A cholesterol-independent membrane microdomain serves as a functional counter-receptor for E-selectin at the Colo201 cell surface and initiates signalling on E-selectin binding.
The present study demonstrates that the functional counter-receptors for E-selectin at the cell surface of Colo201 human colon cancer cells are localized in detergent-insoluble membrane microdomains (DIM). Following isolation of counter-receptors from whole cell lysates using E-selectin-coupled magnetic beads followed by sucrose density gradient separation, both sialyl Lewis a (SLe(a))- and sialyl Lewis x (SLe(x))-carrying glycoproteins which had bound to the E-selectin-beads were distributed in detergent-soluble fractions as well as DIM. In contrast, following isolation of counter-receptors directly from the cell surface, SLe(a)-carrying glycoproteins which had bound to E-selectin-beads at the cell surface were localized only in DIM, together with a Src family kinase, Lyn, while SLe(x)-carrying glycoproteins were not detected in any fraction. The counter-receptors were distributed in a diffuse pattern on the cell surface but clustered following E-selectin binding, leading to the subsequent phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of the cells with methyl-beta-cyclodextrin, a cholesterol-depleting drug, had little effect on either the association of SLe(a)-carrying glycoproteins and Lyn with the domain or ERK phosphorylation. Thus, the functional counter-receptors and Lyn are co-localized in a cholesterol-independent microdomain and create a physiological domain ('glycosynapse') at the cell surface that initiates signalling in cancer cells upon binding to E-selectin.
Suzuki
Chikako
C
The Institute of Glycotechnology, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan.
Kojima
Naoya
N
D001665:Binding Sites; D018395:CA-19-9 Antigen; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D019040:E-Selectin / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005732:Gangliosides / Q000032:analysis; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins / Q000494:pharmacology
D018395:CA-19-9 Antigen; D019040:E-Selectin; D005732:Gangliosides; D011956:Receptors, Cell Surface; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C064564:sialyl Le(a) ganglioside; D002784:Cholesterol; D048049:Extracellular Signal-Regulated MAP Kinases