Ca 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-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 Ca 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 Ca 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 Ca allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTH) 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 Ca allosterism and endosomal cAMP signaling. These findings coupled to defective Ca allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTH suggest that Ca allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.