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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 transmembrane domain GGxxG motif impairs T cell activation.(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4TTMD or CD4TΔbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or  (HB) peptide tethered to I-Ek. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4TTMD 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-Ek (CHO Ek) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).
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pone.0132333.g003: Mutating the CD4 transmembrane domain GGxxG motif impairs T cell activation.(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4TTMD or CD4TΔbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or (HB) peptide tethered to I-Ek. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4TTMD 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-Ek (CHO Ek) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).

Mentions: Having established a readout for the Lck-independent function of CD4, we generated a construct encoding a mutant of CD4T, referred to here as CD4TTMD, in which two of the glycines in the GGxxG motif were mutated to valine (G403V) or leucine (G406L) to introduce bulky side-chains at this surface (Fig 3A). As a negative control, we also generated a CD4T mutated in the region known to bind MHC class II (CD4TΔbind) [13, 14]. To test the function of these mutants we generated 58α-β- T cell hybridomas expressing the 5c.c7 TCR along with CD4T, CD4TTMD, or CD4TΔbind. Cell surface expression of the TCR and CD4 were assessed by flow cytometry to ensure that the cell lines expressed equivalent levels of these proteins in order to assign any phenotype to the mutations rather than unmatched expression (Fig 3B).


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 transmembrane domain GGxxG motif impairs T cell activation.(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4TTMD or CD4TΔbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or  (HB) peptide tethered to I-Ek. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4TTMD 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-Ek (CHO Ek) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).
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pone.0132333.g003: Mutating the CD4 transmembrane domain GGxxG motif impairs T cell activation.(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4TTMD or CD4TΔbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or (HB) peptide tethered to I-Ek. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4TTMD 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-Ek (CHO Ek) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).
Mentions: Having established a readout for the Lck-independent function of CD4, we generated a construct encoding a mutant of CD4T, referred to here as CD4TTMD, in which two of the glycines in the GGxxG motif were mutated to valine (G403V) or leucine (G406L) to introduce bulky side-chains at this surface (Fig 3A). As a negative control, we also generated a CD4T mutated in the region known to bind MHC class II (CD4TΔbind) [13, 14]. To test the function of these mutants we generated 58α-β- T cell hybridomas expressing the 5c.c7 TCR along with CD4T, CD4TTMD, or CD4TΔbind. Cell surface expression of the TCR and CD4 were assessed by flow cytometry to ensure that the cell lines expressed equivalent levels of these proteins in order to assign any phenotype to the mutations rather than unmatched expression (Fig 3B).

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.