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.