Structural basis of receptor‐dependent G protein activation

Abstract

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP/GDP exchange on the α subunit (Gα) of heterotrimeric G proteins. GDP release is rate-limiting for the initiation of all downstream signaling and, until GTP binds, a high-affinity complex between the activated receptor (R*) and nucleotide-free G protein is formed. Despite the important role of the R*-G protein interaction in cell signaling, little is known about the structure of this complex. Biochemical studies suggest the nucleotide binding pocket is nearly 30 Å away from the receptor contacts; how the receptor catalyzes GDP release from this distance is unknown. Over 50 single cysteine substitutions have been introduced into a Gαi1 background to investigate R*-mediated conformational changes using site-directed spin-labeling and electron paramagnetic resonance spectroscopy (EPR). These studies indicate that R* causes a rigid body rotation and translation of the α5 helix, which connects the Gα C-terminus, a well known receptor contact site, to the nucleotide binding pocket at the β 6/α5 loop. No R*-dependent conformational changes have been observed in the N-terminus or Switch II. Double electron-electron resonance EPR distance measurements were made to confirm this movement. This conformational change was not observed when a flexible loop was inserted between the α5 helix and C-terminus to uncouple R*-binding from nucleotide exchange, suggesting that the movement of the α5 helix is necessary for GDP release. Supported by NIH grants EY06062 (H.E.H.), EY005216 (W.L.H.) and GM07347 (W.M.O.), the PhRMA Foundation (W.M.O.) and the Jules Stein Professorship (W.L.H.).