The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.

• MeSH
• Thy-1 Antigens immunology   MeSH
• Cell Membrane immunology   MeSH
• Cholesterol physiology   MeSH
• Detergents MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescent Antibody Technique MeSH
• Rats MeSH
• Humans MeSH
• Mast Cells immunology   MeSH
• Molecular Sequence Data MeSH
• Mice MeSH
• Precipitin Tests MeSH
• Base Sequence MeSH
• src-Family Kinases metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Thy-1 Antigens MeSH
• Cholesterol MeSH
• Detergents MeSH
• lyn protein-tyrosine kinase MeSH Browser
• src-Family Kinases MeSH

The T-lymphocyte co-receptors of MHC glycoproteins CD4 and CD8 are known to be associated with the protein tyrosine kinase Lck via cysteine-containing sequences in the cytoplasmic domains of CD4 and CD8 and in the N-terminal domain of Lck. Here we demonstrate that a fraction of CD4 and CD8 molecules are associated with very large, detergent-resistant complexes containing several glycosylphosphatidylinositol-anchored proteins, (glyco)lipids, and protein tyrosine kinases Lck and Fyn but apparently no other major transmembrane proteins. Association of Lck and Fyn with these large complexes is, in contrast to simple CD4/CD8-Lck complexes, not sensitive to alkylation with iodoacetamide. These large complexes therefore represent an alternative way of association of CD4 and CD8 with the protein tyrosine kinases, which may play a role in signaling through these receptors.

• MeSH
• CD4 Antigens analysis   MeSH
• CD8 Antigens analysis   MeSH
• Cell Membrane chemistry   MeSH
• Glycosylphosphatidylinositols MeSH
• Humans MeSH
• Lymphocytes chemistry   MeSH
• Neoplasm Proteins * MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH
• Protein-Tyrosine Kinases analysis   classification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CD4 Antigens MeSH
• CD8 Antigens MeSH
• FRK protein, human MeSH Browser
• Glycosylphosphatidylinositols MeSH
• Neoplasm Proteins * MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH
• Protein-Tyrosine Kinases MeSH