Molecular basis of G protein-coupled receptor function

根据其药理特性，目前将其分为激动剂、中性拮抗剂和反向激动剂。反向激动剂是指在没有配体的情况下可以降低GPCR介导的G蛋白激活的药物(基础GPCR活性)。目前，人们对反向激动剂在其靶受体中诱导的结构变化的本质知之甚少。 为了阐明这个问题，我们使用了大鼠M3乙酰胆碱受体，一个原型的I类GPCR，作为一个模型系统。为了监测配体诱导的受体结构的变化，我们采用了一种最近开发的原位二硫键交联策略，该策略允许检测受体三维结构中相邻的半胱氨酸残基之间的二硫键形成。这一策略的一个主要优点是，可以检测到存在于天然膜环境中的受体的配体依赖性构象变化，而不需要任何受体纯化和重建步骤。 最近，我们研究了不同类别的M配体(全M受体激动剂和反向M受体激动剂)是否对8螺旋相对于跨膜结构域I(TM I)C末端的相对取向有不同的影响。螺旋8代表TM VII的细胞质α-螺旋延伸，它通过短连接序列连接到该延伸。大量证据表明，螺旋8在生产性受体/G蛋白偶联中起着重要作用。牛视紫红质的高分辨结构表明，螺旋8中的几个残基位于TM I的细胞质末端附近。因此，我们推测，在二硫键交联研究中，替换到TM I这一片段中的半胱氨酸残基可能成为检测配体诱导螺旋8移动的有用报告。 具体地说，我们将成对的Cys残基引入到M3 M受体的一个修改版本中，该版本缺少大多数天然Cys残基，并在第三个细胞内环中包含两个因子Xa裂解位点(我们将这种结构称为‘M3’(3C)-Xa‘受体)。我们先前已经证明，M3‘(3C)-Xa受体具有与野生型M3 M受体相似的配体结合和G蛋白偶联特性。我们获得了20个双Cys突变体M3受体，它们都在Tm I的胞质端(A91-N95)和螺旋8的N-末端片段(K548-R551)中分别含有一个Cys替换和第二个Cys替换。 我们证明了毒扁豆碱激动剂抑制了A91C/T549C和F92C/F550C双半胱氨酸突变M3受体中的二硫键交联(Li等人，J.Biol。化学。2007年7月10日；印刷前的epub)。相反，阿托品和NMS促进了这两个突变受体中二硫键的形成(Li等人，J.Biol。化学。2007年7月10日；印刷前的epub)。因此，我们的数据有力地支持了一个模型，在该模型中，完全毒扁豆碱激动剂引发螺旋8的N-末端从TM I的细胞质末端分离，从而防止在91/549和92/550位引入的半胱氨酸残基之间形成二硫交联键。另一方面，反向毒扁豆碱激动剂被预测可以缩短TMI的细胞质末端和螺旋8的N端之间的距离。 这些发现为毒碱激动剂和反向激动剂相反的生物学效应提供了结构基础。这项研究还提供了关于这两类功能不同的配体在分子水平上诱导的构象如何不同的第一条直接结构信息。鉴于在大多数GPCR之间发现了高度的结构同源性，我们的发现应该具有广泛的普遍相关性。

According to their pharmacological characteristics, GPCR ligands are currently classified into agonists, neutral antagonists, and inverse agonists. Inverse agonists are drugs that can reduce GPCR-mediated G protein activation observed in the absence of ligands (basal GPCR activity). At present, little is known about the nature of the structural changes that inverse agonists induce in their target receptors. To shed light on this issue, we have used the rat M3 muscarinic acetylcholine receptor, a prototypic class I GPCR, as a model system. To monitor ligand-induced changes in receptor structure, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional (3D) structure of the receptor. One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps. We recently examined whether different classes of muscarinic ligands (full versus inverse muscarinic agonists) had different effects on the relative orientation of helix 8 relative to the C-terminus of transmembrane domain I (TM I). Helix 8 represents a cytoplasmic alpha-helical extension of TM VII to which it is connected via a short linker sequence. Considerable evidence suggests that helix 8 plays an important role in productive receptor/G protein coupling. The high-resolution structure of bovine rhodopsin indicates that several residues contained within helix 8 are located close to the cytoplasmic end of TM I. We therefore hypothesized that Cys residues substituted into this segment of TM I might serve as useful reporters to detect potential ligand-induced movements of helix 8 in disulfide cross-linking studies. Specifically, we introduced pairs of Cys residues into a modified version of the M3 muscarinic receptor that lacked most native Cys residues and contained two factor Xa cleavage sites within the third intracellular loop (we referred to this construct as 'M3'(3C)-Xa' receptor). We demonstrated previously that the M3'(3C)-Xa receptor exhibits ligand binding and G protein coupling properties similar to the wild-type M3 muscarinic receptor. We generated twenty double Cys mutant M3 receptors, all of which contained one Cys substitution within the cytoplasmic end of TM I (A91-N95) and a second one within the N-terminal segment of helix 8 (K548-R551). We demonstrated that muscarinic agonists inhibited disulfide cross-linking in the A91C/T549C and F92C/F550C double Cys mutant M3 receptors (Li et al., J. Biol. Chem. 2007 Jul 10; Epub ahead of print). In contrast, atropine and NMS enhanced disulfide bond formation in these two mutant receptors (Li et al., J. Biol. Chem. 2007 Jul 10; Epub ahead of print). Our data therefore strongly support a model in which full muscarinic agonists trigger a separation of the N-terminal segment of helix 8 from the cytoplasmic end of TM I, thus preventing the formation of disulfide cross-links between Cys residues introduced at positions 91/549 and 92/550. On the other hand, inverse muscarinic agonists are predicted to decrease the distance between the cytoplasmic end of TM I and the N-terminal portion of helix 8. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most GPCRs, our findings should be of broad general relevance.