title: Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein.
abstract: CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid 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 CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.
authors.affiliation: Department of Molecular and Cellular Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: J P
authors.initials: JP
authors.lastname: Allison
mesh_terms: D000069594:Abatacept; D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism*; D060908:CTLA-4 Antigen; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D018796:Immunoconjugates; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae
chemical_list: D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D002966:Clathrin; C556707:Ctla4 protein, mouse; D018076:DNA, Complementary; D018796:Immunoconjugates; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C438885:adaptor protein complex 2, mu 1 subunit; D000069594:Abatacept