Gold M R, Jakway J P, DeFranco A L
Department of Microbiology and Immunology, University of California, San Francisco 94143.
J Immunol. 1987 Dec 1;139(11):3604-13.
Cross-linking of membrane immunoglobulin, the B cell receptor for antigen, activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by phospholipase C. This reaction yields two intracellular second messengers, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes an increase in cytoplasmic Ca2+. The experiments reported here demonstrate that activation of phospholipase C by membrane IgM (mIgM) involves a guanine nucleotide-dependent step. Saponin was used to permeabilize WEHI-231 B lymphoma cells and permit direct manipulation of nucleotide and Ca2+ concentrations. Very high levels of Ca2+ (greater than 100 microM) activated the phospholipase maximally without a requirement for cross-linking of mIgM. However, at much lower, physiologically relevant Ca2+ concentrations (100 to 500 nM), receptor-stimulated PtdInsP2 hydrolysis could be demonstrated. The ability of anti-IgM antibodies to activate phospholipase C in permeabilized WEHI-231 cells was greatly increased by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues (guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate), but not by guanosine diphosphate or guanosine diphosphate analogues or by a nonhydrolyzable analogue of adenosine triphosphate. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein analogous to those that couple receptors to adenylate cyclase is involved in the activation of phospholipase C by mIgM in WEHI-231 B lymphoma cells. In order to characterize this putative GTP-binding component, we examined the ability of pertussis toxin and cholera toxin to affect anti-IgM-stimulated inositol phosphate production. These bacterial toxins covalently modify and modulate the activity of various GTP-binding regulatory proteins and in some cell types can block receptor-stimulated PtdInsP2 breakdown. In WEHI-231 B lymphoma cells, neither toxin blocked signaling by mIgM. Thus mIgM appears to be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to both pertussis toxin and cholera toxin.
膜免疫球蛋白（抗原的B细胞受体）的交联激活了磷酸肌醇信号转导途径。该途径的起始事件是磷脂酶C催化磷脂酰肌醇4,5-二磷酸（PtdInsP2）水解。此反应产生两种细胞内第二信使，即激活蛋白激酶C的二酰基甘油和导致细胞质Ca2+浓度升高的肌醇三磷酸。本文报道的实验表明，膜IgM（mIgM）激活磷脂酶C涉及一个依赖鸟嘌呤核苷酸的步骤。皂角苷用于使WEHI-231 B淋巴瘤细胞透化，从而能够直接调控核苷酸和Ca2+浓度。非常高浓度的Ca2+（大于100 microM）可最大程度地激活磷脂酶，而无需mIgM交联。然而，在低得多的、生理相关的Ca2+浓度（100至500 nM）下，可证明受体刺激的PtdInsP2水解。非水解性鸟苷5'-三磷酸（GTP）类似物（鸟苷-5'-O-(3-硫代三磷酸)或5'-鸟苷酰亚胺二磷酸）可大大增强抗IgM抗体在透化的WEHI-231细胞中激活磷脂酶C的能力，但鸟苷二磷酸或鸟苷二磷酸类似物以及三磷酸腺苷的非水解性类似物则无此作用。对GTP类似物的这种特异性与以下假设一致，即类似于那些将受体与腺苷酸环化酶偶联的GTP结合调节蛋白参与了WEHI-231 B淋巴瘤细胞中mIgM对磷脂酶C的激活。为了表征这种假定的GTP结合成分，我们研究了百日咳毒素和霍乱毒素影响抗IgM刺激的肌醇磷酸生成的能力。这些细菌毒素可共价修饰并调节各种GTP结合调节蛋白的活性，并且在某些细胞类型中可阻断受体刺激的PtdInsP2分解。在WEHI-231 B淋巴瘤细胞中，两种毒素均未阻断mIgM的信号传导。因此，mIgM似乎通过一种对百日咳毒素和霍乱毒素均不敏感的依赖GTP的成分与磷酸肌醇信号传导途径偶联。
