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
• antigeny Thy-1 imunologie   MeSH
• buněčná membrána imunologie   MeSH
• cholesterol fyziologie   MeSH
• detergenty MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fluorescenční protilátková technika MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mastocyty imunologie   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• precipitinové testy MeSH
• sekvence nukleotidů MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny Thy-1 MeSH
• cholesterol MeSH
• detergenty MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• skupina kinas odvozených od src-genu MeSH

Rat peritoneal and pleural mast cells and rat basophilic leukemia cells, RBL-2H3, have been previously shown to be activated by Thy-1-specific monoclonal antibodies (mAb). In the present study we investigated the mechanism of Thy-1-mediated activation and compared it with activation induced by cross-linking of the high-affinity IgE receptor. Binding of an IgG Thy-1 x 1-specific mAb, MRCOX7 (OX7), to RBL-2H3 cells and mast cells, and activation of RBL-2H3 by the OX7 were abrogated by pretreatment of the cells with phosphatidyl inositol-specific phospholipase C (PI-PLC). The F(ab')2 fragment of OX7, in contrast to the Fab' fragment, induced cell activation as well as intact OX7 mAb. Cells sensitized with IgE exhibited an increased responsiveness to anti-Thy-1 antibodies suggesting formation of functional complexes of IgE receptor/IgE/Thy-1/anti-Thy-1. Pretreatment of RBL-2H3 cells with cholera toxin potentiated activation induced by IgE+antigen (Ag) and IgE+OX7, but had no effect on activation induced by OX7 antibody alone. Similarly, dexamethasone had no effect on OX7-induced activation but inhibited IgE+Ag- and IgE+OX7-induced activation. Analysis of phosphotyrosine-containing proteins in RBL-2H3 cell lysates revealed that IgE+Ag and IgE+OX7 induced a marked increase in tyrosine phosphorylation of several proteins that were not tyrosine phosphorylated in cells exposed to OX7 mAb alone. Similar results were obtained when RBL-2H3-derived cells, expressing transfected mouse Thy-1.2, were activated with Thy-1.2-specific IgM antibody. The combined data suggest that Thy-1-specific antibodies activate cells by a mechanism that is different from activation induced by cross-linking of high-affinity IgE receptor.

• MeSH
• antigeny povrchové imunologie   MeSH
• antigeny Thy-1 MeSH
• cholerový toxin imunologie   MeSH
• dexamethason imunologie   MeSH
• fosfodiesterasy imunologie   MeSH
• fosfolipasa C fosfoinositidové signalizace MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• mastocyty imunologie   MeSH
• membránové glykoproteiny imunologie   MeSH
• monoklonální protilátky imunologie   MeSH
• reagencia zkříženě vázaná MeSH
• receptory Fc imunologie   MeSH
• receptory IgE imunologie   MeSH
• tyrosin imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• antigeny Thy-1 MeSH
• cholerový toxin MeSH
• dexamethason MeSH
• fosfodiesterasy MeSH
• fosfolipasa C fosfoinositidové signalizace MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• membránové glykoproteiny MeSH
• monoklonální protilátky MeSH
• reagencia zkříženě vázaná MeSH
• receptory Fc MeSH
• receptory IgE MeSH
• tyrosin MeSH

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cells (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with src-related protein-tyrosine kinase p53/p56lyn and other protein-tyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large ( > 10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

• MeSH
• akutní bazofilní leukemie MeSH
• antigeny povrchové izolace a purifikace   metabolismus   MeSH
• antigeny Thy-1 MeSH
• C-terminální Src kinasa MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfodiesterasy MeSH
• gelová chromatografie MeSH
• imunoblotting MeSH
• krysa rodu Rattus MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny izolace a purifikace   metabolismus   MeSH
• molekulová hmotnost MeSH
• monoklonální protilátky MeSH
• nádorové buňky kultivované MeSH
• protoonkogenní proteiny pp60(c-src) izolace a purifikace   metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• skupina kinas odvozených od src-genu * MeSH
• tyrosinkinasy izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• antigeny Thy-1 MeSH
• C-terminální Src kinasa MeSH
• fosfodiesterasy MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• makromolekulární látky MeSH
• membránové glykoproteiny MeSH
• monoklonální protilátky MeSH
• protoonkogenní proteiny pp60(c-src) MeSH
• receptory IgE MeSH
• skupina kinas odvozených od src-genu * MeSH
• tyrosinkinasy MeSH