Electrostatic Interactions with Extracellular Loop 1 Modulate Hormone-Specific Changes in Parathyroid Hormone 1 Receptor Activation and Signaling

Parathyroid hormone 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) expressed in bone and kidney cells that is activated by two endogenous hormones, parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). Hormone activation promotes G protein binding and downstream signaling from the Gs and Gq G protein isoforms. Flexible regions of PTH1R impact the stability of hormone and G protein interactions to modulate the downstream signaling. Extracellular loop 1 (ECL1) is one flexible region of PTH1R, which is abnormally long compared to other family B GPCRs and is implicated in positive allosteric modulation of PTH1R. However, a detailed understanding of how ECL1 interactions regulate PTH1R signaling is unknown. Therefore, we created a PTH1R mutant with ECL1 removed (ΔECL1 PTH1R) to investigate the role of the flexible loop in G protein binding and downstream signaling. Compared to WT, ΔECL1 PTH1R increased Gs interactions with the receptor and downstream signaling induced by PTH while decreasing those induced by PTHrP. However, ΔECL1 PTH1R increased both PTH and PTHrP Gq interactions and signaling compared to those induced by WT. The hormone-specific regulation of Gs signaling depends on charged residues in the C-terminal regions of PTH and PTHrP. The hormones have opposite electrostatic interactions with clusters of negative charges in ECL1, where ECL1 repels negative charges in PTH and stabilizes positive charges in PTHrP. Understanding the mechanism of ECL1 regulation of PTH1R signaling provides a clearer understanding of PTH and PTHrP interactions as future targets for drug development.