Barren Brandy, Natochin Michael, Artemyev Nikolai O
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Mol Vis. 2006 May 12;12:492-8.
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 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.
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.
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.
某些形式的遗传性和光诱导性视网膜变性被认为涉及过度的光转导信号传导。视觉G蛋白转导素的显性负性突变体将是设计针对这类视觉疾病的潜在治疗策略的主要工具。我们想进一步研究转导素α亚基的一种新型突变体R238E，最近有报道称它是视紫红质/转导素/磷酸二酯酶视觉系统的显性负性抑制剂。
将R238E替换引入到一个类似转导素的嵌合Gtα亚基中。使用胰蛋白酶保护试验评估GtαR238E突变体的核苷酸结合状态。通过GTPγS结合试验检测GtαR238E突变体与Gtβγ相互作用、与光激发视紫红质（R）偶联以及进行R刺激的鸟嘌呤核苷酸交换的能力。在GTP水解的稳态和单周转测量中表征突变体Gtα的GTP酶活性及其与RGS蛋白的相互作用。采用利用荧光标记的磷酸二酯酶6（Pγ）γ亚基的结合试验来监测Gtα*R238E的效应器功能。
GtαR238E突变体结合GDP并能够发生AlF4-诱导的激活构象变化。在存在Gtβγ的情况下，GtαR238E与R偶联的能力与Gtα相似。然而，突变体的GTP酶活性明显受损。GtαR238E与GAP蛋白RGS9和RGS16的相互作用减少进一步加剧了这一缺陷。该突变的另一个后果是GtαR238E对Pγ的亲和力降低。
转导素突变体GtαR238E以核苷酸结合状态存在，并且完全能够与R进行激活偶联。该突变导致Gtα*水解GTP的能力以及与磷酸二酯酶6抑制亚基相互作用的能力显著受损。这种表型与最近报道的显性负性抑制剂的表型完全不一致。
