| 1998 |
GADS (Gads) was identified as a novel adaptor protein containing N- and C-terminal SH3 domains flanking a central SH2 domain plus a 120 aa unique region; its SH2 domain binds tyrosine-phosphorylated Shc, Bcr-Abl, and c-kit, while the isolated C-terminal SH3 domain can bind Sos, Cbl, and Sam68 in vitro but full-length Gads does not interact with Sos, indicating the unique region regulates downstream SH3 binding specificity. |
Expression library screen with phosphopeptides, co-immunoprecipitation, SH3/SH2 domain binding assays |
Oncogene |
Medium |
9872323
|
| 1998 |
Mona (GADS ortholog) was cloned via yeast two-hybrid as an interactor of the M-CSF receptor (Fms); it binds Fms at phospho-Tyr697 (the Grb2-binding site) via its SH2 domain and is expressed specifically in spleen and peripheral blood mononuclear cells; overexpression in bone marrow cells strongly reduced M-CSF-dependent macrophage production in vitro, implicating Mona in monocyte/macrophage developmental signaling downstream of Fms. |
Yeast two-hybrid screen, co-immunoprecipitation, overexpression in bone marrow colony assay |
The EMBO journal |
Medium |
9857184
|
| 1999 |
Gads constitutively interacts with SLP-76 via its C-terminal SH3 domain binding a 20 aa proline-rich region of SLP-76, and inducibly associates with tyrosine-phosphorylated LAT via its SH2 domain after TCR cross-linking; overexpression of Gads and SLP-76 synergistically augmented NFAT activation in a manner requiring a functional Gads SH2 domain, demonstrating that Gads bridges LAT and SLP-76 in TCR signaling. |
Co-immunoprecipitation, domain deletion mutants, NFAT reporter assays in T cells |
Current biology : CB |
High |
10021361
|
| 1999 |
Grf40 (GADS) binds SLP-76 via its C-terminal SH3 domain with higher affinity than Grb2, and binds LAT via its SH2 domain; overexpression of wild-type Grf40 augmented SLP-76-dependent IL-2 promoter and NFAT activation upon TCR stimulation, whereas deletion of the C-terminal SH3 domain abolished this, and deletion of the SH2 domain acted as dominant-negative, inhibiting signaling more strongly than SH2-deleted Grb2. |
Co-immunoprecipitation, domain deletion mutants, IL-2 promoter and NFAT luciferase reporter assays in Jurkat cells |
The Journal of experimental medicine |
High |
10224278
|
| 2000 |
Three distal LAT tyrosines (Tyr171, Tyr191, Tyr226) are responsible for Grb2 binding; Tyr171 and Tyr191 (but not Tyr226) are necessary for Gads binding; mutation of all three distal tyrosines abolished PLC-γ1 binding to LAT, establishing the specific phosphotyrosine docking sites used by Gads on LAT. |
Tyrosine-to-phenylalanine LAT mutants expressed in LAT-deficient Jurkat cells, co-immunoprecipitation, Ca2+ flux, ERK and NF-AT activation assays |
The Journal of biological chemistry |
High |
10811803
|
| 2000 |
GADS couples HPK1 to the activated TCR: HPK1 inducibly associates with Gads after TCR activation, becomes tyrosine phosphorylated, and HPK1 kinase activity is upregulated; interaction requires the Gads C-terminal SH3 domain and the fourth proline-rich region of HPK1; expression of a Gads SH2 mutant or deletion of HPK1 proline-rich region 4 inhibits TCR-induced HPK1 tyrosine phosphorylation. |
Expression library screen, co-immunoprecipitation, kinase assays, domain deletion/mutation mapping in DO11.10 T cell hybridoma |
Journal of immunology (Baltimore, Md. : 1950) |
Medium |
10903746
|
| 2000 |
LAT, Gads, and Grb2 are collectively required for SLP-76 recruitment into glycolipid-enriched microdomains (GEMs/lipid rafts) after antigen receptor cross-linking; in Zap-70+BLNK− B cells reconstituted with SLP-76 and LAT+Gads, SLP-76 entered GEMs and BCR function was restored; Grb2 overexpression could substitute for Gads in this membrane compartmentation. |
Sucrose density fractionation to isolate GEMs, reconstitution in BLNK-deficient B cells, functional BCR signaling assays |
The Journal of experimental medicine |
Medium |
10993915
|
| 2001 |
Gads-deficient mice generated by gene targeting show a severe block in thymocyte proliferation, impaired positive and negative selection, and failure to respond to CD3 cross-linking in vivo; immunoprecipitation experiments revealed that the SLP-76–LAT association is uncoupled in GADS-deficient thymocytes, demonstrating that GADS is the critical adaptor bridging these two proteins in vivo. |
Gene targeting (knockout mouse), immunoprecipitation, in vivo CD3 cross-linking, flow cytometry analysis of T cell development |
Science (New York, N.Y.) |
High |
11239162
|
| 2001 |
Gads contains a caspase cleavage site in its unique region; induction of apoptosis in lymphocytes results in caspase-3-mediated Gads cleavage within 60 min; a point mutation at the caspase-3 site prevented cleavage in vitro and in vivo; the resulting cleavage products retain binding to SLP-76 and LAT but expression of cleavage products in Jurkat cells inhibited NFAT activation upon TCR cross-linking, indicating cleavage uncouples Gads function. |
Apoptosis induction, caspase inhibitor treatment, site-directed mutagenesis of cleavage site, in vitro caspase cleavage assay, NFAT reporter assay |
Oncogene |
High |
11313864
|
| 2001 |
CD95/Fas ligation leads to caspase-3-dependent cleavage of GrpL/Gads, removing its C-terminal SH3 domain and preventing SLP-76 recruitment to the membrane; a truncated GrpL transfected into Jurkat T cells blocked TCR-induced NFAT activation, linking CD95-mediated Gads cleavage to desensitization of antigen receptor signaling. |
CD95 ligation, caspase inhibitors, co-immunoprecipitation, truncated Gads transfection, Ca2+ mobilization assay, NFAT reporter |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
11391000
|
| 2001 |
Grap2 interacts with HPK1 via the Grap2 C-terminal SH3 domain and the second proline-rich motif of HPK1 in vitro and in Jurkat T cells; co-expression of Grap2 with HPK1 increased HPK1 kinase activity and had an additive effect on HPK1-mediated JNK activation and c-Jun transcriptional activation and IL-2 reporter activity. |
Co-immunoprecipitation in Jurkat cells, in vitro binding assay, kinase assay, JNK and IL-2 reporter assays |
Oncogene |
Medium |
11313918
|
| 2002 |
The Gads C-terminal SH3 domain binds a non-proline-based R-X-X-K motif in SLP-76 (residues R237, K240) with high affinity (Kd = 240 ± 45 nM), approximately 40-fold higher than the analogous Grb2 C-SH3 binding; single point mutations R237 or K240 in SLP-76 abolished Gads–SLP-76 association in vivo and impaired SLP-76 function; a chimeric Grb2 bearing the Gads C-SH3 domain partially substituted for Gads in TCR signaling. |
Peptide arrays, surface plasmon resonance/affinity measurements, co-immunoprecipitation with SLP-76 point mutants, chimeric protein functional assay |
Current biology : CB |
High |
12176364
|
| 2002 |
Mona/Gads associates specifically with Gab3 (but not Gab2) during monocyte/macrophage differentiation via the Mona C-terminal SH3 domain and the atypical proline-rich domain of Gab3; M-CSFR mutations Y697F and Y807F both reduce Mona induction; Mona and Gab3 are co-induced and form multimolecular complexes during M-CSF-stimulated macrophage differentiation of bone marrow cells. |
GST pull-down, co-immunoprecipitation, M-CSFR point mutants, bone marrow differentiation assay |
Molecular and cellular biology |
Medium |
11997510
|
| 2003 |
The NMR solution structure of the Gads C-terminal SH3 domain in complex with an SLP-76 RSTK-containing peptide was determined; the SLP-76 peptide engages four distinct binding pockets and adopts a right-handed 3(10) helix at the RSTK locus—a unique mode distinct from the canonical polyproline type II helix of classical SH3 ligands. Supporting mutagenesis and peptide binding data validated the structural findings. |
NMR structure determination, mutagenesis, peptide binding assays |
Molecular cell |
High |
12620234
|
| 2003 |
The crystal structure of the Mona/Gads C-terminal SH3 domain in complex with SLP-76 peptide was solved to 1.7 Å; the SLP-76 peptide lacks a canonical PxxP motif, forms a clamp around the SH3 β-barrel with the central RxxK forming a 3(10) helix inserted into a negatively charged double pocket, explaining the uniquely high binding affinity. Additionally, the SH3C displayed ion-dependent dimerization in crystal and in solution. |
X-ray crystallography (1.7 Å resolution), solution dimerization assay |
The EMBO journal |
High |
12773374
|
| 2003 |
Gads/Grb2-mediated association with LAT is critical for the inhibitory function of Gab2 in T cells: Gab2 associates with LAT upon TCR stimulation via its MBD domain containing a PXXXR Grb2 SH3 binding motif; through this association Gab2 is recruited to lipid rafts and exerts inhibitory function; T cells from Gab2-deficient mice showed enhanced proliferative responses. |
Co-immunoprecipitation, Gab2 mutant analysis, lipid raft fractionation, transgenic and knockout mouse T cell functional assays |
Molecular and cellular biology |
Medium |
12640133
|
| 2004 |
Crystal structures of Gads-SH2 bound to phosphopeptides representing LAT Tyr171, Tyr191, and Tyr226 were determined at 1.8–1.9 Å; calorimetry showed Gads-SH2 binds LAT pTyr171 and pTyr191 with higher affinity than other sites; the structural basis for this selectivity (and for the broader promiscuity of Grb2) was revealed, showing Gads prefers asparagine at +2 only at certain sites. |
X-ray crystallography (1.8–1.9 Å), isothermal titration calorimetry |
The EMBO journal |
High |
15029250
|
| 2004 |
The Mona/Gads C-terminal SH3 domain binds HPK1 via a mode combining an RXXK charge interaction with a complementary PXXP motif in HPK1; ITC and X-ray crystallography characterized this interaction, revealing it differs substantially from the SLP-76 binding mode and highlighting the versatility of SH3 domains. |
Isothermal titration calorimetry, X-ray crystallography |
The Journal of biological chemistry |
High |
15100220
|
| 2005 |
The transcription factors Spi-1 and Spi-B directly regulate Grap2 expression in B cells through a functional binding element in the downstream Grap2 promoter; ectopic Grap2 expression in Grap2-deficient B cells reduced BLNK recruitment to Igα and altered phosphorylation of specific BCR signaling substrates in a manner dependent on the Grap2 proline-rich and SH2 domains. |
Promoter deletion analysis, EMSA, ectopic Grap2 expression in Grap2-deficient B cells, co-immunoprecipitation of BLNK/Igα |
Gene |
Medium |
15936902
|
| 2006 |
The Gads C-terminal SH3 domain crystal structure at 1.54 Å in complex with SLP-76 peptide 233-PSIDRSTKP-241 reveals the minimal binding site and, notably, a unique homophilic SH3–SH3 interaction present in solution in the presence of the SLP-76 peptide, suggesting a novel mechanism for increasing signaling specificity. |
X-ray crystallography (1.54 Å), solution studies |
The international journal of biochemistry & cell biology |
High |
17010654
|
| 2006 |
Gads is essential for specific CD28-mediated NF-κB activation: binding of Gads to CD28 requires the whole CD28 cytoplasmic domain including both N-terminal and C-terminal PXXP motifs (not just YMNM); mutations of these PXXP motifs reduced Gads association and specifically impaired NF-κB reporter activity while maintaining NFAT/AP-1 activity; a Gads dominant-negative significantly inhibited NF-κB but not NFAT/AP-1. |
Mutagenesis of CD28 cytoplasmic domain, co-immunoprecipitation, NF-κB and NFAT/AP-1 reporter assays, dominant-negative Gads expression |
Journal of immunology (Baltimore, Md. : 1950) |
Medium |
16818765
|
| 2006 |
Disruption of the Gads–SLP-76 interaction (by SLP-76 mutation or expression of a Gads-binding SLP-76 peptide) inhibits FcεRI-induced translocation and dynamic clustering of SLP-76 at the plasma membrane in mast cells, and impairs FcεRI-induced calcium flux, degranulation, and cytokine secretion; demonstrating that the Gads–SLP-76 interaction is critical for appropriate SLP-76 subcellular localization. |
Confocal real-time live imaging, SLP-76 site-directed mutagenesis, dominant-negative peptide expression, calcium flux, degranulation, and cytokine assays in RBL cells and bone marrow-derived mast cells |
Molecular and cellular biology |
High |
16479002
|
| 2007 |
The affinity of the Gads C-SH3 domain for SLP-76 is physiologically important: residues surrounding the RxxK motif are largely optimized for Gads C-SH3 binding, yielding Kd = 8–20 nM; Gads-mediated TCR signaling in Jurkat cells declines with decreasing affinity; very high SLP-76 specificity for Gads C-SH3 is maintained, and TCR signaling tolerates potential crossreactivity only if very high affinity is preserved. |
Surface plasmon resonance/binding affinity measurements with mutant peptides, functional TCR signaling assays in Jurkat cells |
The EMBO journal |
High |
17235283
|
| 2007 |
Low-resolution SAXS structure of full-length GADS in solution shows it is monomeric and more compact than expected for a protein with a long unstructured region; the glutamine/proline-rich unique region retains significant structural order, suggesting it is not fully disordered. |
Small-angle X-ray scattering, gel filtration, ab initio and rigid body modeling |
Biophysical journal |
Medium |
17993503
|
| 2008 |
Gads is required for efficient GPVI- and CLEC-2-induced platelet aggregation and secretion at submaximal stimulation, but is dispensable for stronger stimulation and for integrin αIIbβ3- or GPIb-IX-V-induced spreading; in contrast, LAT is required for full activation over a wider agonist range. Gads-deficient platelets constitutively associate SLP-76 with Gads. |
Gads-deficient mouse platelets, aggregation and secretion assays, flow cytometry, shear flow assay |
Journal of thrombosis and haemostasis : JTH |
Medium |
18826392
|
| 2008 |
CD28 stimulation triggers NF-κB activation through a CARMA1–PKCθ–Grb2/Gads axis; Grb2/Gads binding (but not PI3K binding) to CD28 is required for Bcl10-induced NF-κB activation downstream of CD28; PKCθ and CARMA1 are required for CD28-mediated NF-κB activation independently of TCR signaling. |
Bcl10 overexpression in Jurkat cells, dominant-negative PKCθ and CARMA1, CD28/TCR cross-linking, NF-κB reporter assay |
International immunology |
Medium |
18829987
|
| 2010 |
Bcr-Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/SLP-76/Nck1 adaptor protein pathway; GADS binding to Bcr-Abl requires Bcr-Abl tyrosine kinase activity and is imatinib-sensitive; GADS/SLP-76/Nck1 co-localize in cortical actin at membrane blebs; knockdown of each adaptor disrupts actin cytoskeleton and membrane blebbing. |
Functional interaction proteomics, co-immunoprecipitation, imatinib treatment, siRNA knockdown, confocal microscopy |
Cellular signalling |
Medium |
20079431
|
| 2013 |
GADS associates with BCR-ABL specifically through pY177 and mediates a unique BCR-ABL–SLP-76 complex in BCR-ABL-positive cell lines and B-ALL patient samples; GADS-deficient bone marrow transduced with BCR-ABL develops only short-latency myeloid disease (not lymphoid disease), demonstrating that GADS is specifically required for BCR-ABL-driven lymphoid leukemia. |
Co-immunoprecipitation with BCR-ABL mutants, retroviral transduction of Gads−/− bone marrow, bone marrow transplantation model, patient sample immunoprecipitation |
Leukemia |
High |
23399893
|
| 2014 |
GADS is required for TCR-induced calcium influx and cytokine (IL-2, IFN-γ) release in human CD4+ T cells, acting by promoting recruitment of SLP-76 and PLC-γ1 to the LAT complex; however, GADS deficiency does not impair TCR-induced cellular adhesion in human CD4+ T cells or murine CD8+ T cells. |
GADS siRNA knockdown in HuT78 human T cells and primary murine CD8+ T cells, calcium flux assay, cytokine ELISA, LAT complex immunoprecipitation, adhesion assay |
Cellular signalling |
Medium |
25636200
|
| 2014 |
GADS acts as a dose-dependent amplifier of TCR signaling, conferring responsiveness to weak TCR stimuli leading to PLC-γ1 phosphorylation and calcium flux; TALEN-derived Gads-deficient Jurkat cells show that Gads is dispensable for TCR-induced SLP-76 phosphorylation but required for CD69 expression; phosphorylation of Gads at T262 was identified by mass spectrometry, and T262 mutation increased TCR responsiveness. |
TALEN-based genome editing, mass spectrometry phosphoproteomics, Ca2+ flux, CD69 expression, PLC-γ1 phosphorylation assays |
Cellular signalling |
Medium |
25452106
|
| 2016 |
GADS interacts with FLT3 (wild-type and oncogenic FLT3-ITD) via pY955 and pY969 sites on FLT3; GADS expression enhances FLT3-ITD-induced cell proliferation, colony formation, and tumor formation in a xenograft model; GADS expression upregulates MYC and mTORC1 target genes and enhances FLT3-mediated phosphorylation of AKT, ERK1/2, p38, and STAT5. |
Co-immunoprecipitation, FLT3 phosphopeptide mapping, colony formation assay, xenograft mouse model, western blotting for downstream signaling |
Oncotarget |
Medium |
26895103
|
| 2017 |
Gads undergoes SH2 domain-mediated dimerization via an interface distinct from the pTyr-binding pocket; dimerization promotes cooperative, preferential binding of paired Gads to dually phosphorylated LAT; this cooperativity enables Gads to discriminate between dually and singly phosphorylated LAT; mutagenesis of the dimerization interface reduced cooperativity and abrogated Gads signaling in T cells and mast cells. |
Co-immunoprecipitation, surface plasmon resonance, mathematical modeling of cooperativity, mutagenesis of dimerization interface, T cell and mast cell functional assays |
Science signaling |
High |
28951535
|
| 2017 |
GADS is recruited to the phosphorylated CD6 cytoplasmic domain Y629 via its SH2 domain; this interaction, together with SLP-76 recruitment to Y662, is required for CD6-mediated T cell costimulation; both Y629F and Y662F mutations abolished costimulation, consistent with bivalent GADS/SLP-76 complex recruitment being required. |
Biochemical pulldown and co-immunoprecipitation, CD6 tyrosine mutants, functional costimulation assays in Jurkat and primary human T cells |
Molecular and cellular biology |
Medium |
28289074
|
| 2020 |
Grb2 and Gads have critically redundant roles in platelet (hem)ITAM signaling: megakaryocyte/platelet-specific Grb2/Gads double-knockout mice show virtually abolished (hem)ITAM signaling, severely impaired phosphorylation of key cascade molecules, and impaired hemostasis and arterial thrombosis, exceeding defects of either single KO; however, CLEC-2-dependent blood-lymphatic vessel separation was not affected, indicating this function is ITAM-independent. |
Conditional double-knockout mouse genetics, platelet aggregation and secretion assays, phosphorylation western blots, in vivo hemostasis and thrombosis models |
Platelets |
High |
31948362
|
| 2021 |
Itk phosphorylates Gads at Y45 within the N-terminal SH3 domain in a TCR-inducible manner in human T cells and primary mouse T cells; Y45 phosphorylation depends on Gads–SLP-76 interaction and on dimerization-dependent Gads binding to phospho-LAT; monomeric, unphosphorylated Gads mediates an RE/AP inhibitory activity that is relieved upon Gads dimerization and Y45 phosphorylation; this mechanism enforces TCR/CD28 interdependence for RE/AP-mediated IL-2 transcription. |
Mass spectrometry identification of pY45, phospho-specific antibody validation, domain mutant analysis, Gads-deficient cell reconstitution, RE/AP reporter assays in Jurkat and primary T cells |
Journal of immunology (Baltimore, Md. : 1950) |
High |
33931484
|