Research ArticleImmunology

Dimerization of the adaptor Gads facilitates antigen receptor signaling by promoting the cooperative binding of Gads to the adaptor LAT

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Science Signaling  26 Sep 2017:
Vol. 10, Issue 498, eaal1482
DOI: 10.1126/scisignal.aal1482
  • Fig. 1 Spontaneous Gads dimerization occurs through its SH2 domain.

    (A) Full-length MBP-Gads protein was resolved by size exclusion chromatography on a Superdex 200 10/300 GL column. The labeled peaks correspond to the monomer (1) and dimer (2). Data are representative of at least five independent experiments. (B) The thermal stability of purified MBP-Gads protein was determined by nano-DSF. Triplicate samples of MBP-Gads from peak 1 (monomer, green) and peak 2 (dimer, purple) were heated at a rate of 1°C/min while intrinsic tryptophan fluorescence was measured. The calculated Tm values are indicated. (C) Domain organization of wild-type (WT) and the indicated mutant MBP-Gads proteins. All constructs consistently resolved into two peaks (fig. S1D). (D) The His-tagged Gads SH2 domain was resolved by size exclusion chromatography on a Superdex 75 10/300 GL column. Peaks correspond to monomer (1) and dimer (2). Data are representative of at least three independent experiments. (E) Full-length MBP-Gads protein (left) or MBP-Gads SH2 domain protein (right) from the dimeric fraction shown in (D) was incubated at 37°C for the indicated times before undergoing size exclusion chromatography. Data are representative of three independent experiments. (F) Left: Triplicate samples of monomeric (peak 1, green) or dimeric (peak 2, purple) His-SH2 domain were analyzed by nano-DSF. Right: The resulting parameters of the monomer (Mon.) and dimer (Dim.). (G) Self-association of full-length Gads in yeast cells was demonstrated with the RRS, in which interaction of bait and prey proteins rescues yeast growth at the restrictive temperature of 36°C. The well-characterized interaction of Gads and SLP-76 served as a positive control. Data are representative of three independent experiments.

  • Fig. 2 Identification of the Gads SH2 domain dimerization interface.

    (A) Left: Structure of the murine Gads SH2 domain cocrystallized with a short peptide encompassing LAT-pY171 [PDB: 1R1P, (37)]. The adjacent SH2 units (cyan and green) are each bound to a pLAT peptide (red). The dotted box indicates the putative dimerization interface. Right: Enlarged view of part of the dimerization interface, highlighting the position of F92 (shown in space-filling form), D91, and R109. (B) The calculated binding free energy (ΔΔGbind) of amino acid residues that were predicted to destabilize (ΔΔGbind > 0) or stabilize (ΔΔGbind < 0) the Gads SH2 domain dimer interface when mutated to alanine, as determined by computational alanine scanning. (C and D) Purified MBP-Gads proteins bearing the indicated point mutations in either the SH2 domain alone (C) or the full-length (FL) Gads protein (D) were resolved by size exclusion chromatography. Data are representative of three independent experiments. (E) Representative isotherms for the interaction of the indicated monomeric MBP-Gads SH2 domains with the pY171-LAT peptide. Data analysis was performed with AFFINImeter software using a 1:1 stoichiometry binding model. Data are the mean Ka and ΔH values ± SEM obtained upon linked-parameter analysis of three independent measurements for each Gads construct.

  • Fig. 3 Preferentially paired binding of the Gads SH2 domain to its dual target sites on LAT.

    (A) The two possible modes of Gads binding to 2pY-LAT. (B) Altered fast protein liquid chromatography (FPLC) mobility of the Gads SH2 domain upon binding to single- or dual-pLAT peptides. MBP-Gads SH2 domain (0.7 μM) from the monomeric (red) or dimeric (blue) fraction was incubated on ice for 15 min alone (solid line) or in the presence of 5 μM pY171-LAT (dashed line) or 2pY-LAT (dotted line) peptides before being resolved by size exclusion chromatography. Data are representative of at least five independent experiments. (C) Full-length MBP-Gads protein (0.7 μM) from the monomeric fraction was incubated for 15 min at 37°C either alone (solid line) or in the presence of 5 μM 2pY-LAT peptide (dotted line) before being resolved by size exclusion chromatography. Data are representative of three independent experiments. (D) Stabilization of the dimeric form of the Gads SH2 domain upon binding to LAT. MBP-Gads SH2 domain (20 μM) from the dimeric fraction was incubated for 30 min on ice (blue) or at 37°C for 15 min (red, solid line), followed by an additional 15 min at 37°C in the presence of 40 μM 2pY-LAT (red, dotted line) before undergoing size exclusion chromatography. Data are representative of three independent experiments.

  • Fig. 4 Gads dimerization interface supports discrimination between single- and dual-pLAT.

    (A) Modes of Gads binding to competing LAT peptides, 2pY-LAT and pY171-LAT. Paired binding to 2pY-LAT can proceed sequentially (blue arrows) or by capture of transient Gads dimers (black arrows). Positive cooperativity occurs if the second Gads molecule binds with higher affinity than the first, resulting in preferentially paired binding. (B) Purified monomeric MBP-Gads SH2 (0.7 μM), either WT or F92D, was incubated for 10 min at 37°C with 2pY-LAT (5 μM), in the absence (black) or presence (solid red) of pY171-LAT competitor peptide (10 μM), and resolved by size exclusion chromatography. The red curve was deconvoluted into its constituent dimeric (dotted red) and monomeric (dashed red) components, using the Solve Excel plugin. Data are representative of three independent experiments. (C) Competitive binding experiments were performed in triplicate and analyzed as in (B) using the indicated concentrations of competing pY171-LAT. Data are the average percentage of Gads protein found in the dimeric fraction. Standard deviations were too small to depict. The P values for all F92D data points, compared to the wild type, were <10−8. (D) Purified monomeric full-length MBP-Gads (2.5 μM, gray), WT, F92D, or F92A,R109A, was incubated for 10 min at 37°C with 2pY-LAT (25 μM), in the absence (black) or presence (red) of pY171-LAT competitor (50 μM). Data are representative of three independent experiments.

  • Fig. 5 Gads dimerization is required for TCR signaling.

    (A to C) dG32 cells were stably transduced with viruses expressing GFP or the indicated alleles of twin-strep–tagged Gads-GFP, and cells within a broad (A) or narrow, homogeneous range of GFP (B and C) were isolated by fluorescence-activated cell sorting (FACS). (A) CellTrace Violet–barcoded cells were stimulated in quadruplicate overnight with anti-TCR or phorbol 12-myristate 13-acetate (PMA) and then stained with anti-CD69. Median TCR-induced CD69 cell surface abundance was normalized to that induced by PMA within each of the GFP ranges shown at the left. Data are representative of four independent experiments. (B and C) Cells were stimulated for 1 min with anti-TCR (C305) or mock-stimulated and lysed. (B) Strep-Tactin beads were used to purify twin-strep–tagged Gads from the lysates of 60 × 106 cells, and associated SLP-76 and pTyr132-pLAT were detected by Western blotting analysis. Right: The ratio of the abundance of pLAT to that of Gads from two (F92A,R109A), three (F92D), or four (WT) experiments, normalized to that of TCR-stimulated WT cells from the same experiment. (C) Cell lysates were analyzed by Western blotting with the indicated antibodies. Right: The intensity of the band corresponding to phosphorylated PLC from four (F92A,R109A and vector), seven (F92D), or eight (WT) experiments, normalized to that of TCR-stimulated WT cells from the same experiment. In all panels, error bars indicate SD. The P values were determined for the comparison of TCR-stimulated cells to TCR-stimulated vector (A) or WT-reconstituted cells (B and C). *P < 0.05, **P < 0.005, ***P < 0.0005.

  • Fig. 6 F92D mutation disrupts FcεRI signaling.

    (A to C) Fully differentiated BMMCs, derived from wild type (WT), Gads-deficient [knockout (KO)], or retrovirally reconstituted KO bone marrow cells (KO + WT or KO + F92D), were barcoded, sensitized with IgE (anti-DNP), and stimulated at 37°C with DNP-HSA. Responses were analyzed by FACS while gating on matched, narrow regions of Gads-GFP. (A) Left: Changes in intracellular calcium, with DNP-HSA (0.6 ng/ml) added at 60 s. Cells above the baseline during the last 200 s were considered to be responsive. Right: Percentage of responding cells as a function of stimulant concentration. Data are representative of three independent experiments. (B) Left: BMMCs were left unstimulated (filled histogram) or stimulated for 15 min with DNP-HSA (1.2 ng/ml, solid line, WT; dashed line, F92D) and then fixed and stained with anti–CD63-PE. The indicated gate defined the CD63+ responding cells. Middle: CD63+ responding cells, as a function of Gads-GFP abundance (data are the average of triplicate measurements; error bars indicate SD; dotted lines indicate the response of WT and KO cells in the same experiment). Right: CD63+ responding cells as a function of stimulant concentration. Data are representative of five independent experiments. (C) Left: Cells were unstimulated (filled histograms) or stimulated for 4.5 hours with DNP-HSA (0.6 ng/ml, black line), and the IL-6+ responding cells, defined by the indicated gate, were identified by intracellular staining. Right: IL-6+ responding cells, as a function of stimulant concentration. Data are representative of two independent experiments.

  • Table 1 Binding parameters from the mathematical model of the binding of the Gads SH2 domain to 2pY-LAT.

    The indicated parameters were calculated from a mathematical model, based on standard equilibrium binding equations, as described in the Supplementary Materials.

    Binding constant
    at pTyr171 (Kd3) (nM)
    First sequential constant
    for binding of a single
    Gads at either site
    (Kd1 = 0.5 × Kd3) (nM)
    Second sequential binding
    constant (Kd2) (nM)
    Fold increase in
    site-specific binding
    affinity at second
    binding event due to
    cooperativity (Kd3/Kd2)
    WT SH217788.50.95186.5
    F92D SH24722365.486.7
    SourceSet equal to
    pTyr171 dissociation
    constant from Fig. 2E
    On the basis of an
    assumption of equivalence
    of the two binding sites
    Calculated from mathematical model of competitive
    binding data from Fig. 4C

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/498/eaal1482/DC1

    Mathematical modeling

    Fig. S1. Molecular weights of the monomeric and dimeric forms of Gads.

    Fig. S2. Thermal stabilities of the monomeric and dimeric forms of MBP-Gads SH2.

    Fig. S3. Model of the SH2 domain dimerization interface.

    Fig. S4. The R109D, R109A, and F92A single mutations are not sufficient to disrupt Gads SH2 dimerization.

    Fig. S5. Thermal stabilities of nondimerizing Gads mutants.

    Fig. S6. Multiple sequence alignment of the C-terminal regions of LAT molecules from 15 mammalian species.

    Fig. S7. Cell surface FcεRI abundance is independent of Gads.

    Fig. S8. FcεRI-induced cell surface expression of the degranulation marker CD107a depends on the Gads dimerization interface.

    Table S1. Summary of the abbreviations used in the mathematical model.

    Table S2. Calculated kinetic constants.

    References (60)

  • Supplementary Materials for:

    Dimerization of the adaptor Gads facilitates antigen receptor signaling by promoting the cooperative binding of Gads to the adaptor LAT

    Sigalit Sukenik, Maria P. Frushicheva, Cecilia Waknin-Lellouche, Enas Hallumi, Talia Ifrach, Rose Shalah, Dvora Beach, Reuven Avidan, Ilana Oz, Evgeny Libman, Ami Aronheim, Oded Lewinson, Deborah Yablonski*

    *Corresponding author. Email: debya{at}tx.technion.ac.il

    This PDF file includes:

    • Mathematical modeling
    • Fig. S1. Molecular weights of the monomeric and dimeric forms of Gads.
    • Fig. S2. Thermal stabilities of the monomeric and dimeric forms of MBP-Gads SH2.
    • Fig. S3. Model of the SH2 domain dimerization interface.
    • Fig. S4. The R109D, R109A, and F92A single mutations are not sufficient to disrupt Gads SH2 dimerization.
    • Fig. S5. Thermal stabilities of nondimerizing Gads mutants.
    • Fig. S6. Multiple sequence alignment of the C-terminal regions of LAT molecules from 15 mammalian species.
    • Fig. S7. Cell surface FcεRI abundance is independent of Gads.
    • Fig. S8. FcεRI-induced cell surface expression of the degranulation marker CD107a depends on the Gads dimerization interface.
    • Table S1. Summary of the abbreviations used in the mathematical model.
    • Table S2. Calculated kinetic constants.
    • Reference (60)

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    Citation: S. Sukenik, M. P. Frushicheva, C. Waknin-Lellouche, E. Hallumi, T. Ifrach, R. Shalah, D. Beach, R. Avidan, I. Oz, E. Libman, A. Aronheim, O. Lewinson, D. Yablonski, Dimerization of the adaptor Gads facilitates antigen receptor signaling by promoting the cooperative binding of Gads to the adaptor LAT. Sci. Signal. 10, eaal1482 (2017).

    © 2017 American Association for the Advancement of Science

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