Todeschini Adriane Regina, Dos Santos Jose Nilson, Handa Kazuko, Hakomori Sen-itiroh
Division of Biomembrane Research, Pacific Northwest Research Institute, and Department of Pathobiology, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1925-30. doi: 10.1073/pnas.0709619104. Epub 2008 Feb 6.
Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through "cross-talk" of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway.
在HCV29细胞及其他上皮细胞的糖突触微结构域中，与四跨膜蛋白CD82复合的神经节苷脂GM2可抑制肝细胞生长因子诱导的cMet酪氨酸激酶活性。此外，HCV29细胞与细胞外基质蛋白的黏附还可通过整合素与cMet的“串扰”激活cMet激酶，进而抑制细胞迁移和生长。目前的研究表明，细胞迁移和生长受GM2、GM3或GM2/GM3复合物表达的显著影响，基于以下一系列观察结果，这些复合物会影响各类细胞的cMet激酶活性：（i）在二氧化硅纳米球上共包被GM2和GM3培养的表达CD82的细胞，相较于单独用GM2或GM3培养的细胞，或与其他糖鞘脂一起培养的细胞，表现出更强且更一致的迁移抑制作用。（ii）通过电喷雾电离（ESI）质谱以及与针对GM2/GM3二聚体结构的单克隆抗体8E11的特异性反应证明，在Ca2+存在的情况下，GM2 - GM3形成异二聚体。（iii）表达cMet和CD82的细胞表现出与HGF诱导的cMet激活相关的迁移增强。用包被在纳米球上的GM2/GM3二聚体培养细胞，可强烈抑制cMet和迁移。（iv）在无HGF的情况下，HCV29或YTS - 1/CD82细胞与层粘连蛋白-5包被的平板的黏附激活了cMet激酶，而GM2/GM3二聚体抑制黏附诱导的cMet激酶活性并抑制细胞迁移。（v）如在（i）、（iii）和（iv）中所述的受抑制的细胞迁移，通过添加阻断GM2/GM3二聚体与CD82相互作用的单克隆抗体8E11恢复至正常水平。响应于GM2/GM3二聚体与CD82的相互作用，通过Src和MAP激酶的信号传导与cMet激酶紧密相关地被激活或抑制。因此，一种与CD82复合的此前未被表征的GM2/GM3异二聚体通过CD82 - cMet或整合素 - cMet途径抑制细胞迁移。
