OR30-3 Structural Basis of Activation and Sustained Signaling by the PTH Receptor

Abstract

Parathyroid hormone (PTH) receptor (PTHR) is a medically important family B G Protein Coupled Receptor (GPCR) that primarily couples to Gs/cAMP and Gq/Ca2+ signaling pathways and has a central role in regulating Ca2+ homeostasis and bone turnover. PTHR is activated by two endogenous hormones, endocrine PTH and paracrine PTH related protein (PTHrP), but only PTH mediates prolonged activation of the receptor and sustained cAMP responses after receptor internalization to the endosomes. To investigate the structural basis of prolonged activation of PTHR, we employed cryo-EM and solved the high resolution (3Å) structure of PTHR coupled to Gs and activated by long-acting PTH (LA-PTH), an analog of PTH that remarkably sustains endosomal PTHR signaling and induces significantly prolonged elevation in blood calcium in mice and monkeys. LA-PTH binds the receptor as a single extended α-helix with its N-terminus inserted deep into the transmembrane domain (TMD) of the receptor, while positioning the extracellular domain (ECD) of PTHR perpendicular to the cell membrane. The receptor activating N-terminal half of LA-PTH forms a dense network of hydrophobic and polar interactions with receptor’s TMD resulting in a tight binding and giving structural basis for remarkably sustained activation of PTHR by LA-PTH. Binding of LA-PTH activates the receptor by inducing partial unwinding of the C-terminus of transmembrane helix 6 (TM6) and a sharp kink at the middle of this helix, a hallmark of family B GPCR activation. This results in a pronounced outward movement of TM6, opening the cytosolic cavity of the receptor, which allows subsequent G protein coupling. Additionally, our cryo-EM data revealed structural dynamics of PTHR demonstrated by the ability of receptor ECD to adopt multiple conformational states. Together, these results reveal the structural determinants of PTHR signaling and provide a molecular framework for structure-based design of novel therapeutics for osteoporosis and hypocalcaemia treatment.