Mechanistic Basis of Calcium Sensing Receptor Signaling

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ABSTRACT: Mechanistic Basis of Calcium Sensing Receptor Signaling The calcium sensing receptor (CaSR) is the master regulator of calcium metabolism in human and represents an outstanding drug target for the treatment of parathyroid disorders that develop in patients with chronic kidney diseases (CKDs). For patients with renal disfunction that develop hyperparathyroidism, calcimimetic drugs that act as positive allosteric modulators (PAMs) of the CaSR are the favored therapeutics. PAMs, such as cinacalcet, evocalcet and etelcalcetide, are approved treatment for CKD; however, their clinical use is limited due to their adverse side effects. By elucidating the dynamic structural mechanisms of receptor activation, its modulation by small-molecule modulators, and the specificity of G protein activation, we seek to understand in detail the CaSR signaling mechanism and enable the rational design of improved therapeutics modulating receptor function. CaSR is a family C member of G protein-coupled receptors (GPCRs), which also include the metabotropic glutamate receptors (mGlus) and the metabotropic gamma aminobutyric acid receptor (GABAB). Like other members of this family, CaSR functions as an obligate homodimer with an N-terminal extracellular domain (ECD) responsible for ligand binding, linked to the seven-transmembrane (7TM) domain. We have recently determined cryo-electron microscopy (cryoEM) structures of the near-full-length human CaSR homodimer in active and inactive states, revealing how ECD rearrangement upon Ca2+ binding induces the activation of the 7TMs and how allosteric modulators engage the receptor. Our results illustrate an essential asymmetry in the active state where each CaSR protomer is stabilized by a PAM molecule bound to each 7TM in two distinct conformations leading to the activation of only one transmembrane region, priming it for G protein coupling. Here we propose to extend these studies in order to characterize the mechanism and specificity of G protein activation by CaSR, its dynamics, as well as the detailed action of allosteric modulators with distinct pharmacological interest. Specifically, we seek to apply: structure-based mutagenesis coupled with cell signaling assays that monitor the effects of allosteric modulators; cryoEM structural studies of CaSR alone and in complex with allosteric modulators and distinct G proteins in a near native lipid environment; and single-molecule fluorescence resonance energy transfer (smFRET) complemented by double electron-electron (DEER) spectroscopy to reveal the dynamics of receptor and G protein activation as well as its modulation by different allosteric ligands. Collectively, the proposed structural, cellular, biochemical and biophysical experiments aim to provide a full mechanistic framework for transmembrane signaling by CaSR and will guide the future development of novel drugs targeting this receptor.