Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchan ge on the G protein alpha -subunit (G alpha) followed by the release of G a lpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognit ion domain. In this study, we tested the hypothesis that the GPCR-induced c onformational change is communicated from the G alpha C-terminus, via the a lpha5 helix, to the nucleotide-binding beta6/alpha5 loop causing GDP releas e. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcit ed rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha -helical segment was inserted between the 11 C-term inal residues and the alpha5 helix of G alpha (t)-like chimeric G alpha, G alpha (ti). The mutant G alpha subunits with the Gly-loop (G alpha L-ti) an d the extended alpha5 helix (G alpha H-ti) retained intact interactions wit h G beta gamma (t), and displayed modestly reduced binding to R*. G alpha H -ti was capable of efficient activation by R*. In contrast, R* failed to ac tivate G alpha L-ti, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provi de evidence for the role of G alpha C-terminus/alpha5 helix/beta6/alpha5 lo op route as a dominant channel for transmission of the GPCR-induced conform ational change leading to G protein activation.