Regulation and function of the CD3gamma DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro.
Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3gamma. Thus, phosphorylation of CD3gamma serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3gamma internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3gamma molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.
Dietrich
J
J
Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, DK-2200 Copenhagen, Denmark.
Kastrup
J
J
Nielsen
B L
BL
Odum
N
N
Geisler
C
C
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000596:Amino Acids; D001665:Binding Sites; D017252:CD3 Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism; D018699:Coated Vesicles / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000596:Amino Acids; D017252:CD3 Complex; D015704:CD4 Antigens; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins