MON-538 Ca(2+)Allostery in PTH-Receptor Signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for two endogenous ligands, PTH and its related peptide (PTHrP). Binding of PTHrP to the PTHR results in only transient cAMP production that is limited to the plasma membrane, while PTH induces sustained cAMP responses derived from endosomes following internalization of the ligand-receptor complex. The underlying mechanism and regulation of these kinetically-distinct signaling events are poorly understood. Here we show that extracellular Ca(2+) acts as a positive allosteric modulator of the PTHR that promotes sustained cAMP production. Equilibrium and kinetic analysis of ligand binding and receptor activation revealed that extracellular Ca(2+) increases the residence time of ligands on the receptor, consequently increasing the duration of receptor activation and cAMP production. Additionally, we found that this sensitivity to Ca(2+) is lost for the PTH mutant R25C, identified as a cause of hypocalcemia in human patients. Using mass spectrometry (MS) approaches, we identified acidic clusters in the first extracellular loop of PTHR that serve a critical role in mediating Ca(2+) allostery and sustained cAMP signaling. These findings provide insights toward understanding the determinants of PTHR-mediated signaling events and how these processes are regulated. Sources of Research Support: the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH)