Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes
Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes
复制标题
DOI：
10.1074/jbc.m109.038109
复制
发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Dong, Maoqing
中科院分区：
文献类型：
--
作者：
Chen, Quan;Pinon, Delia I.;Dong, Maoqing
胰高血糖素样肽1（GLP 1）受体是家族B G蛋白偶联受体的成员，是治疗2型糖尿病的重要药物靶点。尽管最近的解决方案的氨基末端结构域的这种受体和几个密切的家庭成员的结构，了解天然配体GLP 1结合其完整的受体的分子基础仍然有限。本研究的目的是探索GLP 1的羧基末端内的特定受体残基与其受体正常对接之间的空间近似。因此，我们开发并表征了两种高亲和力的完全激动剂光不稳定GLP 1探针，其在位置24和35具有用于共价连接的位点。两种探针都特异性地和饱和地标记受体。随后对纯化的野生型和突变型GLP 1受体进行了化学和蛋白酶裂解肽图分析，发现受体氨基端质膜区的Arg（131）-Lys（136）片段含有24位探针的标记位点，并通过放射化学测序确定该探针标记的特异性受体残基为Glu（133）。类似地，在受体氨基末端结构域的相同区域内的邻近残基Glu（125）被鉴定为位置35探针的标记位点。这些数据代表了第一次直接证明GLP 1和其完整的受体之间的空间近似对接，提供了两个重要的限制，这种相互作用的建模。这将扩大我们对天然激动剂配体与GLP 1受体结合的分子基础的理解，并可能与其他家族成员相关。
The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.