Leucine-based Receptor Sorting Motifs Are Dependent on the Spacing Relative to the Plasma Membrane

Carsten Geisler,Jes Dietrich,Bodil L Nielsen,Jesper Kastrup,Jens Peter H Lauritsen,Niels Ødum,Mette D Christensen

doi:10.1074/jbc.273.33.21316

Carsten Geisler, Jes Dietrich + Show 5 more

Open Access

https://doi.org/10.1074/jbc.273.33.21316

Copy DOI

Abstract

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic residue of leucine-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs. Thus, our observations suggest that the position, the exact sequence, and surrounding residues are major determinants of the function of leucine-based receptor sorting motifs.

Highlights

At least two types of leucine-based receptor sorting motifs can be described
protein kinase C (PKC)-induced internalization of the T cell receptor (TCR) is mediated via the Leu-based motif S126DKQTLL132 in the cytoplasmic tail of the TCR subunit CD3␥ [5, 7]
We found that a minimal spacing of 7 residues between the plasma membrane (PM) and Ser126 was required for phosphorylation and activation of the CD3␥ Leu-based motif in the TCR

Summary

EXPERIMENTAL PROCEDURES

Cells and Antibodies—JGN cells, a TCR cell-surface negative variant of the human T cell line Jurkat that does not synthesize CD3␥ [19], were cultured in RPMI 1640 medium supplemented with 2 ϫ 105 units/liter penicillin (Leo Pharmaceutical Products, Ballerup, Denmark), 50 mg/liter streptomycin (Merck, Darmstadt, Germany), and 10% (v/v) FCS (Life Technologies, Inc., Paisley, United Kingdom) at 37 °C in 5% CO2. The phosphorylated CD3␥ chain with a molecular mass of 26 –30 kDa was coprecipitated with CD3⑀ (20 kDa) using anti-CD3⑀ mAb UCHT1 and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. To determine the internalization rates of TCR and chimeric CD4/ CD3␥ molecules, cells were incubated in RPMI 1640 medium and 10% FCS at a cell density of 2 ϫ 105 cells/ml at 37 or 4 °C with PEconjugated anti-CD3⑀ or anti-CD4 mAb, respectively. Labeled cells were lysed in 1% Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 8 mM iodoacetamide, and 1% Nonidet P-40), precipitated with rat anti-mouse CD4 mAb and rabbit anti-rat Ig, and subsequently analyzed by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels under nonreducing conditions. Autoradiography of the dried gels was performed using Hyperfilm-MP (Amersham International). 14C-Labeled proteins from Amersham International were used as molecular mass markers

RESULTS

DISCUSSION

Iia Iib