Functional heterogeneity of Thy‐1 membrane microdomains in rat basophilic leukemia cells

Antibody‐mediated cross‐linking of Thy‐1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy‐1, like some other glycosylphosphatidylinositol (GPI)‐anchored proteins, forms detergent‐insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 106 Thy‐1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy‐1 complexes. Using sucrose density gradient ultracentrifugation of detergent‐lysed RBL cells we found that the density of Thy‐1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy‐1 and Lyn PTK complexes. Cross‐linking of surface Thy‐1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium‐density fractions. Thy‐1 in low‐density fractions was relatively resistant to cleavage with phosphatidylinositol‐specific phospholipase C (PI‐PLC). Interestingly, removal of only a small fraction of surface Thy‐1 by PI‐PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X‐100 lysates were fractionated at 12 000 × g, about 50 % of Thy‐1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy‐1 exhibited an increased sensitivity to PI‐PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy‐1 was relatively homogeneously distributed over the plasma membrane, whereas the PI‐PLC‐resistant Thy‐1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy‐1 with increased sensitivity to PI‐PLC are directly involved in coupling Thy‐1 aggregation to transmembrane signaling.