Calcium-dependent ligand binding and g-protein signaling of family B GPCR parathyroid hormone 1 receptor purified in nanodiscs

GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca²⁺ is a weak agonist of PTH1R, and Ca²⁺ in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.