Fig. 2: The inter-subunit disulfide bonds block the constitutive activity of CaSR.
From: Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges
a Close-up view of the upper loop where the two cysteine residues Cys129 (orange circle) and Cys131 (yellow circle) critical for the formation of two inter-subunit disulfide bonds (PDB: 7M3E). Of note, in this structure, only the two Cys131 forms a disulfide bridge are shown, meanwhile in another structure (PDB: 7DTV) this is the two Cys129 that forms a disulfide bridge. This variability between CaSR structures is most probably due to the high flexibility of this upper loop. b IP1 accumulation induced by CaCl2 in HEK-293 cells transfected with the CaSR WT or indicated mutants and the corresponding potencies (n = 3, pEC50 = −logEC50). c In both Flag-tagged WT receptor and indicated mutants, basal IP1 accumulation is proportional to the amount of receptors at the cell surface measured by ELISA. Data are mean ± SD from a typical experiment performed in triplicates (n = 3). d Effect of NAM NPS-2143 (10 μM, pretreated for 1.5 h) on the basal IP1 accumulation measured for the WT and indicated mutants (n = 4). e Intracellular calcium release for the WT and indicated mutants stimulated by PAM R568 (n = 5) or ago-PAM AC265347 (n = 4) in the absence of ligands. f Basal IP1 accumulation measured for the WT and indicated mutants (n = 4). g Schemes illustrating the link between the number of inter-subunit disulfide bonds and the basal activity of CaSR. Data above are mean ± SEM of at least three biologically independent experiments each performed in triplicates and normalized to mock (c, d, e, f) or the WT (b). Significance was analyzed using one-way ANOVA with Dunnett’s multiple comparisons (b, f) or two-way ANOVA with Sidak’s multiple comparisons (d) with ****P ≤ 0.0001, **P ≤ 0.01, ns for P > 0.05 versus the mock (d, f) or the WT (b), and ####P ≤ 0.0001, ###P ≤ 0.001, ns for P > 0.05 compared with indicated groups.
