Illuminating the allosteric modulation of the calcium-sensing receptor

Abstract

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca²⁺ alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca²⁺ activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

Keywords: G protein-coupled receptor; allosteric modulator; amino acids; calcium; nutrient sensing.

Fig. 1.

A conformational FRET-based sensor to investigate the structural dynamics of the CaSR ECD. (A) Orthosteric binding sites for calcium remain unclear and controversial. Many genetic mutations have been found in patients with calcemic disorders, and they are located in the different regions of the receptor. Besides, autoantibodies have been identified in rare autoimmune diseases. (B) Crystal structures of the extracellular domain of class C GPCR dimers in the resting forms (Left, CaSR PDB 5K5T biological assembly 2, mGluR5 PDB 6N52, and GABA_BR PDB 4MQE) and in the active forms in the presence of l-Trp, l-quisqualate, or GABA (Right, CaSR PDB 5K5S, mGluR5 PDB 6N51, and GABA_BR PDB 4MS3, respectively). The residues used to measure distances [D23 for CaSR, R26 for mGluR5, R50 for GABA_B1b (Right) and S53 for GABA_B2 (Left), respectively] are highlighted as orange spheres. (C) Cartoon illustrating the full-length SNAP-CaSR labeled with the Lumi4-Tb donor and the green fluorescent acceptor, with high FRET signal in the absence of agonist and a lower FRET signal in the presence of agonist. (D) FRET signal between the two VFTs after cell surface labeling of SNAP-CaSR-expressing cells with fluorophores as indicated in C, in the presence of a saturating concentration of calcium (20 mM CaCl₂, injection at t = 30 s) and after calcium removal (t = 60 to 90 s). Data are mean ± SEM of a typical experiment performed in replicates. The control with 20 mM CaCl₂ (dotted red line) or buffer alone (dotted blue line) are shown for the same period of time (0 to 90 s). (E–H) Correlation (H) between the potencies (pEC₅₀) of different agonists on CaSR determined by FRET sensor assay (E), inositol monophosphate (IP₁) accumulation assay (F), and intracellular calcium (Ca²⁺_i) release assay (G). Data in E–G are mean ± SEM of at least three independent experiments performed in triplicates and normalized to the maximum response of CaCl₂. Data in H are mean ± SEM from the fitted curves for each individual experiment in E–G. (I) FRET signal measured for CaCl₂ in the presence of either PAM (10 μM NPS R-568) or NAM (10 μM NPS 2143). Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to the maximum response of the control. (J) FRET signal measured for the indicated mutants in the absence or presence of 20 mM CaCl₂. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to WT. Data are analyzed using two-way ANOVA with Tukey’s multiple comparisons test to determine significance, with ****P ≤ 0.0001.

Fig. 2.

The 7TM interface rearrangement is revealed by cysteine cross-linking and FRET. (A) Schematic representation of CaSR WT, mutant C129A-C131A (CACA, to remove the endogenous disulfide bonds between the two VFTs of CaSR dimer) and 7TM cysteine mutants with CACA background. (B) A 3D model of the CaSR 7TM in lateral and top view. Residues substituted by cysteine are highlighted as yellow spheres (α carbon), and the well cross-linked ones are highlighted in red. (C) Cysteine cross-linked mutants screened by TR-FRET in the absence or presence of 20 mM CaCl₂ with and without CuP treatment. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to WT (data with and without CuP are normalized separately). Data are analyzed using two-way ANOVA with Tukey’s multiple comparisons test to determine significance within each mutant group, with ****P ≤ 0.0001 and **P ≤ 0.01. (D) Analysis of cell surface CaSR subunits of the cysteine mutants (CACA as control) in SDS/PAGE experiments under nonreducing conditions, after treatment (+) or without treatment (−) with CuP. Changes of dimer ratio induced by CuP treatment for WT, the CACA control, and every indicated mutant are quantified and shown. Quantitative data are mean ± SEM of at least four independent experiments (n = 4 to 12) while the blot for each mutant is representative of one of these experiments. Data are analyzed using one-way ANOVA with Dunnett’s multiple comparisons test to determine significance (compared with CACA control), with ****P ≤ 0.0001, **P ≤ 0.01, and ns P > 0.05. (E) IP₁ accumulation for WT and the indicated TM6 mutants after treatment (+) or without treatment (−) with CuP, in the absence or presence of 20 mM CaCl₂. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to WT (data with or without CuP are normalized separately). Two-way ANOVA with Tukey’s multiple comparisons test within each mutant group, with ****P ≤ 0.0001. (F) Model highlighting the TMs involved in the dimerization of CaSR in the resting state and in the active state.

Fig. 3.

Allosteric modulation by l-AA is clarified with a cell-free assay. (A) Cartoon illustrating the development of FRET-based CaSR biosensor in a cell-free assay based on cellular membrane preparations as indicated. (B) FRET signal measured on nondialyzed membranes with the indicated ligands. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to the maximum response of CaCl₂. (C) Correlation between the FRET potencies (pEC₅₀) of the indicated agonists determined on cells and nondialyzed membranes. (D) FRET measurement performed with CaCl₂ on nondialyzed and dialyzed membranes in the absence or presence of 10 mM l-Phe. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to the basal of control and the maximum response of l-Phe. (E) FRET potencies (pEC₅₀) of calcium on cells and on nondialyzed (−) or dialyzed (+) membranes in the absence or presence of 10 mM l-Phe. Data are mean ± SEM from at least eight independent experiments (n = 8 to 24). Two-way ANOVA with Tukey’s multiple comparisons, with ****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05, and ns P > 0.05. (F) Basal FRET in the presence of the indicated l-AA at 10 mM (for Tyr-1 mM is used due to low solubility) or 10 μM TNCA. Data are mean ± SEM of at least three independent experiments (n = 3 to 13) performed in triplicates and normalized to the control. One-way ANOVA with Dunnett’s multiple comparisons test (compared with control) with ****P ≤ 0.0001, *P ≤ 0.05, and ns P > 0.05. (G) FRET potencies (pEC₅₀) of calcium on dialyzed membranes in the presence of the indicated amino acid (same concentrations as in F). Data are mean ± SEM from at least three independent experiments (n = 3 to 13). One-way ANOVA with Dunnett’s multiple comparisons test (compared with control) with ****P ≤ 0.0001 and ns P > 0.05. (H) FRET signal change induced by the indicated l-AA performed on dialyzed membranes in the presence of 5 mM CaCl₂. Data are mean ± SEM of at least three independent experiments performed in triplicates. Data are normalized to the basal conditions (5 mM CaCl₂ without l-AA) and the maximum response induced by l-AAs.

Fig. 4.

Allosteric modulation induced by chloride ions in CaSR VFT. (A) Three chloride binding sites were reported in the CaSR structure (as shown in PDB 5FBK, sites b, c, and g refer to SI Appendix, Fig. S1). (B and C) FRET measurement (B) and Ca²⁺_i release (C) for calcium gluconate on CaSR performed in a buffer with the indicated concentrations of chloride ions. Either Ca²⁺ concentration (Left) or Cl⁻ concentration (in buffer, Right) is used as the x axis. Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum response in the buffer with the highest Cl⁻ concentration.

Fig. 5.

Binding of two calcium ions nearby l-AA stabilizes the active state. (A) Cartoon illustrating the possible calcium binding at the lobe 2 interface which was previously proposed to be important for receptor activation. (B) Ca²⁺_i release for the indicated mutants in this lobe 2 interface. (C) Possible Ca²⁺ binding in the l-AA binding pocket. (D–F) Proposed Ca²⁺ binding site 1 in the VFT hinge as illustrated by the cartoon (D), the 3D model of this site based on the crystal structure of the VFT (PDB 5K5S) (E), and Ca²⁺_i release data for the indicated mutant in this site (F). Ca²⁺ is proposed to be bound to S170, D190, Q193, Y218, E297, and one water molecule found in the crystal structure. (G–I) Similar analysis for the proposed calcium binding site 2 in the VFT adjacent to l-AA, and Ca²⁺_i release data for the indicated mutants. Ca²⁺ is proposed to be bound to the lobe 2 residues D216, S272, D275, and one water molecule found in the crystal structure (bridging this Ca²⁺ and bound l-Trp). (J–L) Combination of the two functional calcium binding sites 1 and 2 adjacent to the bound l-AA, top view of the l-AA surrounded by the two Ca²⁺ and Ca²⁺_i release data for the indicated mutants. In the 3D model, interactions are shown as dashed lines (green for H-bonds, gray for metal bonds). Data in B, F, I, and L are mean ± SEM of at least three independent experiments performed in triplicates and normalized to WT. Mock indicates mock transfected cells with empty pRK5 vector.

Fig. 6.

Model for the activation of CaSR. (A) A 3D model of two possible functional calcium sites 1 and 2 near the bound l-AA based on the crystal structure of the VFT (PDB 5K5S). (B) VFT close state is proposed to be stabilized by calcium ions in the presence of ambient l-AA (cell-based conditions), but also by calcium ions alone in the absence of l-AA (cell-free conditions) during activation. The ambient l-AAs bound to CaSR VFT contribute to the high calcium potency and enable the receptor to sense low concentrations of calcium ions. But this high sensitivity to calcium is reduced when l-AA is lost. (C) FRET measurement performed with CaCl₂ on dialyzed membranes in the absence or presence of one-fold l-AA mixture containing l-AA concentrations mimicking the human fasting plasma. Data are mean ± SEM of at least three independent experiments performed in triplicates and normalized to the basal of control and the maximum response of l-AA mixture. (D) FRET signal change induced by different folds of the l-AA mixture (C) performed in dialyzed membranes in the presence of CaCl₂. Data are mean ± SEM of at least three independent experiments performed in triplicates. Data are normalized to the basal (without Ca²⁺ or l-AA) and the maximum response induced by Ca²⁺ in the presence of l-AA mixture. The vertical dotted line represents the related total concentration 2.82 mM of l-AAs used in one-fold l-AA mixture. (E) Molecular mechanism of activation of the CaSR upon l-AA and calcium binding. Binding of calcium ions in the VFT binding pocket most probably occupied by l-AA in physiological conditions, is expected to stabilize VFT closure and their relative rearrangement. Then it would induce CRD interactions and 7TM interface reorientation through allosteric propagation of the conformation changes. In the active state, TM6s will be at the dimer interface, a conformation required to stabilize at least one of the 7TMs in the active state for G protein activation.