Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells.
Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains.
Infectious Disease Unit, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115, USA.
K R
KR
Solomon
M A
MA
Mallory
R W
RW
Finberg
D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018958:CD55 Antigens / Q000737:chemistry / Q000302:isolation & purification; D018957:CD59 Antigens / Q000737:chemistry / Q000302:isolation & purification; D002460:Cell Line; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000032:analysis*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017830:Octoxynol; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011092:Polyethylene Glycols; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018958:CD55 Antigens; D018957:CD59 Antigens; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010750:Phosphoproteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40