Demonstration of a Specific Site of Covalent Labeling of the Human Motilin Receptor Using a Biologically Active Photolabile Motilin Analog
Demonstration of a Specific Site of Covalent Labeling of the Human Motilin Receptor Using a Biologically Active Photolabile Motilin Analog
复制标题
DOI：
10.1124/jpet.104.081562
复制
发表时间：
2005-06
影响因子：
3.5
通讯作者：
B. Matsuura;M. Dong;B. Coulie;D. Pinon;L. Miller
中科院分区：
文献类型：
--
作者：
B. Matsuura;M. Dong;B. Coulie;D. Pinon;L. Miller
胃动素受体属于一类G蛋白偶联受体，还包括生长激素促分泌素和胃饥饿素受体。这些代表了临床上有用的药物治疗靶点。它们潜在的独特结构及其结合的分子基础尚不清楚。我们以前报道的初始亲和标记的区域内的受体（溴化氰片段从第一个延伸到第二个细胞外环）使用的位置1光不稳定的胃动素类似物。为了扩展我们对胃动素结合的分子基础的理解，我们已经开发了另外的放射性碘标记胃动素类似物探针，其在位置5具有共价连接位点。这是一种完全激动剂，特异性地和高亲和力地与胃动素受体结合，并有效地与其受体建立单一共价键。标记的野生型和突变型胃动素受体结构的顺序化学和酶促裂解建立了标记区域在第三胞外环内。使用放射化学Edman降解测序将其进一步定位于Phe332。这些数据提供了第一个空间近似约束，可用于该肽配体与其受体的对接。我们希望，一系列这样的限制可以被确定，以提供足够的结构信息，开始，以阐明这种激动剂结合受体的构象，并最终在合理设计药物作用于这个重要的目标是有用的。
The motilin receptor belongs to a group of class I G protein-coupled receptors that also includes the growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (a cyanogen bromide fragment extending from the first to the second extracellular loop) using a position 1 photolabile motilin analog. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analog probe having site of covalent attachment in position 5. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild-type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe332 using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be used in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor and to ultimately be useful in the rational design of drugs acting at this important target.