Legler D F, Loetscher M, Jones S A, Dahinden C A, Arock M, Moser B
Theodor-Kocher Institute, University of Bern, Switzerland.
Eur J Immunol. 1996 Apr;26(4):753-8. doi: 10.1002/eji.1830260405.
The proteolytic cleavage product of complement component 3, (C3a), is like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors of C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I-C3a with high-(Kd1 = 2.1-4.8 nM) or low-affinity (Kd2 = 30-150 nM), and both receptors are expressed at high level: 3 x 10(5)-6 x 10(5) C3aR1/cell and 5 x 10(5)-2.3 x 10(6) C3aR2/cell. Results from cross-linking experiments with 125I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54-61 kDa (p57) and 86-107 kDa (p97) could be covalently modified with 125I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GRO alpha, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1 alpha, MIP-1 beta and I309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes.
补体成分3（C3）的蛋白水解裂解产物C3a与C4a和C5a一样，是一种强效过敏毒素，可诱导吞噬细胞产生炎症介质。值得注意的是，肥大细胞对C3a作出反应，释放包括组胺在内的血管活性物质。我们已经研究了C3a在人白血病肥大细胞系HMC-1中的功能和受体结合情况。与白细胞中的趋化激动剂类似，C3a可诱导HMC-1细胞胞质游离钙浓度迅速升高。乙二醇双四乙酸（EGTA）不会减弱这种反应，表明可动员的Ca2+来自细胞内储存库。HMC-1细胞中的C3a受体部分与百日咳博德特氏菌毒素敏感的G蛋白偶联，因此似乎属于需要G蛋白进行信号转导的蛇形受体家族。HMC-1细胞表达两种类型的C3a受体，C3aR1和C3aR2，它们分别以高亲和力（Kd1 = 2.1 - 4.8 nM）或低亲和力（Kd2 = 30 - 150 nM）结合125I-C3a，并且两种受体均高水平表达：3×105 - 6×105个C3aR1/细胞和5×105 - 2.3×106个C3aR2/细胞。用125I-C3a进行交联实验的结果完全证实了HMC-1细胞中存在两种不同类型的C3a受体。两种表观分子量分别为54 - 61 kDa（p57）和86 - 107 kDa（p97）的膜蛋白可被125I-C3a共价修饰，并且这种交联可被过量的未标记C3a抑制。许多已知的白细胞激动剂，包括13种趋化因子（IL-8、NAP-2、GROα、ENA-78、IP10、PF4、MCP-1、2和3、RANTES、MIP-1α、MIP-1β和I309）、三种神经肽（神经肽Y、生长抑素和降钙素）以及C5a，均不能激活HMC-1细胞，这表明C3a是该细胞系表达特异性受体的少数蛋白质配体之一。对C3a的明显选择性以及C3a受体的丰富表达使得HMC-1细胞系成为克隆受体基因的理想选择。
