Microdomains of GPI-anchored proteins in living cells revealed by crosslinking.
There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane. These putative microdomains have been implicated in processes such as sorting in polarized cells and signal transduction. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.
Department of Cell Biology, Max Delbrück Centre for Molecular Medicine, Berlin-Buch, Germany.
T
T
Friedrichson
T V
TV
Kurzchalia
D000818:Animals; D018958:CD55 Antigens / Q000737:chemistry*; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D013006:Growth Hormone / Q000737:chemistry*; D051381:Rats
D018958:CD55 Antigens; D003432:Cross-Linking Reagents; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D013006:Growth Hormone