  = p<0.05, **  = p<0.01, ***  = p<0.001. Data are shown as mean ± SEM and representative of three individual conducted experiments using five replicates.

Figure 3. Difference in CT_(a)R-mediated β-arrestin recruitment by prolonged continuous hCT and sCT stimulation.

A) Data show β-arrestin recruitment as a function of ligand concentration. U2OS CALCR cells were stimulated with sCT or hCT at a dose range of 0.1 nM to 100 nM for a prolonged period of time, ranging from 1, 4, 24, 48 to 72 hours. Ligands and medium were only added at the experiment initiation. B) Single dose of 100 nM sCT, 100 nM hCT and Vehicle shown in (A) plotted as a function of time. Asterisk (*) indicate significant difference between AUC sCT and AUC hCT, p<0.05 was considered to be significant. *  = p<0.05, **  = p<0.01, ***  = p<0.001. Data are shown as mean ± SEM of 3 experiments performed with five replicates.

Figure 4. Prolonged ERK activation/phosphorylation in Cos-7 CT_(a)R cells by sCT, but not hCT.

Cos-7 CT_(a)R Cells were stimulated for 5 min, 10 min, 30 min, 120 min, 24 hours or 48 hours with either Vehicle (A), 100 nM hCT (B) or 100 nM sCT (C). At each time point, the cells were lysed and protein content extracted. The protein extracts was then analyzed by western blotting investigating ligand-mediated phospho-ERK1/2 activation. Total ERK1/2 was used as a protein loading control.

Figure 5. Acid wash decrease, but do not attenuate sCT mediated prolonged cAMP and β-arrestin signaling.

Effects of acid wash on cAMP production (A) and β-arrestin recruitment (B). U2OS CALCR cells were initially stimulated for 60 min with sCT or hCT using U2OS CALCR cells, then acid washed for 2×2 min and cultured for another 3, 6, 24 or 48 hours in fresh ligand free culture medium, and then assayed at end incubation. In parallel, standard assay groups of sCT and hCT were included as controls. C) Internalization rate of ¹²⁵I-sCT and ¹²⁵I-hCT stimulated CT_(a) receptors in U2OS CALCR. Cells were pre-stimulated for 45 min at 4°C with 0.25 nM ¹²⁵I-sCT or 0.25 nM ¹²⁵I-hCT for ensure receptor occupancy, then incubating for an additional 0, 15, 30, 60 and 120 min at 37°C to assess internalization. Asterisk (*) indicates significant difference between AUC sCT, Not Washed and AUC sCT, Acid Wash. Asterisk (#) indicates significant difference between AUC hCT, Not Washed and AUC hCT, Acid Wash. p<0.05 was considered to be significant. *  = p<0.05, **  = p<0.01, ***  = p<0.001. cAMP data are shown as mean ± SEM and representative of three individual conducted experiments with five replicates. β-arrestin data are shown as mean ± SEM of 3 experiments performed with five replicates. Internalization data are shown as mean ± SEM and representative of two individual conducted experiments in triplicates.

Figure 6. Continuous stimulation attenuates additional prolonged responses in inverse proportion to ligand concentration.

U2OS CALCR cells were stimulated with sCT for 4, 24, 48, 72 or 96(A) or hCT for 4, 24, 48 or 72 hours (B) in a similar prolonged setup as demonstrated in Figure 2. In parallel, one set of cells was stimulated for 96h (sCT) or 72h (hCT), following which the culture medium was replaced with fresh medium with ligands and incubated for another 4, 24 or 48 hours. Ligand concentration sCT or hCT: 10 nM, 1 nM, 0.1 nM or vehicle. The vertical arrow indicates time of medium-ligand replacement.

Figure 7. Prolonged ligand dissociation kinetics on membranes preparations and live cells.

A) Total binding of ¹²⁵I-sCT and ¹²⁵I-hCT ligand bound to U2OS CALCR cells at different time points (0, 2, 4, 8, 24, 48 and 72 hours). B₀ is total ¹²⁵I-ligand bound to cells at t = 0 and B is ¹²⁵I-ligand bound. B) Specific activity of ¹²⁵I-sCT and ¹²⁵I-hCT ligand released into the supernatant by cells in (B) at different time points (0, 2, 4, 8, 24, 48 and 72 hours). C) ¹²⁵I-sCT and ¹²⁵I-hCT ligand dissociation was investigated using isolated U2OS CALCR membrane preparations. The dissociation from the membranes was measured after 1, 2, 4, 8, 24, 48 and 72 hours with or without the presence of 20 μM GTPγS to determine G-protein dependency. B₀ is total ¹²⁵I-ligand bound to membranes at t = 0 and B is ¹²⁵I-ligand bound.B₀ is total ¹²⁵I-ligand bound to cells at t = 0 and S is ¹²⁵I-ligand in the medium. Live cell assays were conducted at 37°C and membrane assays were conducted at room temperature.