Mutation R238E in transducin-alpha yields a GTPase and effector-deficient, but not dominant-negative, G-protein alpha-subunit.

PURPOSE

Certain forms of inherited and light-induced retinal degenerations are believed to involve excessive phototransduction signaling. A dominant-negative mutant of the visual G-protein, transducin, would represent a major tool in designing potential therapeutical strategies for this group of visual diseases. We thought to further investigate a novel mutant of the transducin-alpha subunit, R238E, that was recently reported to be a dominant-negative inhibitor of the rhodopsin/transducin/PDE visual system.

METHODS

The R238E substitution was introduced into a tranducin-like chimeric Gtalpha*-subunit. The nucleotide-bound state of the GtalphaR238E mutant was assessed using the trypsin-protection assay. The ability of the GtalphaR238E mutant to interact with Gtbetagamma, couple to photoexcited rhodopsin (R*), and undergo R*-stimulated guanine nucleotide exchange was examined by a GTPgammaS binding assay. The GTPase activity of the mutant Gtalpha* and its interaction with RGS proteins was characterized in the steady-state and single turnover measurements of GTP hydrolysis. A binding assay utilizing the fluorescently-labeled gamma-subunit of PDE6 (Pgamma) was employed to monitor the effector function of Gtalpha*R238E.

RESULTS

The GtalphaR238E mutant bound GDP and was capable of the AlF4--induced activational conformational change. The capacity of GtalphaR238E to couple to R* in the presence of Gtbetagamma was similar to that of Gtalpha*. However, the mutant GTPase activity was markedly impaired. This defect was further exacerbated by the diminished interactions of GtalphaR238E with the GAP proteins, RGS9 and RGS16. Another consequence of the mutation was the reduction in GtalphaR238E's affinity for Pgamma.

CONCLUSIONS

Transducin mutant GtalphaR238E exists in a nucleotide-bound state and is fully capable of activational coupling to R. This mutation results in a significant impairment of Gtalpha*'s ability to hydrolyze GTP and interact with the inhibitory subunit of PDE6. This phenotype is entirely inconsistent with that of a dominant-negative inhibitor as recently reported.