Figure 5. Positive allosteric modulation of the four CaSR heterodimers of interest.

HEK293T cells transiently transfected with a HA-tagged CaSR construct (or pEGFPN1) and a myc-tagged CaSR construct (or pEGFPN1) in a 1:1 ratio were tested. Specifically, cells co-expressing (a) HA-S170A and myc-F801A, (b) HA-S170A/E837A and myc-F801A/E837A, (c) HA-S170A and myc-F801A/E837A and (d) HA-S170A/E837A and myc-F801A were tested. The function of these heterodimers was assessed using the IP-One assay, which measures increases in d-myo-inositol monophosphate (IP₁) upon activation of the G_q signaling pathway. The endogenous agonist Ca²⁺ was tested in increasing concentrations in the presence and absence of 0.05 μM, 0.1 μM and 1 μM NPS R-568, respectively. (e) Effect of 1 μM NPS R-568 on the EC₅₀ of Ca²⁺ on WT HA-CaSR and the S170A:F801A and S170A:F801A/E837A heterodimers. EC₅₀ values are shown as fold decrease of the EC₅₀ value of Ca²⁺ alone for each of the constructs. EC₅₀: the ligand concentration that is required for eliciting 50% of the maximum response. Data are means ± S.E.M. of three independent experiments performed in triplicates. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-test (ns, P > 0.05). (f,g) The function of each mutant upon co-expression with pEGFPN1 was assessed by testing (f) 10 mM Ca²⁺ or (g) 23 mM Ca²⁺ in the presence and absence of 0.05 μM, 0.1 μM and 1 μM NPS R-568, respectively. In (g), statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-test in which the Ca²⁺ response for each mutant was compared to the Ca²⁺ response of S170A (*P  < 0.05) (h) Ca²⁺ concentration-response curves of HA-S170A were generated in the presence and absence of 0.05 μM, 0.1 μM and 1 μM NPS R-568, respectively. Data obtained in Fig. 5a–d,h from stimulation with 4.6 mM Ca²⁺ is depicted in (i). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-test in which responses in the presence of PAM were compared to the Ca²⁺-mediated response alone for each mutant homodimer or heterodimer (*P  < 0.05, ***P < 0.001). All data are shown as fold increase over the basal level of IP₁ upon incubation in ligand buffer and are means ± S.E.M. of three independent experiments performed in triplicates.