EGF induces coalescence of different lipid rafts.
The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.
Hofman
Erik G
EG
Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
Ruonala
Mika O
MO
Bader
Arjen N
AN
van den Heuvel
Dave
D
Voortman
Jarno
J
Roovers
Rob C
RC
Verkleij
Arie J
AJ
Gerritsen
Hans C
HC
van Bergen En Henegouwen
Paul M P
PM
D000818:Animals; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005677:G(M1) Ganglioside / Q000032:analysis / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D015398:Signal Transduction
D017261:Glycosylphosphatidylinositols; D049452:Green Fluorescent Proteins; D005677:G(M1) Ganglioside; D004815:Epidermal Growth Factor; D066246:ErbB Receptors