Xenbase is experiencing technical problems. Users may experience limited functionality. We are working to rectify these issues.
Click on this message to dismiss it.
Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2013 Jan 01;84:e60903. doi: 10.1371/journal.pone.0060903.
Show Gene links
Show Anatomy links
Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.
Kaur S
,
Qureshi OS
,
Sansom DM
.
???displayArticle.abstract???
CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM) within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.
???displayArticle.pubmedLink???
23565286
???displayArticle.pmcLink???PMC3614947 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. Cellular localisation of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were incubated with unlabeled anti-CTLA-4 Ab at 37°C for 1 hour, cooled to 4°C and surface CTLA-4 stained red with anti-mouse Alexa 555. Cells were subsequently fixed, permeabilised and stained with Alexa488 anti-mouse IgG (green) and imaged by confocal microscopy. B. The ratio of plasma membrane to internalised CTLA-4 fluorescence (PM/I) was calculated by outlining cells in ImageJ. C. CHO cells expressing human CTLA-4 were labeled with anti-CTLA-4 PE at 37°C for 30 minutes followed by labeling surface CTLA-4 on ice (4°C) with Alexa647 anti-mouse IgG. Cells were analysed by flow cytometry and data are plotted as cycling CTLA-4 (37°C label) vs surface CTLA-4 (4°C label). D. CHO cells expressing the CTLA-4 chimeras were labeled as described in C and analysed by flow cytometry. Dotted line provides a standard gradient for reference purposes.
Figure 3. Endocytosis rates of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were labeled at 4°C with anti-CTLA-4 to label surface CTLA-4. Cells were then warmed to 37°C to allow endocytosis for the times indicated. Cells were then placed on ice and any remaining surface CTLA-4 detected with Alexa647 anti-mouse IgG. The 647 signal was plotted against time as the fraction remaining compared to 4°C. B. CHO cells expressing CTLA-4 chimeras were labeled as in A but in medium supplemented with sucrose (0.45 M) to prevent endocytosis. C. CHO cells expressing the chimeric CTLA-4 constructs were incubated with a transferrin (Tf) Alexa633 conjugate (Invitrogen) and anti-CTLA-4 PE at 37°C for 45 minutes. Cells were subsequently fixed and analysed by confocal microscopy. The red arrows indicate co-localisation. D. Rate of endocytosis of VGNF mutant was performed as described in A. E. Transferrin uptake of VGNF mutant was performed as described in C and analysed by confocal microscopy.
Figure 4. Degradation of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were incubated in medium or medium supplemented with cycloheximide (CHX) at 37°C for 3 hours. Cells were fixed, permeabilised and stained for total CTLA-4 with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-mouse IgG (green) and nuclei counterstained using DAPI (blue). Cells were analysed by confocal microscopy. B Total CTLA-4 was quantified by outlining cells in ImageJ and MFI plotted (left column). For flow cytometric quantitation (right hand column) cells were stained for total CTLA-4 using anti-CTLA-4 PE after 3 hours of CHX or NH4Cl treatment at 37°C and MFI plotted as a percentage of initial fluorescence. C. CHO cells expressing CTLA-4 chimeras were transfected with CD63-GFP. Cells were incubated in medium supplemented with NH4Cl at 37°C for 3 hours. Cells were fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab and Alexa565 anti-human IgG (red) to stain total CTLA-4 protein and analysed by confocal microscopy. The red arrows indicate co-localisation.
Figure 5. Recycling rates of CTLA-4 chimeras.A. CHO cells expressing WT human CTLA-4 were labeled with mouse anti-CTLA-4 PE at 37°C to detect cycling CTLA-4, washed and any recycling CTLA-4-PE antibody detected by addition of Alexa647 anti-mouse IgG at either 4°C or 37°C. Representative FACS plots are shown for PE-label (cycling CTLA-4) vs Alexa647 label (re-cycling CTLA-4) at the indicated time points. B. Recycling rates are plotted for the chimeras as normalised to the 4°C control.
Figure 6. Recycling efficiency is regulated by the YVKM motif.A. CHO cells expressing WT human CTLA-4 YVKM or YEKM motif were labeled at 4°C with anti-CTLA-4 to label surface CTLA-4. Cells were warmed to 37°C for the time indicated. Cells were then placed on ice and the remaining surface CTLA-4 detected with Alexa647 anti-mouse IgG and plotted over time. B. CHO cells expressing WT human CTLA-4 YVKM or a point mutant YEKM motif were labeled with mouse anti-CTLA-4 PE at 37°C to detect cycling CTLA-4. Recycling protein was detected with Alexa647 anti-mouse IgG at 37°C for the indicated time points. Recycling rates are plotted for the CTLA-4 variants normalised to the 4°C control. C. Representative data comparing human and xenopus CTLA-4 recycling after 30 minutes is shown.
Figure 1. Generation and localisation of CTLA-4 chimeras.
A. C-terminal sequence alignments of selected mammalian CTLA-4 based on sequence data from Ensembl and in ref 12. B. Diagram of human CTLA-4 chimeras containing the extracellular and transmembrane domain of human CTLA-4 and the C-terminus of species shown. C-terminal amino acid sequence alignments of human, chicken, xenopus and trout CTLA-4 are shown below, based on alignments using Clustal W. C. CHO cells expressing CTLA-4 chimeras were incubated with WGA-tetramethylrhodamine at 4°C for 45minutes. Cells were subsequently fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-human IgG (green) to stain total CTLA-4 protein. Cells were analysed by confocal microscopy. D Relative expression of surface (4°C) and total CTLA-4 for each chimera as determined by flow cytometry.
Bernard,
Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4.
2006, Pubmed
Bernard,
Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4.
2006,
Pubmed
Bernard,
Costimulatory receptors in jawed vertebrates: conserved CD28, odd CTLA4 and multiple BTLAs.
2007,
Pubmed
,
Xenbase
Boehm,
Evolution of lymphoid tissues.
2012,
Pubmed
Chuang,
Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression.
1997,
Pubmed
Egen,
Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength.
2002,
Pubmed
Heuser,
Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.
1989,
Pubmed
Iacopetta,
A role for the cytoplasmic domain in transferrin receptor sorting and coated pit formation during endocytosis.
1988,
Pubmed
Iida,
Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells.
2000,
Pubmed
Lane,
Lymphoid tissue inducer cells: pivotal cells in the evolution of CD4 immunity and tolerance?
2012,
Pubmed
Mead,
Exocytosis of CTLA-4 is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated during activation of regulatory T cells.
2005,
Pubmed
Obermüller,
The tyrosine motifs of Lamp 1 and LAP determine their direct and indirect targetting to lysosomes.
2002,
Pubmed
Ohno,
The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals.
1998,
Pubmed
Qureshi,
Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4.
2011,
Pubmed
Qureshi,
Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation.
2012,
Pubmed
Sansom,
The role of CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4) in regulatory T-cell biology.
2006,
Pubmed
Schneider,
Cytolytic T lymphocyte-associated antigen-4 and the TCR zeta/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2.
1999,
Pubmed
Shiratori,
Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2.
1997,
Pubmed
Teft,
A molecular perspective of CTLA-4 function.
2006,
Pubmed
Walker,
The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses.
2011,
Pubmed
Waterhouse,
Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4.
1995,
Pubmed
Zhang,
Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein.
1997,
Pubmed
