Structural insights into the amino-terminus of the secretin receptor: I. Status of cysteine and cystine residues
Structural insights into the amino-terminus of the secretin receptor: I. Status of cysteine and cystine residues
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DOI：
10.1124/mol.58.5.911
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发表时间：
2000-11-01
影响因子：
3.6
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Asmann, YW;Dong, MQ;Miller, LJ
促胰液素受体是 G 蛋白偶联受体 II 类家族的原型，具有长的胞外氨基末端结构域，其中包含 6 个高度保守的 Cys 残基和 1 个仅存在于最密切相关的家族成员中的 Cys 残基 (Cys(11))。该结构域对于功能至关重要，一些半胱氨酸残基组分被认为参与关键的二硫键，尽管这些从未被直接证明。在这里，我们检查每个残基的功能重要性并确定它们在二硫键中的参与。用细胞不可渗透的还原剂处理细胞后，促胰液素结合显着减弱，这支持了重要的细胞外二硫键的存在。为了确定氨基末端结构域是否通过二硫键共价连接至受体体，实施了一种策略，该策略涉及引入酸不稳定的 Asp-Pro 序列，以实现在这些结构域的边界处的特异性切割。在非还原条件下，氨基末端从受体体中释放出来，支持这些结构域之间不存在共价关联。在不存在和存在化学还原的情况下，将[C-14]碘乙酰胺定量掺入受体的分离的氨基末端结构域中，确定游离与总Cys残基的比率为1:7，与三个二硫键一致。仅 Cys(11) 可以耐受将每个氨基末端 Cys 残基诱变为 Ala，这表明这些键连接了保守的 Cys 残基。用细胞不渗透的巯基反应试剂处理表达野生型或 C11A 突变型促胰液素受体的完整细胞进一步支持了这一点。因此，促胰液素受体的功能上重要的氨基末端代表结构上独立的、高度折叠的和二硫键连接的结构域，其模式在整个受体家族中可能是关键且保守的。
The secretin receptor is prototypic of the class II family of G protein-coupled receptors, with a long extracellular amino-terminal domain containing six highly conserved Cys residues and one Cys residue (Cys(11)) that is present only in the most closely related family members. This domain is critical for function, with some component Cys residues believed to be involved in key disulfide bonds, although these have never been directly demonstrated. Here, we examine the functional importance of each of these residues and determine their involvement in disulfide bonds. Secretin binding was markedly diminished after treating cells with cell-impermeant reducing reagents, supporting the presence of important extracellular disulfide bonds. To determine whether the amino-terminal domain was covalently attached to the receptor body by disulfide linkage, a strategy was implemented that involved introduction of an acid-labile Asp-Pro sequence to enable specific cleavage at the boundary of these domains. Under nonreducing conditions, the amino terminus was released from the receptor body, supporting the absence of covalent association between these domains. Quantitative [C-14] iodoacetamide incorporation into the isolated amino-terminal domain of the receptor in the absence and presence of chemical reduction established the ratio of free to total Cys residues as 1:7, consistent with three disulfide bonds. Mutagenesis of each of the amino-terminal Cys residues to Ala was tolerated only for Cys(11), suggesting that these bonds linked the conserved Cys residues. This was further supported by treatment of intact cells expressing wild-type or C11A mutant secretin receptor with a cell-impermeant sulfhydryl-reactive reagent. Thus, the functionally important amino terminus of the secretin receptor represents a structurally independent, highly folded, and disulfide-bonded domain, with a pattern that is likely critical and conserved throughout this receptor family.