Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling
Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling
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DOI：
10.1074/jbc.m110.217901
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发表时间：
2011-05-06
影响因子：
4.8
通讯作者：
Dong, Maoqing
中科院分区：
文献类型：
--
作者：
Miller, Laurence J.;Chen, Quan;Dong, Maoqing
胰高血糖素样肽1（GLP 1）受体是G蛋白偶联受体B家族中的重要药物靶点。其天然激动剂配体GLP 1具有肠促胰岛素样作用，该受体是公认的2型糖尿病治疗靶点。尽管最近解决了GLP 1受体和几个密切的家族成员的氨基末端的结构，GLP 1结合和激活完整受体的分子基础仍然不清楚。我们先前证明了GLP 1及其受体的氨基和羧基末端残基之间的分子近似。在这项工作中，我们研究了空间近似与该肽的中间区域，以深入了解完整的受体和配体-受体复合物的方向。我们制备了两种新的光不稳定探针，它们分别在GLP 1（7-36）的16和20位引入了对苯甲酰基-L-苯丙氨酸。两种探针均特异性地且以高亲和力结合GLP 1受体。这些均为完全有效的激动剂，以浓度依赖性方式刺激携带受体的CHO细胞中的cAMP蓄积。每个探针特异性标记单个受体位点。蛋白酶切割和放射化学测序鉴定了跨膜片段1上方的受体残基Leu（141）作为其位置16探针的标记位点，而位置20探针标记第二胞外环内的受体残基Trp（297）。建立配体残基近似与这个环区域是唯一的家庭成员，并可能有助于定向受体氨基末端结构域相对于其螺旋束区域。
The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino-and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. Wehave prepared two new photolabile probes incorporating a p-benzoyl-L-phenylalanine into positions 16 and 20 of GLP1(7-36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu(141) above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp(297) within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.