Molecular Characterization of the Di-leucine-based Internalization Motif of the T Cell Receptor

doi:10.1074/jbc.271.19.11441

Molecular Characterization of the Di-leucine-based Internalization Motif of the T Cell Receptor (*)

From the Institute of Medical Microbiology and Immunology, University of Copenhagen, DK-2200 Copenhagen, Denmark

^¶Member of “The Biotechnology Centre for Cellular Communication.” To whom correspondence should be sent:
Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Inst., Bldg. 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark.
Tel.: 45 3532 7880; Fax: 45 3532 7881; cgtcr{at}biobase.dk.

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 are required for protein kinase C (PKC)-mediated TCR down-regulation. To identify additional residues required for PKC-mediated phosphorylation of CD3 and for TCR down-regulation, an alanine scanning of CD3 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 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 by PKC. In this process Arg-124, Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of phosphorylated CD3 by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important.

Footnotes

↵^§ Recipient of a Ph.D. scholarship from the University of Copenhagen.
↵* This work was supported by The Novo Nordisk Foundation, The Carlsberg Foundation, The Danish Medical Research Council, Director Ib Henriksens Foundation, and The Danish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TCR

T cell receptor

PKC

protein kinase C

FCS

fetal calf serum

PDB

phorbol 12,13-dibutyrate

MFI

mean fluorescence intensity

WT

wild type

FACS

fluorescence-activated cell sorter.
- Received November 27, 1995.
- Revision received February 13, 1996.