Gether U, Ballesteros J A, Seifert R, Sanders-Bush E, Weinstein H, Kobilka B K
Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305, USA.
J Biol Chem. 1997 Jan 31;272(5):2587-90. doi: 10.1074/jbc.272.5.2587.
Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.
多个结构域中的突变可导致G蛋白偶联受体的激动剂非依赖性组成性激活。然而，组成性开启G蛋白偶联受体的结构和分子变化的本质仍然未知。在此，我们展示了证据，表明β2肾上腺素能受体的组成性激活突变体（CAM）的特征在于结构不稳定性以及对配体结合的夸张构象反应。与野生型受体相比，纯化的受体在37℃下变性速率增加4倍，这可以证明CAM的结构不稳定性。用构象敏感且对半胱氨酸有反应性的荧光团N，N'-二甲基-N-（碘乙酰基）-N'-（7-硝基苯并-2-恶唑-1，3-二唑-4-基）乙二胺标记的纯化CAM的光谱分析进一步表明，激动剂和拮抗剂在CAM中引起的结构变化比在野生型蛋白中更深刻。我们提出，赋予β2肾上腺素能受体组成性活性的突变消除了一些稳定构象限制，使CAM更容易在非活性状态和活性状态之间转换，并使受体更容易变性。
