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


RESULTS
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.


CONCLUSIONS
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.
Barren
Brandy
B
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Natochin
Michael
M
Artemyev
Nikolai O
NO
D000818:Animals; D001120:Arginine; D001667:Binding, Competitive; D002417:Cattle; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases / Q000172:deficiency* / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics* / Q000378:metabolism*; D005799:Genes, Dominant; D018698:Glutamic Acid; D006153:Guanosine Diphosphate / Q000378:metabolism; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency* / Q000378:metabolism; D021122:Protein Subunits / Q000172:deficiency / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D020710:RGS Proteins / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000235:genetics* / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D021122:Protein Subunits; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; C116925:regulator of g-protein signaling 9; D006153:Guanosine Diphosphate; D018698:Glutamic Acid; D012243:Rhodopsin; D001120:Arginine; D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases; D015293:Transducin