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A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

Parrish HL, Glassman CR, Keenen MM, Deshpande NR, Bronnimann MP, Kuhns MS - PLoS ONE (2015)

Bottom Line: CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions.Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation.This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, The University of Arizona College of Medicine, Tucson, Arizona, United States of America.

ABSTRACT
CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster's Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

No MeSH data available.


Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WTGFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).
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pone.0132333.g004: Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WTGFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).

Mentions: Live cells were imaged by total internal reflection fluorescence microscopy (TIRFM) after binding to glass coverslips [20]. This allowed us to limit our analysis to molecules at the cell surface without engaging the TCR or CD4 molecules. FRET efficiency values (FRETE) were quantified by measuring donor recovery after bleaching of the acceptor [29, 30]. In these experiments, ablation averaged greater than 90% for all analyzed populations and did not differ significantly from each other (Fig 4A and 4B). Furthermore, the analyzed cells were matched in their range of donor and acceptor intensities at the cell membrane as well as for their mean GFP and mCherry intensities. As expected, the positive control disulfide-bonded CD28TG/Ch cells had a significantly higher FRETE value compared to the negative control PD1TG/Ch cells (Fig 4C). By comparison, the FRETE values for the CD4TG/Ch cells were significantly higher than the negative control cells, but significantly lower than those of the positive control cells. These data indicate that, while dimerization can occur, a low frequency of CD4 molecules dimerize or multimerize at equilibrium in these cells; thus, homotypic CD4 interactions are likely to be weak (Fig 4C). Of note, no significant difference was observed in the FRETE value between lines expressing CD4WTG/Ch or CD4TG/Ch (Fig 4D). We then compared CD4TTMD-G/Ch cells to CD4TG/Ch cells and did not observe decreased FRETE values, implying that G403 and G406 are not involved in stabilizing CD4 dimerization at steady-state (Fig 4E).


A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

Parrish HL, Glassman CR, Keenen MM, Deshpande NR, Bronnimann MP, Kuhns MS - PLoS ONE (2015)

Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WTGFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493003&req=5

pone.0132333.g004: Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WTGFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).
Mentions: Live cells were imaged by total internal reflection fluorescence microscopy (TIRFM) after binding to glass coverslips [20]. This allowed us to limit our analysis to molecules at the cell surface without engaging the TCR or CD4 molecules. FRET efficiency values (FRETE) were quantified by measuring donor recovery after bleaching of the acceptor [29, 30]. In these experiments, ablation averaged greater than 90% for all analyzed populations and did not differ significantly from each other (Fig 4A and 4B). Furthermore, the analyzed cells were matched in their range of donor and acceptor intensities at the cell membrane as well as for their mean GFP and mCherry intensities. As expected, the positive control disulfide-bonded CD28TG/Ch cells had a significantly higher FRETE value compared to the negative control PD1TG/Ch cells (Fig 4C). By comparison, the FRETE values for the CD4TG/Ch cells were significantly higher than the negative control cells, but significantly lower than those of the positive control cells. These data indicate that, while dimerization can occur, a low frequency of CD4 molecules dimerize or multimerize at equilibrium in these cells; thus, homotypic CD4 interactions are likely to be weak (Fig 4C). Of note, no significant difference was observed in the FRETE value between lines expressing CD4WTG/Ch or CD4TG/Ch (Fig 4D). We then compared CD4TTMD-G/Ch cells to CD4TG/Ch cells and did not observe decreased FRETE values, implying that G403 and G406 are not involved in stabilizing CD4 dimerization at steady-state (Fig 4E).

Bottom Line: CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions.Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation.This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, The University of Arizona College of Medicine, Tucson, Arizona, United States of America.

ABSTRACT
CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster's Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

No MeSH data available.