The Dynamic Process of ²2-Adrenergic Receptor Activation

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ²2-adrenergic receptor (²2AR), a prototypical GPCR. We labeled ²2AR with 13CH3µ-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ²2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ²2ARs ability to engage multiple signaling and regulatory proteins. º NMR using 13CH3-µ-Met reveals dynamics of ²2 adrenergic receptor (²2AR) º NMR and computational approaches show unanticipated conformational states º Conformational heterogeneity is observed in both unliganded and antagonist-bound ²2AR º Agonist alone does not fully stabilize the active conformation of the ²2AR NMR studies reveal that ²2 adrenergic receptor experiences conformational heterogeneity until both agonist and a G protein mimetic bind, shifting the receptor into its active conformation.