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We analyzed the role of gangliosides in the association of the ErbB2 recep-tor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2– GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epi- | ềFEBS Journal Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells Elena Sottocornola1 Roberta Misasi2 Vincenzo Mattei2 3 Laura Ciarlo2 Roberto Gradini2 4 Tina Garofalo2 3 Bruno Berra1 Irma Colombo1 and Maurizio Sorice2 3 1 Institute of GeneralPhysiology and BiologicalChemistry University of Milan Italy 2 Department of ExperimentalMedicine and Pathology University of Rome La Sapienza Italy 3 Laboratory of ExperimentalMedicine and EnvironmentalPathology Rieti Italy 4 INM Neuromed Pozzilli Italy Keywords epidermalgrowth factor receptor ErbB2 GM3 HC11 cells lipid rafts Correspondence M. Sorice Department of Experimental Medicine and Pathology University of Rome La Sapienza viale Regina Elena 324 Rome 00161 Italy Fax 39 6 445 4820 Tel 39 6 499 72675 E-mail maurizio.sorice@uniroma1.it Received 24 December 2005 revised 15 February 2006 accepted 27 February 2006 doi 10.1111 j.1742-4658.2006.05203.x We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase RTK with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2 epidermal growth factor receptor EGFR and Shc-p66 proteins correlated with the ErbB2 signal transduction pathway were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by - -threo-1-phenyl-2-decanoylamino-3-morpho-lino-1-propanol hydrochloride D -PDMP induced a drastic cell-surface redistribution of ErbB2 EGFR and Shc-p66 within the Triton-soluble fractions as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated .
