Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6.
Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6.
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DOI：
10.1124/mol.56.1.175
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发表时间：
1999-07
影响因子：
3.6
通讯作者：
Søren G. F. Rasmussen;A. Jensen;G. Liapakis;P. Ghanouni;J. Javitch;U. Gether
中科院分区：
文献类型：
--
作者：
Søren G. F. Rasmussen;A. Jensen;G. Liapakis;P. Ghanouni;J. Javitch;U. Gether
跨膜片段（TM）3和6的运动在G蛋白偶联受体的激活中起着关键作用。然而，控制这些运动并因此控制受体活化的潜在分子过程仍不清楚。为了阐明β 2肾上腺素能受体（β 2 AR）Asp-Arg-Tyr基序中保守天冬氨酸（Asp-130）的重要性，我们将该残基突变为天冬酰胺（D130 N）以模拟其质子化状态，并突变为丙氨酸（D130 A）以完全去除侧链的功能性。这两种突变体显示的组成型受体激活的证据。在表达D130 N或D130 A的COS-7细胞中，与表达野生型β 2AR的细胞相比，cAMP积累的基础水平明显升高。COS-7细胞膜或纯化的受体在37 ℃下孵育也显示了两种突变体受体的显著结构不稳定性，表明稳定的分子内约束已被破坏。此外，我们获得了证据的构象重排突变的Asp-130。在D130 N中，TM 6中的半胱氨酸Cys-285（在野生型β 2AR中不可接近）变得可被甲硫基磺酸乙基铵（一种带电的巯基反应性试剂）接近。这与TM 6的逆时针旋转或倾斜一致，并首次提供了将DRY基序中天冬氨酸的电荷中和突变与受体的整体构象状态联系起来的结构证据。我们建议，质子化的天冬氨酸导致释放约束的分子内相互作用，导致TM 6的运动，因此，转换的受体的活性状态。
Movements of transmembrane segments (TMs) 3 and 6 play a key role in activation of G protein-coupled receptors. However, the underlying molecular processes that govern these movements, and accordingly control receptor activation, remain unclear. To elucidate the importance of the conserved aspartic acid (Asp-130) in the Asp-Arg-Tyr motif of the beta2 adrenergic receptor (beta2AR), we mutated this residue to asparagine (D130N) to mimic its protonated state, and to alanine (D130A) to fully remove the functionality of the side chain. Both mutants displayed evidence of constitutive receptor activation. In COS-7 cells expressing either D130N or D130A, basal levels of cAMP accumulation were clearly elevated compared with cells expressing the wild-type beta2AR. Incubation of COS-7 cell membranes or purified receptor at 37 degrees C revealed also a marked structural instability of both mutant receptors, suggesting that stabilizing intramolecular constraints had been disrupted. Moreover, we obtained evidence for a conformational rearrangement by mutation of Asp-130. In D130N, a cysteine in TM 6, Cys-285, which is not accessible in the wild-type beta2AR, became accessible to methanethiosulfonate ethylammonium, a charged, sulfhydryl-reactive reagent. This is consistent with a counterclockwise rotation or tilting of TM 6 and provides for the first time structural evidence linking charge-neutralizing mutations of the aspartic acid in the DRY motif to the overall conformational state of the receptor. We propose that protonation of the aspartic acid leads to release of constraining intramolecular interactions, resulting in movements of TM 6 and, thus, conversion of the receptor to the active state.