The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-Immnoprecipitation experiments revealed that CD4 and p56lck were irnrnunopracipitated by IgG anfi-GM3, demonstrating that GM3 tightly binds to the CD4-p56lck complex in human PBL. In order to verify whether GM3 association with CD4 rnolecules may depend on the presence of p55lck, we analyzed this association in U937, a CD4 + and p56lck negative cell line. The Imrnunoprecipitation with anti-GM3 revealed the presence of a 58kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.
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