Project Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR is a G protein-coupled receptor (GPCR) that activates multiple G proteins including stimulatory Gs, which subsequently activates adenylate cyclases and induces the production of cAMP. Previous research has suggested that PTHR signaling through Gs is the major mediator of bone anabolism. Therefore, understanding PTHR-Gs signaling is critical for designing novel drugs to treat bone and mineral-ion diseases, such as osteoporosis and hypocalcemia. Two FDA-approved therapeutics for osteoporosis, PTH(1-34) (teriparatide or PTH) and modified PTHrP(1–34) (abaloparatide or ABL), trigger distinct modes of Gs/cAMP signaling. Both ligands stimulate transient cAMP production at the plasma membrane. In addition, PTH stimulates prolonged cAMP production in endosomes. Synthetic long-acting PTH (LA-PTH) promotes endosomal Gs signaling more than does PTH and increases blood calcium levels in mice and primates. Therefore, LA-PTH is a promising therapeutic for hypocalcemia. While the cellular effects of PTH, LA-PTH, and ABL have been previously studied, the molecular mechanisms of biased signaling through each of these three ligands are not known. In the proposed research, we will investigate the molecular details of PTHR-Gs signaling through two aims. In Aim 1, we will determine atomic structures of ligand-bound PTHR-Gs complexes via cryo-electron microscopy. These structures will reveal PTH-, LA-PTH-, and ABL-specific states of PTHR-Gs. In Aim 2, we will identify structural determinants of ligand-selective PTHR signaling. We will use hydrogen-deuterium exchange (HDX) and chemical crosslinking (CX) coupled to mass spectrometry (MS) to identify PTHR-Gs complex interfaces in the presence of PTH, LA- PTH, and ABL. Furthermore, HDXMS will reveal the structural dynamics of PTHR-Gs interactions. The data from HDXMS and CXMS will be used to design PTHR mutants, whose signaling will be tested in live cells. Structural and functional data gathered from both aims will give insight into the distinct PTHR-Gs conformations necessary for transient and prolonged cAMP production. These data will be used in future work to design novel therapies for osteoporosis and other bone diseases. As such, the proposed research supports the mission of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS).