Yamamura S, Handa K, Hakomori S
Department of Pathobiology, University of Washington and Pacific Northwest Research Foundation, Seattle 98122, USA.
Biochem Biophys Res Commun. 1997 Jul 9;236(1):218-22. doi: 10.1006/bbrc.1997.6933.
B16 melanoma is characterized by high content of GM3 ganglioside, which has been recognized as a melanoma-associated antigen defined by specific monoclonal antibodies. We report now that GM3 is present predominantly (>90%) in the 1% Triton X-100-insoluble, low-density microvesicular fraction ("detergent-insoluble glycosphingolipid-enriched microdomain"; DIGEM) separated on sucrose density-gradient centrifugation. Associated with DIGEM, many signal transducer molecules such as c-Src, FAK, and the low-molecular-weight G-proteins Rho A and H-Ras were also found. Rho A and FAK were found in part, and PLC-beta2 and G alphas were found exclusively, in the high-density fraction. Immunoprecipitation of GM3 present in DIGEM by anti-GM3 antibody DH2, followed by Western blotting, revealed co-precipitation of Rho A and c-Src with GM3. These findings suggest (i) a specific organization of GM3 in close association with Rho A and c-Src within DIGEM at the melanoma cell surface; and (ii) such organizational units may be directly involved in signal transduction, in which glycosphingolipids receive signals which are subsequently transduced by associated transducer molecules.
B16黑色素瘤的特征是含有高含量的GM3神经节苷脂，GM3已被公认为是一种由特异性单克隆抗体定义的黑色素瘤相关抗原。我们现在报告，GM3主要（>90%）存在于通过蔗糖密度梯度离心分离的1% Triton X-100不溶性、低密度微囊泡组分（“富含去污剂不溶性糖鞘脂的微区”；DIGEM）中。与DIGEM相关联，还发现了许多信号转导分子，如c-Src、FAK以及低分子量G蛋白Rho A和H-Ras。在高密度组分中部分发现了Rho A和FAK，并且仅发现了PLC-β2和Gαs。用抗GM3抗体DH2对DIGEM中存在的GM3进行免疫沉淀，随后进行蛋白质印迹分析，结果显示Rho A和c-Src与GM3共沉淀。这些发现表明：（i）在黑色素瘤细胞表面的DIGEM内，GM3与Rho A和c-Src紧密结合存在特定的组织形式；（ii）这样的组织单位可能直接参与信号转导，其中糖鞘脂接收信号，随后由相关的转导分子进行信号转导。
