Malhotra V, Hogg N, Sim R B
Eur J Immunol. 1986 Sep;16(9):1117-23. doi: 10.1002/eji.1830160915.
U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b-binding proteins isolated by affinity chromatography on iC3b-Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b-Sepharose. The polypeptides of 170 and 95 kDa were identified as the alpha and the beta subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150-kDa polypeptide was not immunoprecipitated by antibodies to the alpha subunit of CR3 or LFA-1. However, the 150-kDa polypeptide, together with the 95-kDa polypeptide, was immunoprecipitated with an anti-beta subunit-specific antibody IB4, which immunoprecipitates LFA-1, CR3 and p150,95. These results indicated that the 150-kDa polypeptide is the alpha subunit of the p150,95 antigen. The binding of p150,95 and CR3 to iC3b-Sepharose is specific as neither binds to C3u-Sepharose. A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95-kDa polypeptide from U937 cells was characterized as being directed against the alpha-subunit of the p150,95 antigen. Phorbol myristate acetate-stimulated U937 cells from rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for p150,95 in adherence of iC3b-coated particles. Since there is no rosetting with C3d-bearing particles it is unlikely that p150,95 is a receptor for C3d, a role for p150,95 which has been suggested by others (Wright, S.D., Licht, M.R. and Silverstein, S.C., Fed. Proc. 1984. 43: 413).
在佛波酯肉豆蔻酸酯乙酸盐存在的情况下培养的U937细胞（一种单核细胞系）用¹²⁵I进行表面标记，然后在二价阳离子存在的情况下，通过在iC3b-琼脂糖上进行亲和层析分离iC3b结合蛋白。发现有三种分子量分别为170、150和95 kDa的多肽能特异性结合iC3b-琼脂糖。通过用OKM1单克隆抗体进行免疫沉淀，将170 kDa和95 kDa的多肽鉴定为CR3的α和β亚基。150 kDa的多肽不能被针对CR3或LFA-1的α亚基的抗体免疫沉淀。然而，150 kDa的多肽与95 kDa的多肽一起，能用抗β亚基特异性抗体IB4进行免疫沉淀，该抗体能免疫沉淀LFA-1、CR3和p150,95。这些结果表明150 kDa的多肽是p150,95抗原的α亚基。p150,95和CR3与iC3b-琼脂糖的结合是特异性的，因为它们都不与C3u-琼脂糖结合。一种单克隆抗体3.9，它能从U937细胞中免疫沉淀150 kDa和95 kDa的多肽，其特征是针对p150,95抗原的α亚基。佛波酯肉豆蔻酸酯乙酸盐刺激的U937细胞能与EAC3b和EAiC3b形成玫瑰花结，但不能与EAC3d细胞形成玫瑰花结。单克隆抗体3.9不抑制任何一种类型的玫瑰花结形成，但我们无法排除p150,95在iC3b包被颗粒黏附中的作用。由于不存在与携带C3d颗粒的玫瑰花结形成，所以p150,95不太可能是C3d的受体，其他人曾提出过p150,95的这一作用（赖特，S.D.，利希特，M.R.和西尔弗斯坦，S.C.，《联邦程序》，1984年。43: 413）。
