Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 14, Pages -Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.166538
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Funding
- National Institutes of Health [GM047458]
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Residues comprising the guanine nucleotide-binding sites of the alpha subunits of heterotrimeric (large) G-proteins (G alpha subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both G alpha subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric G alpha subunit (designated alpha(T)*) that is mainly comprised of the alpha subunit of the retinal G-protein transducin and a limited region from the alpha subunit of Gi1. The alpha(T)*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type alpha(T)*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on alpha(T)*(S43N). The alpha(T)*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type alpha(T)*. Thus, GTP-bound alpha(T)*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. alpha(T)*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type alpha(T)*. Activation of the PDE by alpha(T)*(S43N) was unaffected if either R* or beta 1 gamma 1 alone was present, whereas it was inhibited when R* and the beta 1 gamma 1 subunit were added together. Overall, our studies suggest that the S43N substitution on alpha(T)* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound G alpha subunit, and the beta 1 gamma 1 complex.
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