MOLECULAR CHARACTERIZATION OF THE DI-LEUCINE-BASED INTERNALIZATION MOTIF OF THE T-CELL RECEPTOR

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kin ases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gam ma are required for protein kinase C (PKC)-mediated TCR down-regulatio n. To identify additional residues required for PKC-mediated phosphory lation of CD3 gamma and for TCR down-regulation, an alanine scanning o f CD3 gamma was done. Mutations of Arg-124, Ser-126, Lys-128, and Gln- 129 inhibited both phosphorylation and TCR down-regulation, whereas mu tation of Asp-127 only inhibited down-regulation. Further analyses dem onstrated a discrepancy between the ability to be phosphorylated on CD 3 gamma and to down-regulate the TCR in several transfectants. Phospho rylation was not as strictly dependent on the nature and position of t he phosphoacceptor group and basic residues as were the subsequent ste ps involved in TCR down-regulation. Our results suggest that PKC-media ted TCR down-regulation may be regarded as a two-step process. 1) Reco gnition and phosphorylation of CD3 gamma by PKC. In this process Arg-1 24, Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of pho sphorylated CD3 gamma by molecules involved in receptor internalizatio n. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are impor tant.