The human being epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors ubiquitous actors in cell signaling proliferation differentiation and migration. poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer by preventing the autophosphorylation of the intracellular kinase domain in response to SAP155 ligand binding. and values of EGFR-K642G mutant receptor ld/lo?±?GM3 proteoliposomes is not affected to a great extent compared to WT-EGFR (values correspond … GM3 Retains EGFR in the Monomeric State. Chemical cross-linking studies have indicated that GM3 inhibits receptor dimerization in the plasma membrane (12) essential for EGFR activation (24). Using the cross-linker BS3 in our proteoliposomes we showed that GM3 had no effect on receptor dimerization in the absence of ligand in ld proteoliposomes. In contrast in phase-separated proteoliposomes GM3 prevented the formation of cross-linked dimers (Fig.?5) but EGF addition overcame the inhibition. Thus GM3 in phase-separated proteoliposomes seemed to stabilize the monomeric form of EGFR. Fig. 5. Ligand-induced dimerization of EGF receptor in proteoliposomes. Prior to cross-linking EGFR proteoliposomes were incubated with EGF (30?min RT). For chemical cross-linking BS3 was used (50?μM 15 RT). In the absence … Discussion The most important finding of this study is that EGFR activity can be regulated by its lipid environment and in this context is specifically inhibited by interaction with the ganglioside GM3. Previous studies have suggested this possibility (6) but because of cellular complexity and membrane compositional diversity unambiguous assignment of lipid-mediated EGFR modulation has not found its way into the signal transduction canon (8-10). Our findings using purified EGFR reconstituted into proteoliposomes of specific lipid compositions unequivocally demonstrate that whereas the lipid environment does not affect EGF binding interactions between the receptor and membrane lipids lead to changes in EGFR tyrosine kinase function. A three-component lipid mixture consisting of unsaturated PC sphingomyelin and cholesterol in molar ratios that phase separate into coexisting ld and lo domains prevented EGFR autophosphorylation in the absence of EGF while allowing ligand-mediated receptor dimerization and activation. When GM3 was added to the ld/lo proteoliposomes EGFR autophosphorylation OSI-420 was inhibited (Fig.?2) without affecting ligand binding (Fig.?1 and EGF receptor proposed a negative cooperativity in ligand binding (27). Upon binding of the EGF-like domain of Spitz (SpitzEGF) to the Drosophila EGFR the first ligand-binding event induced an asymmetric dimer with only one SpitzEGF bound. The structural data suggested that the unoccupied site on the second EGFR subunit was restrained by the first binding event leading to reduced binding affinity for the second SpitzEGF molecule in the asymmetric dimer. However the isolated human EGFR ectodomain does not form asymmetric dimers like its counterpart (27 28 Thus if the negative cooperativity were to explain heterogeneity in ligand binding the intracellular part of the EGFR would be driving the formation of an asymmetric dimer as previously suggested (29). Our binding studies showed that the OSI-420 full-length human EGFR reconstituted into OSI-420 liposomes failed to display high affinity binding. Although our cross-linking data suggested the presence of preformed dimers (Fig.?5) we observed only one affinity state of the EGFR (Fig.?1 and Neuraminidase New England Biolabs) was added to the samples during the last 30?min incubation with EGF. Supplementary Material Supporting Information: Click here to view. Acknowledgments. The authors thank all past and present members of the Simons laboratory. We thank Anna Shevchenko and OSI-420 Julio Sampaio for their skillful mass spectrometric analysis of our samples. We also thank Li Ying (Nanyang Technological University Singapore) for help with the initial.