Analysis of β2AR-Gsand β2AR-Gicomplex formation by NMR spectroscopy

Abstract

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gsand stimulates cAMP formation. Functional studies have shown that the β2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE2001, re15 (2001)]. A crystal structure of the β2AR-Gscomplex revealed the interaction interface of β2AR-Gsand structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β2AR signaling through Gsand its preferential coupling to Gsover Giis still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β2AR in response to the full agonist BI-167107 and Gsand Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β2AR are qualitatively the same for Gsand Gi1, we detected distinct differences between the β2AR-Gsand the β2AR-Gi1complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β2AR-Gsand β2AR-Gi1complexes in ICL2 may be key determinants for G protein coupling selectivity.