Purification of Family B G Protein-Coupled Receptor using Nanodiscs: Calcium Dependent Ligand Binding and G-Protein Signaling of Parathyroid Hormone 1 Receptor

G protein-coupled receptors (GPCRs) belong to the largest gene family in the human genome. They mediate intracellular signaling in response to external stimuli, making them ideal drug targets. Despite their importance, activation mechanisms of GPCRs, especially those in Families B and C, are poorly understood at the molecular level. This is mostly because GPCRs are difficult to purify to the quantities enough for mechanistic biophysical studies. Here, we develop a purification method using Nanodiscs, which are nanometer-sized lipid bilayer disk-shaped particles. We use Nanodiscs to incorporate GPCRs immediately after membrane solubilization. We then perform chromatographic purification, while the GPCRs are in the lipid environments of Nanodiscs. This approach minimizes the contact time of the GPCRs with detergent, and thereby reduces the chance of denaturation by detergent. We used this method to purify family B parathyroid hormone 1 receptor (PTH1R). Applying fluorescent methods, we showed that the purified PTH1R in Nanodiscs can bind to its native ligand PTH(1-34) and activate G protein. This is the first study in which a Family B GPCR has been examined in a Nanodisc. Doing so reveals for the first time that both binding of native ligand PTH(1-34) and G protein activity of PTH1R depend on calcium concentration in the milli-molar range. This calcium dependence is likely correlated to the function of PTH1R in signaling calcium homeostasis in kidney and bone. The results provide a basis for further investigations to understand the mechanism of administrating PTH(1-34) as treatment for osteoporosis. We conclude that our purification method using Nanodiscs is viable for purifying GPCR and enables observations such as calcium-dependent G protein activity that would have been difficult in cells or membrane preparations.