Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains

Increasing evidence for the organization of cell-surface proteins and lipid s into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 car cinoma cells, using a combination of cell fractionation and immunoprecipita tion techniques. Density-gradient centrifugation of sodium carbonate cell e xtracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fr action, with the biochemical characteristics of detergent-insoluble glycoli pid-rich domains (DIGs). However, ultrastructural analysis of the CLM fract ion revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that. in cont rast with caveolin, EGF receptors were unlikely to be localized to DIG doma ins. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF r eceptor activation occurs in a compartment distinct from caveolae. Similarl y, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membra ne. In addition, by demonstrating that EGF receptor activation is compartme ntalized within low-density, non-caveolar regions of the plasma membrane, i t is also shown that the co-localization of proteins in a CLM fraction is i nsufficient to prove caveolar localization.