Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.
Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.
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DOI：
10.4049/jimmunol.149.8.2600
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发表时间：
1992-10
影响因子：
4.4
通讯作者：
Craig Gerard;Lu Bao;O. Orozco;M. Pearson;D. Kunz;Norma P. Gerard
中科院分区：
文献类型：
--
作者：
Craig Gerard;Lu Bao;O. Orozco;M. Pearson;D. Kunz;Norma P. Gerard
使用最近描述的人C5 a受体cDNA探针分离小鼠C5 a受体基因（Gerard，N. P.，和C.杰勒德1991. Nature 349：614）。与人基因类似，小鼠同源物含有两个外显子，其中5'非翻译区和起始甲硫氨酸密码子存在于外显子1中，分子的其余部分存在于外显子2中。小鼠C5 a受体的可表达cDNA的产生是使用聚合酶链反应和正义寡脱氧核苷酸引物完成的，所述引物包括编码N-连接的糖基化位点的序列的5'端的起始密码子。当转染到人293肾上皮细胞中时，克隆的cDNA指导人C5 a过敏毒素结合位点的表达，结合常数为2.5 +/- 0.3 nM;在相同条件下表达的人C5 a受体的Kd为1.7 +/- 0.2 nM。总的来说，推导的氨基酸序列的受体是65%相同的类似的基因结构。作为七个跨膜段受体的序列的比对揭示了最大的结构多样性（约70%）存在于推定的胞外结构域。相比之下，该7种跨膜受体家族的其他成员之间的物种差异通常仅为10%至20%，即使对于配体相对较小且预期不会与细胞外表面上的位点相互作用的受体也是如此。观察到C5 a受体在跨膜段和预测存在于细胞质中的所有环中（除了一个环）的高度结构鉴定。由于负责74个氨基酸多肽与两种C5 a受体的高亲和力结合的关键结构涉及物种之间保守的特征，这些数据提供了诱变研究的起点，以确定趋化性受体的结合和活化位点的性质。此外，这些数据提供了一种试剂，免疫学和分子遗传学研究的作用，C5 a受体在炎症模型。
The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.