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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.


CD4T contributes to T cell activation.(A) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4WT, CD4T or no CD4. IL-2 secretion from CD4T 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells was normalized to the matched no CD4 controls within the same experiment to determine a relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (B) IL-2 secretion after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4WT controls within the same experiment to determine relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (*p<0.05; Mann-Whitney).
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pone.0132333.g002: CD4T contributes to T cell activation.(A) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4WT, CD4T or no CD4. IL-2 secretion from CD4T 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells was normalized to the matched no CD4 controls within the same experiment to determine a relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (B) IL-2 secretion after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4WT controls within the same experiment to determine relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (*p<0.05; Mann-Whitney).

Mentions: Because GxxxG motifs and glycine patches are often involved in functionally important protein-protein interactions, we hypothesized that this GGxxG motif is important for CD4’s Lck-independent function in TCR signaling [16–18]. To test this hypothesis, we generated constructs encoding a wild type (CD4WT) or C-terminally truncated version of CD4 (CD4T). CD4T lacks the cysteine clasp that mediates interactions with Lck but has been reported to have Lck-independent function that can mediate CD4+ T cell development [6]. This molecule also lacks Cys 421 that has been reported to be palymitoylated, although mutation of this residue does not impact lipid raft localization of human CD4 [31, 32]. To verify that CD4T makes an Lck-independent contribution to T cell activation, we generated 58α-β- T cell hybridomas expressing the 5c.c7 TCR, which recognizes a peptide from moth cytochrome c (MCC 88–93) in the context of I-Ek, along with either CD4WT, CD4T, or no CD4. We observed a significant increase in IL-2 production from cells expressing CD4T relative to cells lacking CD4 expression when stimulated with M12 cells expressing I-Ek tethered to the agonist MCC peptide (Fig 2A). These data indicate that CD4T does contribute to T cell activation in an Lck-independent manner. This response trended lower than that of CD4WT cells, indicating that Lck-association with CD4 enhances T cell activation (Fig 2B).


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)

CD4T contributes to T cell activation.(A) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4WT, CD4T or no CD4. IL-2 secretion from CD4T 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells was normalized to the matched no CD4 controls within the same experiment to determine a relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (B) IL-2 secretion after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4WT controls within the same experiment to determine relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (*p<0.05; Mann-Whitney).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132333.g002: CD4T contributes to T cell activation.(A) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4WT, CD4T or no CD4. IL-2 secretion from CD4T 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells was normalized to the matched no CD4 controls within the same experiment to determine a relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (B) IL-2 secretion after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4WT controls within the same experiment to determine relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (*p<0.05; Mann-Whitney).
Mentions: Because GxxxG motifs and glycine patches are often involved in functionally important protein-protein interactions, we hypothesized that this GGxxG motif is important for CD4’s Lck-independent function in TCR signaling [16–18]. To test this hypothesis, we generated constructs encoding a wild type (CD4WT) or C-terminally truncated version of CD4 (CD4T). CD4T lacks the cysteine clasp that mediates interactions with Lck but has been reported to have Lck-independent function that can mediate CD4+ T cell development [6]. This molecule also lacks Cys 421 that has been reported to be palymitoylated, although mutation of this residue does not impact lipid raft localization of human CD4 [31, 32]. To verify that CD4T makes an Lck-independent contribution to T cell activation, we generated 58α-β- T cell hybridomas expressing the 5c.c7 TCR, which recognizes a peptide from moth cytochrome c (MCC 88–93) in the context of I-Ek, along with either CD4WT, CD4T, or no CD4. We observed a significant increase in IL-2 production from cells expressing CD4T relative to cells lacking CD4 expression when stimulated with M12 cells expressing I-Ek tethered to the agonist MCC peptide (Fig 2A). These data indicate that CD4T does contribute to T cell activation in an Lck-independent manner. This response trended lower than that of CD4WT cells, indicating that Lck-association with CD4 enhances T cell activation (Fig 2B).

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.