Gerard C, Bao L, Orozco O, Pearson M, Kunz D, Gerard N P
Department of Pediatrics, Beth Israel Hospital, Boston, MA.
J Immunol. 1992 Oct 15;149(8):2600-6.
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.
利用最近报道的人C5a受体cDNA探针分离出小鼠C5a受体基因（杰勒德，N.P.，和C.杰勒德。1991。《自然》349:614）。与人类基因类似，小鼠同源基因包含两个外显子，5'非翻译区和起始甲硫氨酸密码子位于外显子1中，分子的其余部分位于外显子2中。使用聚合酶链反应和一个正义寡脱氧核苷酸引物来生成可表达的小鼠C5a受体cDNA，该引物在编码N-连接糖基化位点的序列的5'端包含一个起始密码子。当转染到人293肾上皮细胞中时，克隆的cDNA指导人C5a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C5a受体的Kd为1.7±0.2 nM。总体而言，鉴于相似的基因结构，受体的推导氨基酸序列有65%相同。将序列比对为七跨膜段受体表明，最大的结构多样性（约70%）存在于假定的细胞外结构域中。相比之下，这个七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%，即使对于其配体相对较小且预计不会与细胞外表面位点相互作用的受体也是如此。在跨膜段以及预测存在于细胞质中的除一个环以外的所有环中，观察到C5a受体具有高度的结构同一性。由于负责74个氨基酸多肽与两种C5a受体高亲和力结合的关键结构涉及物种间保守的特征，这些数据为诱变研究提供了起点，以确定趋化受体的结合和激活位点的性质。此外，这些数据为关于C5a受体在炎症模型中的作用的免疫和分子遗传学研究提供了一种试剂。
