INTERACTION OF CTLA-4 WITH AP50, A CLATHRIN-COATED PIT ADAPTER PROTEIN

CTLA-4 plays a critical role in regulating the immune response, It is mainly located in cytoplasmic vesicles and is expressed only transient ly on the surface after T cell activation, In this study, we demonstra te that CTLA-4 is associated with AP50, the medium chain of the clathr in-associated coated pit adaptor protein complex AP2. In a yeast two h ybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTL A-4 and AP50 are cotransfected into human 293T cells, A Y-201 to F-201 mutation in the YVKM intracellular localization motif of the CTLA-4 c ytoplasmic domain significantly diminished its binding to AP50, We als o found that AP50 bound to a CTLA-4 peptide containing unphosphorylate d Y-201 but not to a peptide containing phosphorylated Y-201, Converse ly, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SW (SHP-2) and SHP (SHP-1) bind o nly to the CTLA-4 peptide containing phosphorylated Y-(201). Therefore , the phosphorylation status of Y-201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest t hat AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function: of CTLA -4.