Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble innoncaveolar plasma membrane microdomains defined by reggie-1 and-2

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in :less than or equal to 0.1-mum plasma membrane microdomains of neurons and astrocytes. In astrocyt es, reggie-1 and -2 do not occur in caveolae but clearly outside these stru ctures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidylinositol-anchor ed proteins Thy-1 and F3 that, when activated by antibody cross-linking, se lectively copatch with reggie. Jurkat cells, after crosslinking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This , and the accumulation of reggie proteins in detergent-resistant membrane f ractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 propertie s of raft-associated proteins. It also suggests that reggie-1 and -2 partic ipate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 togethe r with fyn and Thy-1 increase in endolysosomes concurrent with a decrease a t the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomai n signaling centers in neurons and T cells, and the protein complex involve d in signaling becomes subject to degradation.