Mechanism of action of Gq to inhibit Gβγ modulation of Cav2.2 calcium channels:: Probed by the use of receptor-Gα tandems
Mechanism of action of Gq to inhibit Gβγ modulation of Cav2.2 calcium channels:: Probed by the use of receptor-Gα tandems
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DOI：
10.1124/mol.63.4.832
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发表时间：
2003-04-01
影响因子：
3.6
通讯作者：
Dolphin, AC
中科院分区：
文献类型：
--
作者：
Bertaso, F;Ward, RJ;Dolphin, AC
的稳定。通过在α (2A)肾上腺素能受体（α (2A)-R）和异三聚体g蛋白不同α亚基的百日咳抗毒素突变体之间建立联系，g蛋白偶联受体和特定伴侣g蛋白的相互作用成为可能。α (2A)-R- galpha (o)和α (2A)-R- galpha (i)在COS-7细胞中表达时，能够重建激动剂诱导的n型钙通道（Ca(v)2.2）的电压依赖性抑制，类似于野生型α (2A)-R。G(q)与G（i/o）信号通路的相互作用通过表达Galpha(q)或基于Galpha(q)的嵌合结构体来研究，该嵌合结构体含有Galpha(z)的最后五个氨基酸，由α (2A)-R激活。结果发现，野生型α (2A)-R激活的Galpha（qz5）以电压无关的方式抑制Ca(v)2.2电流。此外，Galpha（qz5）抵消了由α (2A)-R-Galpha(o)激活引起的电压依赖性抑制。我们随后研究了Galpha（qz5）行为的基础。我们的证据表明，这是由于Galpha（qz5）激活的下游效应，因为它被磷脂酶Cbeta1的c端结构体阻断。此外，它可能部分通过蛋白激酶C （PKC）激活发生，因为PKC激活剂phorbol dibutyrate在α (2A)-R-Galpha(o)转染的细胞中模拟了Galpha（qz5）的作用。相反，表达α (2A)-R-Galpha(o)和Galpha（qz5）的细胞在PKC抑制剂双吲哚酰马来酰亚胺I （GF 109203X）的存在下表现出部分电压依赖性抑制的恢复。讨论了潜在的磷酸化位点。
The stable. interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the alpha(2A) adrenergic receptor (alpha(2A)-R) and pertussis toxin-resistant mutants of different Galpha subunits of heterotrimeric G-proteins. Both alpha(2A)-R-Galpha(o) and alpha(2A)-R-Galpha(i) proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca(v)2.2) similar to the wild-type alpha(2A)-R when expressed in COS-7 cells. The interaction of G(q) with the G(i/o) signaling pathways was studied by expressing either Galpha(q) or a chimeric construct based on Galpha(q) containing the last five amino acids of Galpha(z), which is activated by alpha(2A)-R. It was found that Galpha(qz5) activated by the wild-type alpha(2A)-R inhibited Ca(v)2.2 currents in a voltage-independent fashion. Furthermore, Galpha(qz5) counteracted the voltage-dependent inhibition resulting from alpha(2A)-R-Galpha(o) activation. We subsequently investigated the basis for the behavior of Galpha(qz5). Our evidence suggests that this occurs as a result of a downstream effect of activation of Galpha(qz5) because it was blocked by C-terminal construct of phospholipase Cbeta1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of Galpha(qz5) in alpha(2A)-R-Galpha(o)-transfected cells. Conversely, cells expressing both alpha(2A)-R-Galpha(o) and Galpha(qz5) exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.