Petry Renate, Craik David, Haaima Gerald, Fromme Bernhard, Klump Horst, Kiefer Wolfgang, Palm Dieter, Millar Robert
Institut für Physikalische Chemie, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
J Med Chem. 2002 Feb 28;45(5):1026-34. doi: 10.1021/jm011036k.
The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R) that confers agonist selectivity for Arg(8) in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L(296), N(297), and R(298). A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.
哺乳动物促性腺激素释放激素受体（GnRH-R）的细胞外环3（ECL3）含有一个酸性氨基酸（小鼠GnRH-R中的Glu(301)），该氨基酸赋予了对哺乳动物GnRH中Arg(8)的激动剂选择性。有人提出，ECL3的特定构象对于使酸性残基的羧基侧链定向以与GnRH的Arg(8)相互作用是必要的，当相邻的Pro突变为Ala时，对Arg(8) GnRH的亲和力降低但对Gln(8) GnRH的亲和力未降低，这支持了这一观点。为了探究环结构域对羧基侧链提出的结构贡献，我们合成了一个模拟小鼠ECL3第293 - 302位残基的模型肽（CGPEMLNRVSEPGC），其中在N端和C端分别对称添加了Cys和Gly残基，允许引入二硫键以模拟ECL3与受体跨膜结构域6和7相连的距离。ECL3肽以低亲和力结合GnRH的能力通过其对表达GnRH-R的细胞中GnRH刺激的肌醇磷酸产生的抑制作用得到证明。ECL3肽的圆二色光谱带表现出主要为无序结构的叠加以及β-折叠结构的部分贡献。同样，来自显微拉曼和傅里叶变换拉曼实验的酰胺I和酰胺III谱带分析表明，环状和线性肽主要为无序构象。核磁共振数据表明，环状肽在大量无序结构中存在一个β-发夹结构。连接发夹两条链的转角位置被确定为三个中心残基L(296)、N(297)和R(298)。在主要为无规卷曲构象的集合中存在少量结构化物种，这表明未结合配体的受体代表多种结构构象体，其中一些有可能与配体接触。我们提出了一种受体激活机制，即激动剂与无活性受体状态的结合诱导并稳定环结构域的特定结构状态，导致跨膜结构域进一步的构象重排以及与G蛋白的信号传导相互作用。受体的Glu(301)与GnRH的Arg(8)相互作用诱导配体形成折叠构象。因此，我们的提议表明配体和受体的构象变化均源于这种相互作用。
