Ji Z, Hadac E M, Henne R M, Patel S A, Lybrand T P, Miller L J
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 1997 Sep 26;272(39):24393-401. doi: 10.1074/jbc.272.39.24393.
Mechanisms of ligand binding and activation of G protein-coupled receptors are particularly important, due to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125I-desaminotyrosyl-Gly-[Nle28,31, pNO2-Phe33]CCK-(26-33), binds specifically, saturably, and with high affinity (Ki = 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate Mr = 85,000-95,000 plasma membrane glycoprotein with core of Mr = 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO2-Phe33 residue of the probe. A model of this ligand-bound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.
由于G蛋白偶联受体广泛表达且具有作为药物靶点的潜力，其配体结合和激活机制尤为重要。对于结合在跨膜螺旋内的小分子配体，配体与这些受体之间的分子相互作用已得到最明确的界定。基于受体诱变的效应，细胞外结构域对肽配体似乎更为重要，其中对结合或活性的干扰可反映变构效应以及直接效应。我们现在采用更直接的方法，即使用一种氨基末端被封闭的光不稳定型胆囊收缩素（CCK）类似物对CCK受体的活性位点进行光亲和标记。该探针125I-去氨基酪氨酰-甘氨酸-[Nle28,31,对硝基苯丙氨酸33]CCK-(26-33)具有特异性、饱和性结合，且亲和力高（Ki = 3.3 nM），并具有完全激动剂活性。这使得它很可能位于受体的天然位置。作为底物，我们使用了过表达功能性重组大鼠A型CCK受体的CHO-CCK受体细胞。通过SDS-聚丙烯酰胺凝胶电泳和放射自显影确定了Mr = 85,000 - 95,000的质膜糖蛋白与Mr = 42,000的核心发生了共价标记。通过溴化氰裂解以及凝胶和/或高压液相色谱分离确定，与跨膜1相邻的单个结构域被标记。通过用胰蛋白酶进一步进行蛋白水解、纯化标记片段，随后进行手动Edman降解和放射化学测序，进一步确定了相互作用位点。这表明Trp39被特异性标记，并且可能位于探针羧基末端对硝基苯丙氨酸33残基附近。已构建了这种配体结合受体的模型，并将用于规划未来的实验，以完善我们对这种相互作用的理解。
