Dual Role of the Second Extracellular Loop of the Cannabinoid Receptor 1: Ligand Binding and Receptor Localization
Dual Role of the Second Extracellular Loop of the Cannabinoid Receptor 1: Ligand Binding and Receptor Localization
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DOI：
10.1124/mol.109.057356
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发表时间：
2009-10-01
影响因子：
3.6
通讯作者：
Kendall, Debra A.
中科院分区：
文献类型：
--
作者：
Ahn, Kwang H.;Bertalovitz, Alexander C.;Kendall, Debra A.
G蛋白偶联受体（GPCR）的七个跨膜α螺旋是这个超家族的标志。螺旋内相互作用对受体组装至关重要，对于结合小分子的GPCR亚类，配体结合也至关重要。大多数研究都集中在确定的配体结合口袋内的螺旋束，而细胞外环的作用仍然不确定。然而，大麻素受体1（CB 1）细胞外环2（EC2）的分子建模表明，EC2准备进行关键的相互作用。为了测试这种可能性，我们采用丙氨酸扫描突变的CB 1 EC2和确定两个不同的区域的配体结合，G蛋白偶联活性，和受体贩运的关键。在EC2的N末端具有突变的受体（W255 A，N256 A）保留在内质网中，不结合激动剂（1 R，3R，4 R）-3-[2-羟基-4-（1，1-二甲基庚基）苯基]-4-（3-羟丙基）环己-1-醇（CP 55940）或反向激动剂N-（哌啶-1-基）-5-（4-氯苯基）-1（2，4-二氯苯基）-4-甲基-1H-吡唑-3-甲酰胺（SR 141716 A）。相反，EC 2的C端区分为激动剂和反向激动剂; P269 A、H270 A和I271 A受体与几种激动剂的结合减弱，但与反向激动剂SR 141716 A、N-（哌啶-1-基）-5-（4-碘苯基）-1-（2，4-二氯苯基）-4-甲基-1H-吡唑-3-甲酰胺（AM 251），和具有野生型受体亲和力的4-[6-甲氧基-2-（4-甲氧基苯基）苯并呋喃-3-羰基]苄腈（LY 320135）。涉及Cys-X-X-X-Ar基序取代的F268 A受体显示出受损的定位和配体结合。在位置268处的其他氨基酸取代揭示了需要高度疏水的残基来实现这两种功能。值得注意的是，F268 W受体被运输到细胞表面，但显示出与P269 A、H270 A和I271 A受体相当的对反向激动剂的差异结合偏好。这些发现与EC2在稳定受体组装和配体结合中的双重作用一致。
The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R, 3R, 4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamide (SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl] benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.