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

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. The calcium-sensing receptor (CaSR) is a key mediator of Ca2+ homeostasis in vivo. An l-Phe binding site at the CaSR hinge region globally enhances its cooperative activation by Ca2+. Communication between the binding sites for Ca2+ and l-Phe is crucial for functional cooperativity of CaSR-mediated signaling. The results provide important insights into the molecular basis of Ca2+ sensing by the CaSR.