Journal article
Mutation R238E in transducin-alpha yields a GTPase and effector-deficient, but not dominant-negative, G-protein alpha-subunit
Molecular vision, Vol.12(56-58), pp.492-498
05/12/2006
PMID: 16735989
Abstract
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.
The R238E substitution was introduced into a tranducin-like chimeric Gtalpha*-subunit. The nucleotide-bound state of the Gtalpha*R238E mutant was assessed using the trypsin-protection assay. The ability of the Gtalpha*R238E 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.
The Gtalpha*R238E mutant bound GDP and was capable of the AlF4--induced activational conformational change. The capacity of Gtalpha*R238E 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 Gtalpha*R238E with the GAP proteins, RGS9 and RGS16. Another consequence of the mutation was the reduction in Gtalpha*R238E's affinity for Pgamma.
Transducin mutant Gtalpha*R238E 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.
Details
- Title: Subtitle
- Mutation R238E in transducin-alpha yields a GTPase and effector-deficient, but not dominant-negative, G-protein alpha-subunit
- Creators
- Brandy Barren - Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USAMichael NatochinNikolai O Artemyev
- Resource Type
- Journal article
- Publication Details
- Molecular vision, Vol.12(56-58), pp.492-498
- Publisher
- United States
- PMID
- 16735989
- ISSN
- 1090-0535
- eISSN
- 1090-0535
- Grant note
- R01EY012682 / NEI NIH HHS R01 EY012682 / NEI NIH HHS
- Language
- English
- Date published
- 05/12/2006
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
- Record Identifier
- 9984025683502771
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