CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.
Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC-mediated TCR down-regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma-negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC-mediated TCR down-regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC-mediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed.
Dietrich
J
J
Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark.
Hou
X
X
Wegener
A M
AM
Geisler
C
C
D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane; D015536:Down-Regulation; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D010768:Phosphoserine; D014443:Tyrosine; D011493:Protein Kinase C; D007930:Leucine