Mutation R238E in transducin-α yields a GTPase andeffector-deficient, but not dominant-negative, G-protein α-subunit

[摘要] Purpose: Certain forms of inherited and light-induced retinaldegenerations are believed to involve excessive phototransductionsignaling. A dominant-negative mutant of the visual G-protein,transducin, would represent a major tool in designing potentialtherapeutical strategies for this group of visual diseases. We thoughtto further investigate a novel mutant of the transducin-α subunit,R238E, that was recently reported to be a dominant-negative inhibitor ofthe rhodopsin/transducin/PDE visual system.Methods: The R238E substitution was introduced into a tranducin-likechimeric Gtα*-subunit. The nucleotide-bound state of theGtα*R238E mutant was assessed using the trypsin-protection assay.The ability of the Gtα*R238E mutant to interact with Gtβγ,couple to photoexcited rhodopsin (R*), and undergo R*-stimulated guaninenucleotide exchange was examined by a GTPγS binding assay. TheGTPase activity of the mutant Gtα* and its interaction with RGSproteins was characterized in the steady-state and single turnovermeasurements of GTP hydrolysis. A binding assay utilizing thefluorescently-labeled γ-subunit of PDE6 (Pγ) was employed tomonitor the effector function of Gtα*R238E.Results: The Gtα*R238E mutant bound GDP and was capable of theAlF4--induced activational conformational change. Thecapacity of Gtα*R238E to couple to R* in the presence ofGtβγ was similar to that of Gtα*. However, the mutantGTPase activity was markedly impaired. This defect was furtherexacerbated by the diminished interactions of Gtα*R238E with theGAP proteins, RGS9 and RGS16. Another consequence of the mutation wasthe reduction in Gtα*R238E's affinity for Pγ.Conclusions: Transducin mutant Gtα*R238E exists in anucleotide-bound state and is fully capable of activational coupling toR*. This mutation results in a significant impairment of Gtα*'sability to hydrolyze GTP and interact with the inhibitory subunit ofPDE6. This phenotype is entirely inconsistent with that of adominant-negative inhibitor as recently reported.

[学科分类] 生物化学/生物物理