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First published online 27 September 2005
doi: 10.1242/jcs.02592

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A three-amino-acid-long HLA-DRß cytoplasmic tail is sufficient to overcome ER retention of invariant-chain p35

Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Canada, H3C 3J7

View larger version (23K): [in a new window]   Fig. 1. Irrelevant cytoplasmic tails on DRß overcome the Iip35 ER retention motif. (A) Schematic representation of the chimeric class II and ß chain combinations expressed in HEK 293T cells. (B) HEK 293T cells were transiently co-transfected with cDNAs for the indicated chimeric molecules and Iip35 LI/ML. After 48 hours, cells were analysed for Ii expression by flow cytometry using BU45 either before (surface) or after (total) permeabilization with saponin. (C) The ratios of surface to total Ii are shown next to the C-terminal amino-acid sequences of the chimeric molecules' cytoplasmic tails. Class-II expression was measured on permeabilized cells using the ISCR3 mAb. The mean fluorescence values (MFVs) for DR TM/ß, DR TM/ß, DR TM/ßcytoDMmut and DR TM/ßcytoDO expression were 280, 267, 275 and 251, respectively. The MFV for untransfected cells was 4 (data not shown).

View larger version (15K): [in a new window]   Fig. 2. Sequence alignment of the cytoplasmic tails of the MHC-class-II molecules ß chains. The C-terminal amino-acid sequence of the classical MHC-class-II molecules DRß, DPß and DQß are shown along with the chimeric molecules DRßcytoDMmut and DRßcytoDO. The conserved Arg232 is indicated.

View larger version (15K): [in a new window]   Fig. 3. Alanine scan of DRß cytoplasmic tail. HEK 293T cells were transiently co-transfected with the indicated mutant molecules and with pREP4 Iip35 LI/ML and tested after 48 hours. Cells were analysed by flow cytometry using BU45 either before (surface Ii) or after (total Ii) permeabilization with saponin. The ratios of surface to total Ii are shown. The total ISCR3 mean fluorescence value (MFV) for all the chimeric class-II molecules was between 242 and 260. The MFV for the secondary antibody alone was 5 (data not shown).

View larger version (27K): [in a new window]   Fig. 4. Truncation of DRß cytoplasmic tail beyond Arg232 hinders ER egress. (A) HEK 293T cells were transiently co-transfected with the indicated chimeric molecules and with pREP4 Iip35 LI/ML and tested after 48 hours. Cells were analysed by flow cytometry using BU45 either before (surface Ii) or after (total Ii) permeabilization with saponin. The ratios of surface to total Ii are shown. The total ISCR3 mean fluorescence value (MFV) for all the chimeric class II molecules was between 223 and 248. The MFV for the secondary antibody alone was 5 (data not shown). (B) Hela cells stably expressing the indicated class II molcules were supertransfected with Iip35 LI/ML cDNA and stained for Ii using BU45.

View larger version (9K): [in a new window]   Fig. 5. Replacement of Tyr230 by an Arg in a truncated DRß molecule partially restores Iip35 egress. HEK 293T cells were transiently co-transfected with the indicated chimeric molecules and with pREP4 Iip35 LI/ML and tested after 48 hours. Cells were analysed by flow cytometry using BU45 either before (surface Ii) or after (total Ii) permeabilization with saponin. The ratios of surface to total Ii are shown. The total ISCR3 mean fluorescence value (MFV) for all the chimeric class II molecules was between 235 and 255. The MFV for the secondary antibody alone was 5 (data not shown).

View larger version (19K): [in a new window]   Fig. 6. Proline residues in the Iip35 cytoplasmic tail are not required for function of the RXR motif and its masking by HLA-DR. (A) N-Terminal amino acid sequence of the Iip35 proline mutants. Positions 1 and 17 are the initiation sites of Iip35 and Iip33, respectively. The arginine residues in the RXR motif are underlined, the leucine-based targeting signals are in black boxes, prolines in Iip33 region are in bold, and Ser6 and Ser8 are boxed. (B) HEK 293T cells were transiently co-transfected with the indicated invariant-chain molecules and with or without DR TM/ß and tested after 48 hours. Cells were analysed by flow cytometry using BU45 either before (surface Ii) or after (total Ii) permeabilization with saponin. The ratios of surface to total Ii are shown. (C) Schematic representation of the p33 cytoplasmic tail structure as determined by nuclear magnetic resonance (Motta et al., 1997). Proline residues that were mutated to alanine are indicated.

View larger version (11K): [in a new window]   Fig. 7. The RXR motif is inactive when close to the membrane. (A) N-Terminal amino-acid sequence of Iip35 LI/ML and the mutants with deletions in this region. Positions 1 and 17 are the initiation sites of Iip35 and Iip33, respectively. The arginine residues in the RXR motif are underlined, the original position of the mutated leucine-based targeting signals are in black boxes, prolines in Iip33 region are in bold and the phosphorylated Ser8 is boxed. (B) The indicated invariant-chain molecules were transiently co-transfected in HEK 293T cells and tested after 48 hours. Cells were analysed by flow cytometry using BU45 either before (surface Ii) or after (total Ii) permeabilization with saponin. The ratios of surface to total Ii are shown.