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Molecular characterization of the di-leucine-based internalization motif of the T cell receptor
J Dietrich1, X Hou, A M Wegener
1Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark.
Abstract:
Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. 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 gamma are required for protein kinase C (PKC)-mediated TCR down-regulation. To identify additional residues required for PKC-mediated phosphorylation of CD3 gamma and for TCR down-regulation, an alanine scanning of CD3 gamma was done. Mutations of Arg-124, Ser-126, Lys-128, and Gln-129 inhibited both phosphorylation and TCR down-regulation, whereas mutation of Asp-127 only inhibited down-regulation. Further analyses demonstrated a discrepancy between the ability to be phosphorylated on CD3 gamma and to down-regulate the TCR in several transfectants. Phosphorylation was not as strictly dependent on the nature and position of the phosphoacceptor group and basic residues as were the subsequent steps involved in TCR down-regulation. Our results suggest that PKC-mediated TCR down-regulation may be regarded as a two-step process. 1) Recognition and phosphorylation of CD3 gamma by PKC. In this process Arg-124, Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of phosphorylated CD3 gamma by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important.
Insights
Protein kinase C (PKC) mediates T cell receptor (TCR) down-regulation through CD3 gamma phosphorylation. Specific residues are crucial for PKC recognition and subsequent receptor internalization, revealing a two-step process.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- T cell receptor (TCR) surface expression is regulated by phosphorylation and down-regulation following protein kinase activation.
- Previous studies identified Ser-126 phosphorylation and a Leu-131/Leu-132 di-leucine motif in CD3 gamma as essential for protein kinase C (PKC)-mediated TCR down-regulation.
Purpose of the Study:
- To identify additional amino acid residues in CD3 gamma critical for PKC-mediated phosphorylation and subsequent TCR down-regulation.
- To elucidate the distinct molecular requirements for CD3 gamma phosphorylation and TCR internalization.
Main Methods:
- Alanine scanning mutagenesis of CD3 gamma was performed to assess the role of specific residues.
- Analysis of phosphorylation status and TCR down-regulation in cells expressing mutated CD3 gamma constructs.
Main Results:
- Mutations at Arg-124, Ser-126, Lys-128, and Gln-129 impaired both CD3 gamma phosphorylation and TCR down-regulation.
- Mutation of Asp-127 specifically inhibited TCR down-regulation, but not phosphorylation.
- A dissociation between CD3 gamma phosphorylation and TCR down-regulation was observed, indicating distinct requirements for each step.
Conclusions:
- PKC-mediated TCR down-regulation is a two-step process: 1) PKC recognition and phosphorylation of CD3 gamma (involving Arg-124, Ser-126, Lys-128, Gln-129). 2) Recognition of phosphorylated CD3 gamma for internalization (involving Ser(P)-126, Asp-127, Leu-131, Leu-132).
- The phosphorylation step is less stringent regarding phosphoacceptor and basic residues compared to the subsequent internalization step.