Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors
Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors
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DOI：
10.1073/pnas.0509763102
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发表时间：
2006-01-03
影响因子：
11.1
通讯作者：
Gilman, AG
中科院分区：
文献类型：
--
作者：
Gibson, SK;Gilman, AG
以前的研究与G蛋白亚基在活细胞中的受体相互作用的特异性依赖于第二信使或其他下游反应的测量。我们已经研究了α 2-肾上腺素能受体（α 2 R）和Gi α和G β亚基亚型的各种组合之间的相互作用的选择性，通过测量HeLa细胞中Gi α-黄色荧光蛋白和青色荧光蛋白-G β嵌合体之间的FRET变化。所有Gi α 1、-2或-3与G β 1、-2或-4的组合在一定程度上被内源性α 2 R激活，如通过FRET中的激动剂依赖性降低所判断的。G蛋白活化的程度由Gia和G β亚基的组合决定，而不是由单个亚基的身份决定。RT-PCR分析和α 2 R亚型的小干扰RNA敲除，随后定量放射性标记的拮抗剂结合，证明HeLa细胞以2：1的比例表达α 2a-和α 2b-肾上腺素能受体亚型。通过α 2aR亚型的过表达增加受体数量，最大限度地减少了Gia和G β亚型偶联偏好之间的差异。每个Gi α、G β和α 2-肾上腺素能受体亚型的分子性质影响α 2-肾上腺素能受体介导的信号传导途径的信号传导效率。
Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between alpha 2-adrenergic receptors (alpha 2R) and various combinations of Gi alpha and G beta subunit isoforms by measuring changes in FRET between Gi alpha-yellow fluorescent protein and cyan fluorescent protein-G beta chimeras in HeLa cells. All combinations of Gi alpha 1, -2, or -3 with G beta 1, -2, or -4 were activated to some degree by endogenous alpha 2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Gia and G beta subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of alpha 2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express alpha 2a- and alpha 2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the alpha 2aR subtype minimized the differences among coupling preferences for Gia and G beta isoforms. The molecular properties of each Gi alpha, G beta, and alpha 2-adrenergic receptor subtype influence signaling efficiency for the a2-adrenergic receptor-mediated signaling pathway.