proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. membranes) GEMs (glycosphingolipid-enriched microdomains) and CGP 57380 glycosynapses [4]. With the exception of glycosynapses they are all based on a classic model of ‘lipid rafts’. The structure of lipid rafts in the intact plasma membrane is usually unknown but an operational definition has been adopted according to which they are cholesterol-dependent and contain components that are isolated as detergent-insoluble membranes [5 6 TERMs (tetraspanin-enriched microdomains) represent a novel type of molecular aggregate that are distinct from all these mentioned above. They could be operationally defined as the membrane complexes maintained after solubilization with moderate detergents such as Brij96 or Brij98 [7 8 Indeed in contrast with classical lipid rafts TERM are sensitive to Triton X-100 and do not contain glycosylphosphatidylinositol-linked proteins [8]. The principal components of TERM are tetraspanins which constitute a large family of four-transmembrane-domain proteins. Although the exact biochemical function of tetraspanins is not well defined it has been established that these proteins play an important role in membrane compartmentalization and dynamics [8-11]. Tetraspanins interact with each other thereby providing a structural platform for the recruitment of other transmembrane and cytoplasmic proteins into TERM [8 11 12 A number of transmembrane receptors are known to associate with tetraspanin microdomains including integrins CGP 57380 [13] CGP 57380 receptor tyrosine kinases and G-protein-coupled receptors [11 12 Importantly association of a particular receptor with TERM may lead either to the enhancement or to the attenuation of its activity [10 14 The contribution of individual tetraspanins to the generation of co-stimulatory or inhibitory effects has not been studied in detail. Gangliosides are essential structural components of the membranes. These glycosphingolipids are involved in the regulation of signalling through the growth factor and adhesion receptors [15-17]. Previous reports established various links between tetraspanins and gangliosides. It was shown that CD9 (and possibly CD82) and GM3 co-operatively down-regulate motility of tumour cells CGP 57380 by attenuating signalling CGP 57380 induced by laminin-integrin interactions [17-19]. The cross-talk between integrins and fibroblast growth factor receptor has also been influenced by the changes in gangliosides and/or tetraspanin expression levels [20]. Moreover our previous results suggested that the activity of EGFR [EGF (epidermal growth factor) receptor] is attenuated by the tetraspanin CD82 through the modification of ganglioside composition at the membrane [21]. Specifically we found that there was a correlation in the expression levels of CD82 and gangliosides in mammary epithelial cells [21]. However the underlying mechanism of the interactions of gangliosides and tetraspanins has not been identified. Most RACGAP1 likely gangliosides provide stability and structural support for TERM. Gangliosides may also control localization of the transmembrane proteins to the segregated microdomains. In the present study we examined the importance of gangliosides for the assembly of TERM. By manipulating ganglioside content pharmacologically and enzymatically we demonstrated that removal of gangliosides affects associations of CD82 with its partners components of TERM. We also found that there is a clear specificity in the effect that ganglioside depletion had on the interactions involving different tetraspanins. Particularly only associations of CD82 (but not CD9) with EGFR α3β1 integrin and other tetraspanins have been affected. Thus our results..