Detergent- and phospholipid-based reconstitution systems have differential effects on constitutive activity of G-protein-coupled receptors.

A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β2-adrenergic receptor (β2AR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted β2AR, the β2AR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using 19F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted β2AR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of β2AR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that β2AR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress β2AR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.