Probing the mechanism of rhodopsin-catalyzed transducin activation

Abstract

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein α-subunit (Geα) followed by the release of GαGTP and Gβγ which, subsequently, activate their targets. The C-terminal regions of Gα subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the Gα C-terminus, via the α5 helix, to the nucleotide-binding β6/α5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn α-helical segment was inserted between the 11 C-terminal residues and the α5 helix of Gαt-like chimeric Gα, Gαti. The mutant Gα subunits with the Glyloop (GαtiL) and the extended α5 helix (GαtiH) retained intact interactions with Gβγt, and displayed modestly reduced binding to R*. GαtiH was capable of efficient activation by R*. In contrast, R* failed to activate GαtiL, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of Gα C-terminus/α5 helix/β6/α5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.