Purification of G-Protein Coupled Receptors Using Nanodiscs

G-Protein Coupled Receptors (GPCRs) are seven-transmembrane (7-TM) proteins and belong to the largest gene family in the human genome. They mediate intracellular signaling in response to extracellular stimuli such as light, small molecules, peptides, etc.GPCRs are expressed in a wide variety of cell types and modulate cellular and physiological responses to the stimuli, which make them ideal drug targets. Despite their importance, GPCRs are not well understood at the molecular level in terms of their activation mechanism because they are notoriously difficulty to purify for quantitative biophysical studies. Due to their 7-TM hydrophobic domain, they need to be purified in detergents, which are often incompatible with their stability. Currently, GPCRs are purified using detergent conditions determined individually for each GPCR using an empirical approach. Thus, only a handful of GPCRs have been purified, which is an impediment to obtaining a molecular understanding of GPCRs. Here, we have developed a method for purifying GPCRs using nanodiscs, which are nanometer sized, disc-shaped, and self-contained lipid bilayer particles. In our method, we incorporate GPCRs straight from the cell membrane of a mammalian expression system into nanodiscs to minimize the amount of time the protein is in contact with detergent. Using this approach, we have successfully purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R). We have investigated the binding of purified PTH1R to its native ligand PTH1 (1-34) using fluorescence anisotropy and obtained a dissociation constant of ∼29 nM, in agreement with previous reports. We propose that our method could be a general approach to purify GPCRs that will enable quantitative biophysical studies to yield a better molecular understanding of their activation mechanisms and interactions with downstream signaling proteins.