Ca²⁺ allostery in PTH-receptor signaling

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains G_S-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca²⁺ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca²⁺ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca²⁺ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTH^R25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca²⁺ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca²⁺ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTH^R25C suggest that Ca²⁺ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.