Murphy P M, Gallin E K, Tiffany H L
Bacterial Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
J Immunol. 1990 Oct 1;145(7):2227-34.
Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.
血小板活化因子（PAF）和补体第五成分C5a的活性裂解产物都是强效过敏毒素和炎症介质。这两种物质在多种细胞类型上作用于不同的鸟嘌呤核苷酸结合调节蛋白偶联受体，从而激活一个信号级联反应，导致细胞内钙库的动员以及诸如中性粒细胞趋化性和平滑肌收缩等功能反应。关于PAF和C5a受体的结构以及它们所使用的细胞内信号通路，人们了解甚少。我们利用非洲爪蟾卵母细胞表达系统，证明在注射了用二丁酰环磷腺苷分化的早幼粒细胞白血病细胞系HL60的mRNA的卵母细胞中获得了C5a和PAF受体活性。通过测量获得的依赖配体的细胞内45Ca2+外流以及通过测量电压钳制卵母细胞中配体激活的跨膜电流来确定活性。C5a受体活性局限于蔗糖梯度分级分离的RNA中的一个单一尖锐峰，该峰对应于2 kb的转录本大小。相比之下，PAF受体活性在大小分级分离的3.5至6 kb的RNA中广泛分布。这表明不同大小的多个转录本可能编码功能性PAF受体。两种配体均以浓度依赖性和结构依赖性方式激活其各自的受体。获得的依赖C5a的钙外流活性被百日咳毒素抑制，而PAF受体活性未被抑制，这表明两种受体与不同的G蛋白偶联。这些数据确立了非洲爪蟾卵母细胞作为研究吞噬细胞C5a受体和PAF受体的分子和功能特性的模型系统。
