Z. Surviladze et al., FUNCTIONAL-HETEROGENEITY OF THY-1 MEMBRANE MICRODOMAINS IN RAT BASOPHILIC LEUKEMIA-CELLS, European Journal of Immunology, 28(6), 1998, pp. 1847-1858
Antibody-mediated cross-linking of Thy-1 glycoprotein on the surface o
f rat mast cells and rat basophilic leukemia (RBL) cells initiates bio
chemical events which culminate in secretion of allergy mediators. Thy
-1, like some other glycosylphosphatidylinositol (GPI)-anchored protei
ns, forms detergent-insoluble complexes containing protein tyrosine ki
nases (PTK) and some other molecules which are implicated in the signa
ling pathway. On the surface of a rat mast cell there are more than 10
(6) Thy-1 molecules; however, it is not known which fraction of them i
s involved in transmembrane signaling, and what exactly is the heterog
eneity of Thy-1 complexes. Using sucrose density gradient ultracentrif
ugation of detergent-lysed RBL cells we found that the density of Thy-
1 complexes depended on the detergent used and the lysis conditions em
ployed. Sepharose 4B gel chromatography fractionation followed by dens
ity gradient ultracentrifugation revealed both size and density hetero
geneity 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 ph
osphatidylinositol-specific phospholipase C (PI-PLC). Interestingly, r
emoval of only a small fraction of surface Thy-1 by PI-PLC abolished t
he cell activation as determined by tyrosine phosphorylation of certai
n proteins. When Triton X-100 lysates were fractionated at 12 000 x 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 tha
t the total Thy-1 was relatively homogeneously distributed over the pl
asma membrane, whereas the PI-PLC-resislant Thy-1 was found mostly in
small clusters. The combined data suggest that specialized membrane mi
crodomains enriched in Thy-1 with increased sensitivity to PI-PLC are
directly involved in coupling Thy-1 aggregation to transmembrane signa
ling.