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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 upon TCR engagement.(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. (C) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data is concatenated from two independently generated cell lines as in Fig 4D. (D) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for Fig 4. 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.g005: Mutating the CD4 TMD GGxxG motif does not impair dimerization upon TCR engagement.(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. (C) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data is concatenated from two independently generated cell lines as in Fig 4D. (D) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for Fig 4. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).

Mentions: Previous studies using human CD4 have shown that CD4 dimerization increased upon TCR engagement [37]. Because we observed no decrease in FRETE values of CD4TTMD-G/Ch cells at steady-state compared to CD4TG/Ch, but observed a functional decrease in T cell activation with CD4TTMD cells compared to CD4T cells, we hypothesized that the TMD glycine patch may contribute to the increase in CD4 dimerization that has been reported when both CD4 and the TCR engage pMHC. To test this hypothesis we imaged live cells using TIRFM on mobile lipid bilayers containing agonist (MCC-Ek) pMHC. To ensure bilayer mobility, lipids were spiked with a lipid with a biotinylated head group. Streptavidin-PE was added to the bilayers and an area was bleached. Emission of PE was measured before and after bleaching to measure the ability of the bleached area to fill in with new PE molecules (Fig 5A). Similar to what we observed in cells with unengaged TCRs, the positive control CD28TG/Ch cells had significantly higher FRETE values than the negative control PD1TG/Ch cells. CD4TG/Ch cells had FRETE values lower than CD28TG/Ch cells but significantly higher than PD1TG/Ch cells (Fig 5B). No difference in FRETE was observed between cells expressing CD4WTG/Ch and CD4TG/Ch (Fig 5C). Likewise, CD4TTMD-G/Ch cells had equivalent FRETE values compared to CD4TG/Ch cells, indicating that even after TCR engagement the CD4 TMD glycine patch is not involved in CD4 dimerization (Fig 5D). Of note, these hybridomas do not form classic immunological synapses on bilayers with a central cluster of TCR-CD3 complexes and we do not observe obvious segregation of TCR-CD3 and CD4 molecules after extended incubation on the bilayers, as has been reported for T cell lines or ex vivo T cell blasts [40, 41]. Since we did not observe any obvious differences in FRET between the various CD4 lines tested we think it unlikely that differences in segregation have impacted our analysis or interpretation.


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 upon TCR engagement.(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. (C) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data is concatenated from two independently generated cell lines as in Fig 4D. (D) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for Fig 4. 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.g005: Mutating the CD4 TMD GGxxG motif does not impair dimerization upon TCR engagement.(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. (C) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data is concatenated from two independently generated cell lines as in Fig 4D. (D) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for Fig 4. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).
Mentions: Previous studies using human CD4 have shown that CD4 dimerization increased upon TCR engagement [37]. Because we observed no decrease in FRETE values of CD4TTMD-G/Ch cells at steady-state compared to CD4TG/Ch, but observed a functional decrease in T cell activation with CD4TTMD cells compared to CD4T cells, we hypothesized that the TMD glycine patch may contribute to the increase in CD4 dimerization that has been reported when both CD4 and the TCR engage pMHC. To test this hypothesis we imaged live cells using TIRFM on mobile lipid bilayers containing agonist (MCC-Ek) pMHC. To ensure bilayer mobility, lipids were spiked with a lipid with a biotinylated head group. Streptavidin-PE was added to the bilayers and an area was bleached. Emission of PE was measured before and after bleaching to measure the ability of the bleached area to fill in with new PE molecules (Fig 5A). Similar to what we observed in cells with unengaged TCRs, the positive control CD28TG/Ch cells had significantly higher FRETE values than the negative control PD1TG/Ch cells. CD4TG/Ch cells had FRETE values lower than CD28TG/Ch cells but significantly higher than PD1TG/Ch cells (Fig 5B). No difference in FRETE was observed between cells expressing CD4WTG/Ch and CD4TG/Ch (Fig 5C). Likewise, CD4TTMD-G/Ch cells had equivalent FRETE values compared to CD4TG/Ch cells, indicating that even after TCR engagement the CD4 TMD glycine patch is not involved in CD4 dimerization (Fig 5D). Of note, these hybridomas do not form classic immunological synapses on bilayers with a central cluster of TCR-CD3 complexes and we do not observe obvious segregation of TCR-CD3 and CD4 molecules after extended incubation on the bilayers, as has been reported for T cell lines or ex vivo T cell blasts [40, 41]. Since we did not observe any obvious differences in FRET between the various CD4 lines tested we think it unlikely that differences in segregation have impacted our analysis or interpretation.

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.