REGULATION AND FUNCTION OF THE CD3-GAMMA DXXXLL MOTIF - A BINDING-SITE FOR ADAPTER PROTEIN-1 AND ADAPTER PROTEIN-2 IN-VITRO

Several receptors are downregulated by internalization after ligand bi nding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction, O ne way T cells regulate TCR expression is by phosphorylation/dephospho rylation of the TCR subunit clusters of differentiation (CD)3 gamma. T hus, phosphorylation of CD3 gamma serine 126 (S126) causes a downregul ation of the TCR. In this study, we have analyzed the CD3 gamma intern alization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3 gamma chimeras; and in vitro by binding CD3 gamma peptides to clathrin-coated vesicle adaptor proteins (APs), We find that the CD3 gamma D(127)xxxLL(131/132 ) 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 monome ric CD4/CD3 gamma molecules independently of S126. An acidic amino aci d is required at position 127 and a leucine (L) is required at positio n 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 CD3 gamma S12 6 in the context of the complete TCR induces a conformational change t hat exposes the DxxxLL sequence for AP binding, Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demon strate that this leads to an impairment of TCR signaling, On the basis of the present results, we propose the existence of at least three di fferent types of L-based receptor sorting motifs.