The calcium-sensing receptor (CaSR) regulates systemic calcium homeostasis by controlling parathyroid hormone (PTH) secretion and renal calcium handling. Calcimimetics and calcilytics modify its sensitivity to extracellular calcium, providing therapeutic approaches to disorders of mineral metabolism. Understanding how these agents influence receptor activation is essential for developing drugs with improved pharmacological properties.

We combined cryo-electron microscopy, functional assays, and structure-based ligand discovery to investigate the mechanisms of CaSR activation and allosteric modulation. Structures of inactive, active, and G-protein-bound receptors reveal that activation reorganizes the CaSR homodimer into an asymmetric transmembrane assembly that primes one subunit for G-protein coupling. Calcimimetics stabilize this active configuration, whereas calcilytics favor an inactive, symmetric arrangement. Coupling to Gi or Gq involves further remodeling of the dimer interface

and distinct interactions with intracellular receptor regions. The structures also reveal mechanisms of endogenous modulation: polyamines stabilize interactions between the two subunits, while L-tryptophan occupies a transmembrane site overlapping the calcimimetic binding pocket.

Using this structural framework, we screened large virtual compound libraries and experimentally optimized new positive allosteric modulators. One lead showed greater potency than cinacalcet in suppressing PTH secretion from isolated parathyroid glands and reduced circulating PTH in mice without inducing hypocalcemia under the conditions tested.

These studies explain how endogenous ligands and allosteric drugs regulate CaSR signaling and establish a foundation for structure-guided drug discovery. The findings suggest that distinct modes of receptor modulation may offer opportunities to suppress PTH secretion while reducing the hypocalcemia associated with current calcimimetics.