A dominant-negative @entity17535 mutant that traps a stable @entity8695 - XXXX - @entity3007 -betagamma complex.

@entity3007 @entity6708 @entity17535 @entity6370 @entity8695 @entity377 @entity2734 @entity3006 @entity1391 @entity72331 @entity563 @entity1889
Residues comprising the @entity6370 -binding sites of the a subunits of heterotrimeric (large) G-proteins ( @entity17535 subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the @entity1391 -binding loop (P-loop) where both @entity17535 subunits and Ras-related G-proteins have a conserved @entity563 or @entity1889 residue. Substitutions for this residue in Ras and related (small) G-proteins yield @entity377 -depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved @entity563 residue in the P-loop to @entity2734 , within a chimeric @entity17535 subunit (designated aT*) that is mainly comprised of the a subunit of the retinal G-protein transducin and a limited region from the a subunit of Gi1. The aT*( @entity72331 ) mutant exhibits a significantly higher rate of intrinsic @entity3006 - @entity3007 exchange compared with wild-type aT*, with light-activated @entity8695 (R*) causing only a moderate increase in the kinetics of @entity377 exchange on aT*( @entity72331 ). The aT*( @entity72331 ) mutant, when bound to either @entity3006 or @entity3007 , was able to significantly slow the rate of R*-catalyzed @entity3006 - @entity3007 exchange on wild-type aT*. Thus, @entity3007 -bound aT*( @entity72331 ), as well as the @entity3006 -bound mutant, is capable of forming a stable complex with R*. aT*( @entity72331 ) activated the @entity6708 phosphodiesterase (PDE) with a dose-response similar to wild-type aT*. Activation of the PDE by aT*( @entity72331 ) was unaffected if either R* or b1y1 alone was present, whereas it was inhibited when R* and the b1y1 subunit were added together. Overall, our studies suggest that the @entity72331 substitution on aT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a @entity3007 -bound @entity17535 subunit, and the b1y1 complex.
