Abstract 1813 Role of flexible loop on cAMP production in parathyroid hormone 1 receptor activation

Parathyroid hormone 1 receptor (PTH1R) is a G-protein coupled receptor (GPCR) that is a crucial regulator of calcium ion homeostasis and bone density. PTH1R is activated by parathyroid hormone (PTH) which is known to activate numerous pathways through heterotrimeric G protein activation. For instance, PTH binding produces cAMP through the activation of the Galphas G protein signaling pathway. Although the specific binding interaction between PTH and PTH1R is known, it is not known how PTH binding modulates PTH1R dynamics and activation states. Further, extracellular Ca2+ is a positive allosteric modulator of PTH1R signaling likely through interactions with extracellular loop 1 (ECL1). The specific role of ECL1 in PTH1R signaling in different physiological conditions is unknown. Therefore, we will use cAMP assays to measure cAMP produced downstream of changing receptor and hormone conditions in an effort to determine the extent of activation and binding in PTH1R across various mutations and conditions. We will be mutating residues 258-260 within ECL1, which is a flexible loop important in allosteric modulation and PTH binding. Preliminary results show two PTH1R constructs with ECL1 mutations, E258A PTH1R and E258AE259AE260A PTH1R both modulate downstream cAMP production as compared to the wild type PTH1R. The results will enhance our understanding of the role of ECL1 in PTH1R signaling and thus the role of flexible loops in modulating receptor binding and activation. This will ultimately aid in the development of new treatments for osteoporosis and broaden our understanding of the conformational dynamics of class B GPCRs. This research is supported by the Lawrence University Research Fellowship and the Chandler Senior Experience Fund.