Isolation of Thy-1 caps and analysis of their phospholipid composition in mouse T-lymphoma cells

In this study we have used a density perturbation method to isolate anti-Thy-1 antibody-induced Thy-1 caps from mouse T-lymphoma cells in the absence of detergents, and then compared the phospholipid composition of these capped membranes with that of uncapped membranes. Initial phospholipid analysis by two-dimensional thin layer chromatography (2-D TLC) reveals a significant increase in the amount of 32P-labeled phosphatidylcholine in the Thy-1 capped membrane. In contrast, no significant changes are observed in the labeling of phosphatidylserine, phosphatidylethanolamine, or the sphingomyelins. Therefore, it is suggested that phosphatidylcholine may be involved in the organization and/or regulation of Thy-1 antigen redistribution. The composition of phosphoinositide in uncapped and capped membranes was analysed separately using one-dimensional thin layer chromatography (1-D TLC) to resolve phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PlP), and phosphatidylinositol 4, 5-bisphosphate (PIP2) from all other phospholipids. This analysis reveals a significant reduction in levels of PIP and PIP2, but not PI, in Thy-1 caps. Through the use of ion exchange column chromatography, we have found an increased production of all three species of inositol phosphates during anti-Thy-1 antibody-induced capping. Inositol 1, 4, 5-triphosphate (IP3) shows the most significant increase, compared to the much smaller increases in inositol 4, 5-bisphosphate (IP2) and inositol monophosphate (IP). These results suggest that the binding of anti-Thy-1 antibody to Thy-1 antigen activates phospholipase C which, in turn, initiates polyphosphoinositide turnover and IP3 production. It is proposed that these observed effects are the result of early signal transducing events which are prerequisite steps in Thy-1 receptor cap formation.