Molecular characterization of the melanin-concentrating hormone/receptor complex: Identification of critical residues involved in binding and activation
Molecular characterization of the melanin-concentrating hormone/receptor complex: Identification of critical residues involved in binding and activation
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DOI：
10.1124/mol.58.1.217
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发表时间：
2000-07-01
影响因子：
3.6
通讯作者：
Graziano, MP
中科院分区：
文献类型：
--
作者：
Macdonald, D;Murgolo, N;Graziano, MP
构建了人类黑色素浓缩激素 (MCH) 肽的分子模型，并将其对接到最近鉴定的人类 MCH 受体的基于细菌视紫红质的螺旋模型中。从该激素-受体复合物中，鉴定了激动剂-受体相互作用的潜在位点，并使用定点诱变来替换预计位于受体结合口袋内的残基。受体第三个跨膜结构域中 Asp(123) (3.32) 的取代导致可检测的 I-125-MCH 结合和 MCH 刺激的 Ca2+ 通量的损失；突变受体的细胞表面表达不受影响。 MCH 配体的 Arg(11) 和 Arg(14) 被确定为与 Asp(123) (3.32) 相互作用的潜在位点。 [Ala(14)]-MCH 在结合和激活野生型 MCH 受体的能力方面与天然 MCH 相当，而 [Ala(11)]-MCH 的结合亲和力降低了 3000 倍，并且可测量的功能活性完全丧失。此外，[Lys(11)]-MCH 和 [D-Arg(11)]-MCH 对受体的亲和力降低。 [Lys(11)]-MCH 被观察为部分激动剂，在 Ca2+ 通量测定中引发约 67% 的天然肽活性，并且 [D-Arg(11)]-MCH 被确定为 K-b 值为 15.8 μM 的功能性拮抗剂。这些数据提供了证据，表明受体激活需要在该位点具有特定立体化学要求的基本部分。我们得出结论，MCH 受体中的 Asp(123) (3.32) 和 MCH 肽中的 Arg(11) 都是形成 MCH 肽/受体复合物所必需的，并提出它们形成对受体功能至关重要的直接相互作用。
A molecular model of the human melanin-concentrating hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor complex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to reside within the receptor binding pocket. Substitution of Asp(123) (3.32) in the third transmembrane domain of the receptor resulted in a loss of detectable I-125-MCH binding and of MCH-stimulated Ca2+ flux; cell surface expression of the mutant receptor was not affected. Arg(11) and Arg(14) of the MCH ligand were identified as potential sites of interaction with Asp(123) (3.32). [Ala(14)]-MCH was equipotent to native MCH in its ability to bind to and activate the wild-type MCH receptor, whereas [Ala(11)]-MCH displayed a 3000-fold reduction in binding affinity and a complete loss of measurable functional activity. Furthermore, [Lys(11)]-MCH and [D-Arg(11)]-MCH displayed reduced affinity for the receptor. [Lys(11)]-MCH was observed to be a partial agonist, eliciting approximately 67% of the native peptide's activity in a Ca2+ flux assay, and [D-Arg(11)]-MCH was determined to be a functional antagonist with a K-b valve of 15.8 mu M. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is needed for receptor activation. We conclude that both Asp(123) (3.32) in the MCH receptor and Arg(11) in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct interaction that is critical for receptor function.