Identification of an L-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium*

Background: The calcium-sensing receptor (CaSR) is a key mediator of Ca2+ homeostasis in vivo. Results: An l-Phe binding site at the CaSR hinge region globally enhances its cooperative activation by Ca2+. Conclusion: Communication between the binding sites for Ca2+ and l-Phe is crucial for functional cooperativity of CaSR-mediated signaling. Significance: The results provide important insights into the molecular basis of Ca2+ sensing by the CaSR. Functional positive cooperative activation of the extracellular calcium ([Ca2+](o))-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2+](o) or amino acids elicits intracellular Ca2+ ([Ca2+](i)) oscillations. Here, we report the central role of predicted Ca2+-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+](o). Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+](o) and l-Phe in eliciting CaSR-mediated [Ca2+](i) oscillations. The heterocommunication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+](o) signaling by positively impacting multiple [Ca2+](o)-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+](o) and amino acids into intracellular signaling events.