Probing the mechanism of rhodopsin-catalyzed transducin activation.
An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 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 alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, 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 G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.
Natochin
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M
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA.
Moussaif
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M
Artemyev
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NO
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D021122:Protein Subunits; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015293:Transducin