{"gene":"GRB10","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1995,"finding":"GRB10 was identified as a new SH2 domain-containing adapter protein by screening expression libraries with the phosphorylated EGF receptor C-terminus; it is highly related to Grb7 and undergoes serine (but not tyrosine) phosphorylation after EGF treatment, resulting in a mobility shift.","method":"Expression library screen, SH2 domain cloning, phosphorylation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — original cloning paper with biochemical characterization, single lab","pmids":["7731717"],"is_preprint":false},{"year":1995,"finding":"GRB10 (SH2 domain) interacts with the RET receptor tyrosine kinase in a kinase-activation-dependent manner, as demonstrated by yeast two-hybrid and GST pulldown assays; this was the first receptor described to use GRB10 as a signaling intermediate.","method":"Yeast two-hybrid, GST fusion protein pulldown, in vivo co-immunoprecipitation with EGFR/Ret chimera","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by GST pulldown and in vivo co-IP, single lab","pmids":["7665556"],"is_preprint":false},{"year":1996,"finding":"GRB10 binds to the insulin receptor and IGF-I receptor via its SH2 domain in a kinase-dependent manner; GRB10 SH2 domain fusion protein microinjection inhibits insulin- and IGF-I-stimulated mitogenesis but not EGF-induced mitogenesis, indicating a positive role in insulin/IGF-I signaling.","method":"Yeast two-hybrid, GST pulldown with purified insulin receptor, microinjection of SH2 domain fusion protein","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1/2 — in vitro binding plus functional microinjection assay, single lab","pmids":["8798417"],"is_preprint":false},{"year":1996,"finding":"Ligand-activated ELK (Eph-related) receptor recruits GRB10 via its SH2 domain in a phosphorylation-dependent manner; Tyr-929 of ELK was identified as required for GRB10 (but not GRB2) interaction.","method":"Yeast two-hybrid, GST-ELKcy pulldown from endothelial cell extracts, site-directed mutagenesis, co-immunoprecipitation after ligand stimulation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including mutagenesis and in vivo co-IP, single lab","pmids":["8798570"],"is_preprint":false},{"year":1996,"finding":"GRB10 SH2 domain interacts with the insulin receptor carboxyl terminus (specifically phospho-Tyr-1322) in an insulin-dependent and kinase-dependent manner; GRB10 does not associate with IRS-1, indicating an IRS-1-independent function.","method":"Yeast two-hybrid, GST fusion protein pulldown from cell lysates, co-precipitation with purified insulin receptor, phosphopeptide binding","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal in vitro and cell-based methods, phosphopeptide specificity mapped","pmids":["8621530"],"is_preprint":false},{"year":1996,"finding":"GRB10 was identified as a direct binding partner of the IGF-I receptor intracellular domain via yeast two-hybrid; binding requires a catalytically active receptor and maps to residues 1229–1245 of the IGF-IR; GRB10 co-precipitates with IGF-IR in cell lysates.","method":"Yeast two-hybrid, co-immunoprecipitation, IGF-IR mutant analysis","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by co-IP with mutational mapping, single lab","pmids":["8764099"],"is_preprint":false},{"year":1996,"finding":"GRB10 interacts with the IGF-I receptor via its SH2 domain in a kinase-dependent manner; yeast two-hybrid also identified GRB10 as binding to IRS-1 and Shc binding partners of the IGF-IR, and GRB10 interaction does not require the juxtamembrane Tyr-950.","method":"Yeast two-hybrid interaction trap, reporter gene activation assay","journal":"Molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid with kinase-dead receptor controls, single lab","pmids":["8776723"],"is_preprint":false},{"year":1997,"finding":"GRB10 protein translocates from cytosol to membrane upon insulin stimulation; its SH2 domain binds at least two sites in the insulin receptor (kinase activation loop > juxtamembrane); c-Abl SH3 domain (but not Fyn, PI3K p85, or Grb2 SH3) binds GRB10; GRB10 also binds PDGF and EGF receptors.","method":"Cell fractionation/localization, GST pulldown, phosphopeptide binding, co-immunoprecipitation, synthetic mutant receptor analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including localization, binding site mapping, and interactor specificity","pmids":["9006901"],"is_preprint":false},{"year":1997,"finding":"GRB10 associates preferentially with the insulin receptor compared to the IGF-I receptor in intact mouse fibroblasts; association is hormone-activated and sustained 5–10 min after insulin stimulation.","method":"Co-immunoprecipitation from R- cells (IGF-IR knockout) and transfected R-IR or R+ cells","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — clean receptor-specific co-IP system using knockout cell lines, single lab","pmids":["9062339"],"is_preprint":false},{"year":1998,"finding":"GRB10 contains a second novel receptor-binding domain (BPS domain, ~50 amino acids between the PH and SH2 domains) that interacts with the insulin receptor and IGF-I receptor in a kinase-dependent manner requiring the activation loop tyrosines (Y1150/Y1151); the SH2 and BPS domains cooperate to determine receptor binding specificity.","method":"Domain deletion/mutagenesis analysis, GST pulldown, yeast two-hybrid with mutant receptors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis of both receptor and adaptor combined with in vitro binding, single lab but rigorous","pmids":["9506989"],"is_preprint":false},{"year":1998,"finding":"GRB10 SH2 domain interacts in a phosphotyrosine-independent manner with Raf-1 and MEK1 kinases; interaction with MEK1 requires insulin treatment and follows MAP kinase activation; overexpression of GRB10 SH2 domain mutants promotes apoptosis, reversed by co-expression of wild-type GRB10.","method":"Yeast two-hybrid (MEK1 as bait), random mutagenesis of SH2 domain, co-expression in HTC-IR and COS-7 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — two-hybrid plus mutagenesis plus cell-based apoptosis assay, single lab","pmids":["9553107"],"is_preprint":false},{"year":1998,"finding":"GRB10 was identified as a regulator of growth hormone (GH) signaling: GRB10 associates with the GH receptor and Jak2 under GH stimulation, and inhibits transcription of SRE- and GH-response element-containing reporter genes but not STAT5-dependent reporters.","method":"Modified receptor target cloning procedure, co-transfection co-immunoprecipitation in 293 cells, transcriptional reporter assays in Huh-7 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — binding confirmed by co-IP with functional reporter assays, single lab","pmids":["9632636"],"is_preprint":false},{"year":1998,"finding":"GRB10 was identified as a binding partner of BCR-ABL via its SH2 domain at a Bcr autophosphorylation site distinct from the Grb2 binding site; interaction is kinase-activation-dependent; a BCR-ABL mutant lacking the GRB10 binding site has reduced capacity to induce IL-3 independence and focus formation.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation in CML cells, temperature-sensitive BCR-ABL system, transformation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods plus functional consequence via mutant BCR-ABL, single lab","pmids":["9747873"],"is_preprint":false},{"year":1998,"finding":"GRB10/GrbIR is phosphorylated by the non-receptor tyrosine kinase Tec (but not by Syk, Jak2, or insulin receptor) in a transient expression system; GRB10 expression suppresses Tec-driven activation of the c-fos promoter, acting as a downstream effector/suppressor of Tec signaling.","method":"Yeast two-hybrid, transient transfection in HEK293 cells, tyrosine phosphorylation assay, transcriptional reporter assay","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 — two-hybrid plus in-cell phosphorylation and functional reporter, single lab","pmids":["9753425"],"is_preprint":false},{"year":1999,"finding":"Endogenous GRB10 is predominantly localized to mitochondria (by immunofluorescence and subcellular fractionation); small pools relocate to plasma membrane and actin-rich ruffles after IGF-I or serum treatment; endogenous GRB10 and Raf-1 co-immunoprecipitate from mitochondrial fractions, with interaction enhanced by UV-activated Raf-1, suggesting GRB10 regulates mitochondrial Raf-1 anti-apoptotic activity.","method":"Immunofluorescence microscopy, subcellular fractionation, co-immunoprecipitation from mitochondrial extract, yeast two-hybrid mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by two independent methods plus co-IP from fractionated mitochondria, single lab","pmids":["10585452"],"is_preprint":false},{"year":1999,"finding":"GRB10 functions as a positive stimulatory signaling adapter in PDGF-BB-, IGF-I-, and insulin-mediated mitogenesis; Tyr-771 of PDGFRβ mediates GRB10 SH2 domain association; microinjection and cell-permeable peptide mimetics of the GRB10 SH2 domain inhibit DNA synthesis; overexpression increases cell proliferation.","method":"Ecdysone-inducible expression, microinjection, cell-permeable peptide mimetics (antennapedia-fused), DNA synthesis assay, cell number counts","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — four independent experimental strategies consistently supporting same conclusion, single lab","pmids":["10454568"],"is_preprint":false},{"year":2000,"finding":"GRB10 is phosphorylated on tyrosine (specifically Tyr-67) by Src family kinases (Src and Fyn) but not by the insulin receptor kinase itself; this Src/Fyn-mediated phosphorylation negatively regulates GRB10 binding to the insulin receptor.","method":"Pharmacological inhibition (herbimycin A), dominant-negative and constitutively active Src/Fyn expression, purified kinase in vitro assay, site-directed mutagenesis (Y67G), co-immunoprecipitation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis plus multiple cell-based approaches, single lab but multiple orthogonal methods","pmids":["10871840"],"is_preprint":false},{"year":2001,"finding":"The BPS domain of GRB10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and IGF-1 receptor in vitro; inhibition depends on activation-loop phosphorylation but the BPS domain does not bind directly to phosphotyrosine.","method":"In vitro kinase assay with purified recombinant proteins, peptide competition experiments","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro kinase inhibition assay with purified components and peptide competition, mechanistically defined","pmids":["11287005"],"is_preprint":false},{"year":2001,"finding":"GRB10 is a positive regulator of VEGF-R2 (KDR) signaling: overexpression of GRB10 increases KDR protein levels and tyrosine phosphorylation and activates MAP kinase; GRB10 undergoes VEGF-induced tyrosine phosphorylation partly through Src, requiring an intact SH2 domain; GRB10's positive effect on KDR is SH2-domain-independent.","method":"Transfection in HUVEC and 293 cells, co-immunoprecipitation, mutant expression, immunoblotting","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP and mutational analysis in relevant cell types, single lab","pmids":["11494124"],"is_preprint":false},{"year":2002,"finding":"GRB10 inhibits the IRS/PI3K/Akt signaling pathway by physically blocking IRS-1/IRS-2 access to the insulin receptor; overexpression reduces insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation and Akt phosphorylation; yeast tri-hybrid studies show GRB10 SH2 domain is required for blocking IRS-IR association; GRB10 does not reduce IR catalytic activity toward activation loop and juxtamembrane tyrosines.","method":"Stable overexpression in CHO/IR cells and adipocytes, yeast tri-hybrid, RNAi knockdown, immunoblotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple cell systems plus yeast tri-hybrid, mechanism defined at SH2-domain level, replicated with RNAi","pmids":["12493740"],"is_preprint":false},{"year":2002,"finding":"GRB10 forms a constitutive complex with Akt; overexpression of GRB10 and c-kit synergistically activates Akt in a wortmannin-sensitive, PI3K-activity-independent manner; both PH and SH2 domains of GRB10 are required for Akt activation; GRB10 can rescue deficient Akt activation by a c-kit mutant lacking the PI3K binding site.","method":"Yeast two-hybrid (c-kit as bait), co-immunoprecipitation, overexpression in Ba/F3 cells, kinase assay, IL-3-independence growth assay, domain deletion mutants","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — constitutive complex by co-IP plus functional rescue experiment with domain mutants, single lab","pmids":["11809791"],"is_preprint":false},{"year":2003,"finding":"GRB10 forms a complex with the E3 ubiquitin ligase Nedd4 and the IGF-IR; GRB10 acts as adapter bringing Nedd4 to the IGF-IR, promoting ligand-dependent IGF-IR ubiquitination, increased internalization, and shortened receptor half-life via both proteasomal and lysosomal pathways; the GRB10 SH2 domain is required for this effect.","method":"Co-immunoprecipitation (triple complex), overexpression of catalytically inactive Nedd4 mutant, SH2 domain deletion mutant, pulse-chase half-life assay, proteasome/lysosome inhibitors, dansylcadaverine treatment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1/2 — mechanistic dissection with catalytically-dead enzyme mutant, domain deletion, and multiple degradation pathway inhibitors, replicated by later studies","pmids":["12697834"],"is_preprint":false},{"year":2003,"finding":"GRB10 N-terminus interacts with two novel proteins GIGYF1 and GIGYF2 via their GYF domains binding to tandem proline-rich regions in GRB10; IGF-I stimulation increases GIGYF1 binding to GRB10 and transient binding of both to IGF-IR; overexpression of GIGYF1-GRB10-binding fragment increases IGF-I-stimulated receptor tyrosine phosphorylation.","method":"Yeast two-hybrid, co-immunoprecipitation, mutation analysis, overexpression in R+ fibroblasts","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by co-IP in cells, functional consequence shown, single lab","pmids":["12771153"],"is_preprint":false},{"year":2003,"finding":"GRB10 negatively regulates insulin-stimulated MAPK signaling by blocking Shc tyrosine phosphorylation; GRB10 overexpression reduces MAPK and Shc phosphorylation; the inhibitory effect requires the GRB10 SH2 domain; RNAi knockdown of GRB10 enhances MAPK, Shc, and Akt phosphorylation.","method":"Overexpression in CHO/IR cells and 3T3-L1 adipocytes, SH2 domain deletion, RNAi knockdown in HeLa/IR cells, immunoblotting","journal":"Molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — overexpression and RNAi with SH2 domain mutant converge on same mechanism, single lab","pmids":["14615605"],"is_preprint":false},{"year":2003,"finding":"Mouse GRB10 links the insulin receptor to p85 PI3-kinase directly (without involving IRS proteins), regulating PI3K activity and downstream metabolic insulin responses (glycogen synthesis, glucose/amino acid transport, lipogenesis, Akt/PKB, GSK, and glycogen synthase); dominant-negative GRB10 SH2 domain eliminates metabolic insulin responses in 3T3-L1 adipocytes.","method":"Co-immunoprecipitation of GRB10-p85 complex, dominant-negative domain peptides, metabolic assays in differentiated adipocytes and L6 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding demonstrated plus multiple metabolic readouts with dominant-negative peptides, single lab","pmids":["12783867"],"is_preprint":false},{"year":2003,"finding":"Crystal structure of the GRB10 SH2 domain at 1.65 Å reveals a non-covalent homodimer under physiologic conditions; the dimer interface involves residues flanking the C-terminal alpha helix conserved in the Grb7/10/14 family; structural features (Val-522, Asp-500) favor binding to dimeric phosphotyrosine sequences such as the insulin and IGF-1R activation loops.","method":"X-ray crystallography, analytical ultracentrifugation (solution dimerization), structural analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure at high resolution with biochemical validation of dimerization","pmids":["12551896"],"is_preprint":false},{"year":2003,"finding":"GRB10 disruption in mice (maternal allele) results in ~30% overgrowth of embryo and placenta by an IGF-2-independent mechanism; genetic epistasis with Igf2 mutation shows GRB10 acts on a distinct fetal growth axis.","method":"Gene-trap knockout, genetic cross with Igf2 mutant mice (epistasis), body weight measurements","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in defined knockout/double-mutant animals, replicated across multiple studies","pmids":["12829789"],"is_preprint":false},{"year":2004,"finding":"GRB10 constitutively associates with Nedd4 and prevents Nedd4-mediated degradation of VEGF-R2; Nedd4 overexpression causes VEGF-R2 disappearance, but co-expression with GRB10 restores VEGF-R2 levels; VEGF-R2 is ubiquitinated but Nedd4 is not the direct E3 ligase for VEGF-R2 ubiquitination.","method":"Co-immunoprecipitation, overexpression of Nedd4 and GRB10 in cells, ubiquitination assay, Nedd4 catalytic mutant (C854S), MG132 treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus catalytic mutant plus rescue experiment, single lab","pmids":["15060076"],"is_preprint":false},{"year":2005,"finding":"RNAi knockdown of endogenous GRB10 enhances IGF-I-stimulated IRS phosphorylation, Akt/PKB, and ERK1/2, and increases DNA synthesis; GRB10 knockdown paradoxically decreases IGF-IR autophosphorylation, an effect partially reversed by phosphatase inhibitor pervanadate, suggesting GRB10 protects the activated receptor from phosphatases.","method":"siRNA knockdown, immunoblotting of downstream signaling, DNA synthesis assay, pervanadate pretreatment","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA with multiple downstream readouts and pharmacological test of mechanism, single lab","pmids":["16037382"],"is_preprint":false},{"year":2005,"finding":"Transgenic overexpression of Meg1/Grb10 in mice causes postnatal growth retardation and hyperinsulinemic insulin resistance in vivo, confirming that GRB10 negatively regulates both IGF1R- and IR-dependent signaling pathways in vivo.","method":"Transgenic mouse lines (4 independent lines), glucose tolerance test, insulin tolerance test, body weight measurement","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 — four independent transgenic lines with consistent phenotype, in vivo metabolic testing","pmids":["15752742"],"is_preprint":false},{"year":2005,"finding":"Phosphorylation of GRB10 (at Ser-428) by Akt creates a binding site for 14-3-3 proteins; Akt directly binds GRB10 and phosphorylates it in vitro; only the phosphorylated form of GRB10 co-immunoprecipitates with endogenous 14-3-3.","method":"Yeast two-hybrid (14-3-3 as binding partner), co-immunoprecipitation, in vitro Akt kinase assay, site-directed mutagenesis (S428A)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis plus co-IP, identifies writer (Akt) and reader (14-3-3)","pmids":["15722337"],"is_preprint":false},{"year":2006,"finding":"GRB10 mediates insulin-stimulated proteasomal degradation of the insulin receptor; suppression of GRB10 by RNAi leads to increased IR protein levels and reduced insulin-stimulated IR ubiquitination; overexpression reduces IR levels without affecting IR mRNA; IR reduction is blocked by MG132 but not chloroquine.","method":"RNAi knockdown, stable overexpression, qRT-PCR (mRNA unchanged), ubiquitination assay, proteasome/lysosome inhibitors","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 — bidirectional genetic manipulation plus pharmacological inhibition with ubiquitination assay, single lab","pmids":["16434550"],"is_preprint":false},{"year":2007,"finding":"Peripheral (maternal allele) knockout of Grb10 in mice leads to enhanced insulin-stimulated Akt and MAPK phosphorylation in skeletal muscle and fat, and increased whole-body insulin sensitivity by hyperinsulinemic-euglycemic clamp, establishing GRB10 as an in vivo negative regulator of insulin signaling.","method":"Gene-trap knockout mice (maternal inheritance), hyperinsulinemic-euglycemic clamp, insulin-stimulated kinase phosphorylation in tissues","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO with gold-standard in vivo clamp measurements, independently confirmed by other groups","pmids":["17620412"],"is_preprint":false},{"year":2007,"finding":"Grb10-deficient (Grb10Δ2-4) mice show improved glucose tolerance and insulin sensitivity, and tissue-specific changes in IR tyrosine phosphorylation consistent with GRB10 blocking phosphatase access to IR activation loop; IRS-1 tyrosine phosphorylation is also enhanced, supporting attenuation of IR→IRS-1 signal transmission.","method":"Knockout mouse (Grb10Δ2-4), glucose and insulin tolerance tests, tissue IR phosphorylation analysis, IRS-1 phosphorylation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO with mechanistic tissue-level signaling analysis, replicated across labs","pmids":["17562854"],"is_preprint":false},{"year":2007,"finding":"GRB10 and active Raf-1 promote Bad-dependent cell survival; both Grb10 and Raf-1 knockout cells show enhanced apoptosis in response to Bad; GRB10 requires its SH2, proline-rich, and PH domains plus Akt phosphorylation site (and consequent 14-3-3 binding) for anti-apoptotic function; Raf-1 requires its kinase activity and Ras-associated domain binding to GRB10 SH2.","method":"Knockout MEFs (Grb10 and Raf-1 deficient), siRNA, mutagenesis of GRB10 domains, signaling inhibitors, kinase assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple domain mutants in KO cells with convergent results, single lab","pmids":["17535812"],"is_preprint":false},{"year":2007,"finding":"GRB10 interacts with the Wnt co-receptor LRP6 intracellular domain and negatively regulates canonical Wnt signaling; GRB10 overexpression attenuates Wnt3a-induced β-catenin accumulation and TCF reporter activity; GRB10 interferes with Axin binding to LRP6; RNAi knockdown of GRB10 stimulates Wnt signaling.","method":"Co-immunoprecipitation of GRB10-LRP6, TCF reporter assays, RNAi knockdown, β-catenin accumulation assay, Axin binding competition assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus RNAi plus reporter assay and mechanistic Axin competition, single lab","pmids":["17376403"],"is_preprint":false},{"year":2008,"finding":"The GRB10/Nedd4 complex mediates multiubiquitination (not polyubiquitination) of the IGF-IR upon ligand stimulation, which is required for receptor internalization; GRB10 and Nedd4 associate with IGF-IR in early endosomes and caveosomes but are not degraded and are directed to recycling endosomes.","method":"Ubiquitin chain analysis, clathrin-dependent and -independent internalization assays, confocal microscopy, subcellular fractionation, co-immunoprecipitation from endosomal fractions","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple trafficking assays plus subcellular fractionation and localization, single lab","pmids":["18286479"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of the Grb10 RA-PH tandem domain at 2.6 Å reveals an integrated dimeric structural unit (RA+PH+linker); biochemical studies show Grb14 (family member) binds activated Ras via its RA domain; these domains illuminate membrane-recruitment mechanisms shared with MIG-10, RIAM, lamellipodin, and Pico.","method":"X-ray crystallography, biochemical binding assays for Ras interaction","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical validation, mechanistically defines domain architecture","pmids":["19648926"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the NEDD4 C2 domain – GRB10 SH2 domain complex at 2.0 Å shows three interaction interfaces, with the main interface being an antiparallel β-sheet; NEDD4 C2 binds at non-classical sites on the SH2 surface far from the phosphotyrosine pocket (phosphotyrosine-independent); GRB10 SH2 can simultaneously bind NEDD4 C2 and IGF-1R kinase domain.","method":"X-ray crystallography, structural analysis, binding interface characterization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure mechanistically explaining how GRB10 bridges NEDD4 and IGF-1R","pmids":["20980250"],"is_preprint":false},{"year":2011,"finding":"mTORC1-mediated phosphorylation of GRB10 stabilizes GRB10 protein, leading to feedback inhibition of both the PI3K and ERK-MAPK pathways; identified by large-scale quantitative phosphoproteomics as a direct mTORC1 substrate.","method":"Quantitative phosphoproteomics (large-scale), mTORC1 substrate characterization, rapamycin treatment, loss-of-function experiments","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — large-scale phosphoproteomics plus functional characterization, highly cited, replicated by others","pmids":["21659605"],"is_preprint":false},{"year":2011,"finding":"Within the brain, Grb10 is expressed from the paternal allele; ablation of paternal Grb10 specifically increases social dominance behavior in mice; maternal allele ablation causes peripheral overgrowth—demonstrating tissue-specific allelic function controlling distinct physiological processes.","method":"Allele-specific knockout mice, behavioral testing (social dominance, allogrooming), body weight measurement","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — allele-specific knockout with specific behavioral and growth phenotypes, replicated across multiple behavioral paradigms","pmids":["21270893"],"is_preprint":false},{"year":2012,"finding":"GRB10 deletion in mice increases myofiber number (not fiber size) in skeletal muscle; the hypermuscular phenotype originates during embryonic development; Grb10-deficient neonatal muscle shows upregulated functional gene signatures for myogenic signaling and proliferation.","method":"Grb10 knockout mice, histomorphometry (fiber number and cross-sectional area), neonatal limb measurements, gene expression profiling","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — knockout with specific morphological and transcriptional phenotypic readouts, single lab","pmids":["22623587"],"is_preprint":false},{"year":2012,"finding":"GRB10 physically associates with FLT3 at phospho-tyrosines 572 and 793 in a ligand-dependent manner, and constitutively with oncogenic FLT3-ITD; GRB10 enhances FLT3-induced Akt phosphorylation by directly interacting with p85 PI3K subunit; GRB10 depletion reduces Akt phosphorylation.","method":"Co-immunoprecipitation, receptor phosphorylation site mapping (Y572F/Y793F mutants), p85 pulldown, siRNA knockdown, cell proliferation and colony formation assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with mutational mapping of binding site plus functional signaling and cell growth assays, single lab","pmids":["23246379"],"is_preprint":false},{"year":2014,"finding":"mTOR-mediated phosphorylation of GRB10 at Ser501/503 switches its binding preference from the insulin receptor to raptor (mTOR complex component), leading to raptor-mTOR dissociation and mTORC1 downregulation; fat-specific GRB10 disruption increases mTORC1 signaling, suppresses lipolysis, and reduces thermogenesis; these effects are abolished by rapamycin.","method":"mTORC1 substrate phosphorylation assay (Ser501/503), co-immunoprecipitation of GRB10-raptor complex, fat-specific Grb10 knockout mice, in vivo rapamycin treatment, lipolysis assay, thermogenic gene expression","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 1/2 — phospho-site switching mechanism with co-IP, tissue-specific KO, and in vivo rescue with rapamycin; multiple orthogonal methods","pmids":["24746805"],"is_preprint":false},{"year":2014,"finding":"GRB10 associates with IRS-2, NEDD4.2, IL-4Rα, and γc after IL-4 stimulation in macrophages; GRB10 knockdown enhances Tyr(P)-IRS-2 and promotes M2 macrophage gene expression (CD200R, CCL22, MMP12, TGM2); IL-4Rα and γc are ubiquitinated after IL-4 treatment, suggesting GRB10 regulates IL-4 receptor complex degradation through NEDD4.2.","method":"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, M2 gene expression analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP of multi-protein complex plus siRNA with gene expression readout, single lab","pmids":["27742835"],"is_preprint":false},{"year":2016,"finding":"Deletion of the maternal allele of Grb10 in mice substantially increases hematopoietic stem cell (HSC) long-term repopulating capacity and accelerates HSC regeneration after irradiation; enhanced HSC regeneration is dependent on activation of the Akt/mTORC1 pathway; Grb10-deficient HSCs show increased proliferation with upregulation of CDK4 and Cyclin E.","method":"Maternal allele-specific Grb10 knockout mice, competitive transplantation, total body irradiation, flow cytometry, pathway inhibitor studies (Akt/mTORC1), cell cycle marker analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — allele-specific KO with competitive transplantation and pharmacological pathway validation, single lab","pmids":["27806297"],"is_preprint":false},{"year":2018,"finding":"Ablation of Grb10 specifically in muscle (muscle-specific KO using α-skeletal actin-Cre) is sufficient to enlarge muscle fibers and increase insulin-stimulated glucose uptake and phospho-Akt in muscle, confirming a cell-autonomous role of GRB10 as modulator of proximal insulin receptor signaling in muscle.","method":"Muscle-specific conditional knockout (Cre-lox), hyperinsulinemic-euglycemic clamp, immunoblotting of insulin signaling","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific conditional KO with in vivo clamp measurement, cell-autonomous function established","pmids":["29370381"],"is_preprint":false},{"year":2018,"finding":"GRB10 expression is silenced in adult mouse liver but can be reactivated by acute ER stress (tunicamycin or short-term high-fat diet) via ATF4-mediated transcriptional induction; liver-specific GRB10 KO suppresses lipogenic gene expression and ER-stress-induced hepatosteatosis, establishing GRB10 as a mediator of acute ER stress-induced hepatic lipid dysregulation.","method":"Liver-specific GRB10 knockout, ER stress induction (tunicamycin, HFD), ATF4 ChIP/transcription analysis, lipogenic gene expression, hepatic lipid assays","journal":"Journal of molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — tissue-specific KO with mechanistic ATF4 transcriptional link, single lab","pmids":["29555819"],"is_preprint":false},{"year":2019,"finding":"mTORC1 phosphorylates GRB10 at Ser476 in human skeletal muscle cells; insulin and amino acids independently and additively stimulate this phosphorylation; mTORC1 controls PI3K/Akt signaling through GRB10-mediated modulation of insulin receptor protein abundance; rapamycin blocks Grb10 Ser476 phosphorylation and reduces GRB10 protein levels with corresponding increase in IR.","method":"Grb10 knockdown in primary human myotubes, rapamycin treatment, phospho-Ser476 immunoblotting, glucose uptake assay, IR protein quantification","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — human primary cell knockdown with rapamycin pharmacology and defined phosphorylation site, single lab","pmids":["31794259"],"is_preprint":false},{"year":2023,"finding":"GRB10 in hypothalamic AgRP and POMC neurons interacts with the leptin receptor and enhances leptin signaling; GRB10 ablation in AgRP neurons promotes weight gain while overexpression reduces body weight; GRB10 in AgRP neurons enhances leptin inhibition via ATP-sensitive K+ channels and in POMC neurons via transient receptor potential channels.","method":"Neuron-specific conditional knockout and overexpression (AAV-mediated), electrophysiology (patch-clamp), co-immunoprecipitation of GRB10-leptin receptor, body weight and food intake measurements","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 — neuron-specific bidirectional manipulation plus electrophysiology plus co-IP, multiple cell-type specific phenotypes defined","pmids":["36593271"],"is_preprint":false}],"current_model":"GRB10 is a multi-domain adapter protein (containing RA, PH, BPS, and SH2 domains) that functions primarily as a negative regulator of insulin and IGF-1 receptor signaling: its BPS domain acts as a pseudosubstrate inhibitor of the IR/IGF-1R kinase, its SH2 domain physically blocks IRS protein access to the receptor, and it recruits the E3 ubiquitin ligase NEDD4 to promote receptor ubiquitination and degradation; additionally, mTORC1 phosphorylates GRB10 (at Ser501/503 in mice, Ser476 in humans) to stabilize it and mediate feedback inhibition of both PI3K and MAPK pathways, while in adipose tissue this phosphorylation switches GRB10 binding from the insulin receptor to raptor to downregulate mTORC1, and in hypothalamic neurons GRB10 instead enhances leptin receptor signaling to promote energy homeostasis."},"narrative":{"teleology":[{"year":1995,"claim":"Identification of GRB10 as a new SH2-containing adaptor protein that binds activated receptor tyrosine kinases established it as a candidate signaling intermediate downstream of growth factor receptors.","evidence":"Expression library screening with phosphorylated EGF receptor; yeast two-hybrid and GST pulldowns with RET receptor","pmids":["7731717","7665556"],"confidence":"Medium","gaps":["No functional consequence of GRB10–RTK interaction determined","Signaling role (positive vs. negative) unknown"]},{"year":1996,"claim":"Mapping GRB10 SH2 domain binding to specific phosphotyrosines on the insulin receptor and IGF-1R defined the molecular basis of its receptor specificity and raised the question of whether GRB10 promotes or inhibits insulin/IGF signaling.","evidence":"Yeast two-hybrid, GST pulldowns, phosphopeptide binding (IR pY1322), microinjection of SH2 domain fusion proteins; multiple independent labs","pmids":["8621530","8798417","8764099"],"confidence":"High","gaps":["Positive vs. negative signaling role debated","BPS domain not yet discovered","In vivo relevance unknown"]},{"year":1998,"claim":"Discovery of the BPS domain as a second receptor-binding module that cooperates with the SH2 domain revealed a bipartite receptor engagement mechanism unique to the Grb7/10/14 family.","evidence":"Domain deletion/mutagenesis with GST pulldown and yeast two-hybrid using mutant IR/IGF-1R (activation loop tyrosine mutants)","pmids":["9506989"],"confidence":"High","gaps":["BPS domain mechanism of action (pseudosubstrate inhibition) not yet demonstrated","Structural basis undefined"]},{"year":2001,"claim":"Demonstration that the BPS domain directly inhibits IR/IGF-1R kinase activity toward substrates in a reconstituted system established GRB10 as a bona fide kinase inhibitor, not merely a docking partner.","evidence":"In vitro kinase assay with purified recombinant BPS domain and IR/IGF-1R kinase domains, peptide competition","pmids":["11287005"],"confidence":"High","gaps":["In vivo relevance of BPS-mediated inhibition not yet shown","Relative contribution of BPS vs. SH2 in intact cells unclear"]},{"year":2002,"claim":"Resolving the positive-vs-negative debate: GRB10 SH2 domain physically blocks IRS-1/IRS-2 access to the insulin receptor, attenuating PI3K/Akt signaling, while RNAi depletion enhances insulin signaling—establishing GRB10 as a negative regulator.","evidence":"Stable overexpression and RNAi knockdown in CHO/IR cells and adipocytes, yeast tri-hybrid showing competitive exclusion of IRS","pmids":["12493740"],"confidence":"High","gaps":["In vivo confirmation in whole animals pending","Contribution to MAPK pathway inhibition not yet addressed"]},{"year":2003,"claim":"GRB10 was shown to recruit the E3 ligase NEDD4 to IGF-1R, promoting receptor ubiquitination, internalization, and degradation—revealing a second inhibitory mechanism beyond kinase/substrate blockade.","evidence":"Triple-complex co-IP (GRB10–NEDD4–IGF-1R), catalytically inactive NEDD4 mutant, pulse-chase receptor half-life, proteasome/lysosome inhibitors","pmids":["12697834"],"confidence":"High","gaps":["Ubiquitin chain topology on IGF-1R not characterized","Whether NEDD4 recruitment also targets IR in vivo uncertain"]},{"year":2003,"claim":"Crystal structures of the SH2 domain (homodimer at 1.65 Å) revealed how the Grb7/10/14 family recognizes dimeric phosphotyrosine motifs on the IR/IGF-1R activation loop, providing the first structural framework for GRB10-receptor interaction.","evidence":"X-ray crystallography, analytical ultracentrifugation","pmids":["12551896"],"confidence":"High","gaps":["Full-length GRB10 structure unavailable","BPS domain structure not yet solved for GRB10"]},{"year":2003,"claim":"Maternal-allele Grb10 knockout mice exhibited ~30% embryonic overgrowth, genetically establishing GRB10 as a maternally imprinted growth suppressor acting on a pathway distinct from IGF-2.","evidence":"Gene-trap knockout crossed with Igf2 mutant mice (epistasis analysis), body/placenta weight measurements","pmids":["12829789"],"confidence":"High","gaps":["Tissue-specific contributions to overgrowth not dissected","Receptor target in placenta not defined"]},{"year":2007,"claim":"Whole-body insulin sensitivity measurements in Grb10-deficient mice using hyperinsulinemic-euglycemic clamps confirmed that GRB10 constrains insulin signaling in vivo, with enhanced Akt and MAPK phosphorylation in muscle and fat.","evidence":"Gene-trap knockout mice, hyperinsulinemic-euglycemic clamp, tissue-level signaling immunoblots","pmids":["17620412","17562854"],"confidence":"High","gaps":["Cell-autonomous vs. systemic effects not separated","Brain-specific functions of paternal allele not yet known"]},{"year":2010,"claim":"The crystal structure of the NEDD4 C2–GRB10 SH2 complex showed that NEDD4 binds GRB10 at sites distinct from the phosphotyrosine pocket, explaining how GRB10 can simultaneously engage both NEDD4 and the receptor kinase domain to bridge them.","evidence":"X-ray crystallography at 2.0 Å resolution, structural interface analysis","pmids":["20980250"],"confidence":"High","gaps":["No full ternary complex structure (GRB10–NEDD4–receptor)","Stoichiometry and dynamics in vivo not resolved"]},{"year":2011,"claim":"Identification of GRB10 as a direct mTORC1 substrate whose phosphorylation-dependent stabilization mediates feedback inhibition of both PI3K and MAPK pathways placed GRB10 at a critical node linking nutrient sensing to growth factor signaling.","evidence":"Large-scale quantitative phosphoproteomics, rapamycin treatment, loss-of-function experiments","pmids":["21659605"],"confidence":"High","gaps":["Specific phosphorylation sites in human GRB10 partially mapped","Phosphatase(s) that dephosphorylate GRB10 unknown"]},{"year":2011,"claim":"Demonstration that the paternal Grb10 allele is selectively expressed in brain and controls social dominance behavior revealed an unexpected imprinting-based functional partition: maternal allele governs peripheral growth, paternal allele governs behavior.","evidence":"Allele-specific knockout mice, behavioral testing panels (social dominance, allogrooming)","pmids":["21270893"],"confidence":"High","gaps":["Molecular mechanism of GRB10 in social behavior unknown","Receptor partners in brain neurons not identified at this time"]},{"year":2014,"claim":"Discovery that mTOR-mediated phosphorylation switches GRB10 binding from insulin receptor to raptor, causing mTORC1 dissociation and downregulation in adipose tissue, revealed a phosphorylation-dependent partner-switching mechanism integrating GRB10 into bidirectional mTORC1 feedback.","evidence":"Phospho-site mutant analysis, co-IP of GRB10–raptor complex, fat-specific Grb10 KO mice, rapamycin rescue in vivo","pmids":["24746805"],"confidence":"High","gaps":["Whether raptor-switching occurs in all tissues or is fat-specific","Structural basis of phospho-dependent partner switch unknown"]},{"year":2018,"claim":"Muscle-specific GRB10 deletion confirmed cell-autonomous control of insulin-stimulated glucose uptake and fiber size, establishing that the metabolic and growth phenotypes of global knockouts originate in muscle itself.","evidence":"Muscle-specific conditional knockout (α-skeletal actin-Cre), hyperinsulinemic-euglycemic clamp, insulin signaling immunoblots","pmids":["29370381"],"confidence":"High","gaps":["Developmental vs. postnatal contribution to muscle hypertrophy not fully resolved","Whether BPS or SH2 domain is dominant in muscle unclear"]},{"year":2023,"claim":"GRB10 was found to interact with the leptin receptor in hypothalamic neurons and enhance leptin signaling, broadening its role from a growth factor signaling inhibitor to a positive modulator of energy homeostasis in the CNS.","evidence":"Neuron-specific conditional KO and AAV-mediated overexpression in AgRP/POMC neurons, patch-clamp electrophysiology, GRB10–leptin receptor co-IP, body weight/food intake measurements","pmids":["36593271"],"confidence":"High","gaps":["Mechanism by which GRB10 enhances (rather than inhibits) leptin receptor signaling not fully defined","Whether NEDD4 or BPS domain mechanisms are involved in leptin signaling unknown"]},{"year":null,"claim":"A full-length structural model of GRB10 in complex with activated receptor and NEDD4 is lacking, and the molecular basis for context-dependent switching between positive and negative signaling roles remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length GRB10 structure","Mechanism determining positive (leptin/VEGFR/c-Kit) vs. negative (IR/IGF-1R) signaling roles unknown","Upstream signals controlling allele-specific expression in brain vs. periphery not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,4,9,19,21,38,44]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[17,19,23,31,39,43]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,14]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[14]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[36]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,4,19,23,30,39,43,49]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[17,21,31,38]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[26,40,41]}],"complexes":["GRB10–NEDD4 complex","mTORC1 (via raptor interaction)"],"partners":["NEDD4","INSR","IGF1R","RPTOR","LEPR","RAF1","GIGYF1","GIGYF2"],"other_free_text":[]},"mechanistic_narrative":"GRB10 is a multi-domain adaptor protein that functions as a central negative regulator of receptor tyrosine kinase signaling, particularly insulin receptor (IR) and IGF-1 receptor (IGF-1R), integrating growth factor inputs with nutrient-sensing pathways to control growth, metabolism, and cell survival. Its BPS domain acts as a pseudosubstrate inhibitor of IR/IGF-1R kinase activity [PMID:11287005], while its SH2 domain physically blocks IRS protein access to activated receptors, thereby attenuating PI3K/Akt and MAPK signaling [PMID:12493740, PMID:14615605]; simultaneously, GRB10 recruits the E3 ubiquitin ligase NEDD4 to promote receptor multiubiquitination, internalization, and degradation [PMID:12697834, PMID:18286479]. mTORC1 phosphorylates GRB10 to stabilize it, creating a feedback loop that restrains PI3K and MAPK pathways; in adipose tissue, this phosphorylation switches GRB10 binding from IR to raptor, thereby downregulating mTORC1 itself [PMID:21659605, PMID:24746805]. In vivo, maternal-allele GRB10 loss causes fetal overgrowth and enhanced insulin sensitivity, while paternal-allele loss in brain increases social dominance behavior, and in hypothalamic neurons GRB10 enhances leptin receptor signaling to regulate energy homeostasis [PMID:12829789, PMID:21270893, PMID:36593271]."},"prefetch_data":{"uniprot":{"accession":"Q13322","full_name":"Growth factor receptor-bound protein 10","aliases":["GRB10 adapter protein","Insulin receptor-binding protein Grb-IR"],"length_aa":594,"mass_kda":67.2,"function":"Adapter protein which modulates coupling of a number of cell surface receptor kinases with specific signaling pathways. Binds to, and suppress signals from, activated receptors tyrosine kinases, including the insulin (INSR) and insulin-like growth factor (IGF1R) receptors. The inhibitory effect can be achieved by 2 mechanisms: interference with the signaling pathway and increased receptor degradation. Delays and reduces AKT1 phosphorylation in response to insulin stimulation. Blocks association between INSR and IRS1 and IRS2 and prevents insulin-stimulated IRS1 and IRS2 tyrosine phosphorylation. Recruits NEDD4 to IGF1R, leading to IGF1R ubiquitination, increased internalization and degradation by both the proteasomal and lysosomal pathways. May play a role in mediating insulin-stimulated ubiquitination of INSR, leading to proteasomal degradation. Negatively regulates Wnt signaling by interacting with LRP6 intracellular portion and interfering with the binding of AXIN1 to LRP6. Positive regulator of the KDR/VEGFR-2 signaling pathway. May inhibit NEDD4-mediated degradation of KDR/VEGFR-2","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q13322/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GRB10","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GRB10","total_profiled":1310},"omim":[{"mim_id":"618905","title":"SILVER-RUSSELL SYNDROME 2; SRS2","url":"https://www.omim.org/entry/618905"},{"mim_id":"612064","title":"GRB10-INTERACTING GYF PROTEIN 1; GIGYF1","url":"https://www.omim.org/entry/612064"},{"mim_id":"612003","title":"GRB10-INTERACTING GYF PROTEIN 2; GIGYF2","url":"https://www.omim.org/entry/612003"},{"mim_id":"610317","title":"CORDON-BLEU WH2 REPEAT PROTEIN; COBL","url":"https://www.omim.org/entry/610317"},{"mim_id":"609658","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 5; NLRP5","url":"https://www.omim.org/entry/609658"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GRB10"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q13322","domains":[{"cath_id":"3.10.20.90","chopping":"168-248","consensus_level":"high","plddt":95.2256,"start":168,"end":248},{"cath_id":"2.30.29.30","chopping":"254-414","consensus_level":"high","plddt":89.308,"start":254,"end":414},{"cath_id":"3.30.505.10","chopping":"488-589","consensus_level":"high","plddt":94.9057,"start":488,"end":589}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13322","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13322-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13322-F1-predicted_aligned_error_v6.png","plddt_mean":73.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GRB10","jax_strain_url":"https://www.jax.org/strain/search?query=GRB10"},"sequence":{"accession":"Q13322","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13322.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13322/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13322"}},"corpus_meta":[{"pmid":"21659605","id":"PMC_21659605","title":"Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.","date":"2011","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/21659605","citation_count":707,"is_preprint":false},{"pmid":"12829789","id":"PMC_12829789","title":"Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism.","date":"2003","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12829789","citation_count":219,"is_preprint":false},{"pmid":"12697834","id":"PMC_12697834","title":"The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12697834","citation_count":204,"is_preprint":false},{"pmid":"21270893","id":"PMC_21270893","title":"Distinct physiological and behavioural functions for parental alleles of imprinted Grb10.","date":"2011","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/21270893","citation_count":156,"is_preprint":false},{"pmid":"10861285","id":"PMC_10861285","title":"Human GRB10 is imprinted and expressed from the paternal and maternal allele in a highly tissue- and isoform-specific fashion.","date":"2000","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10861285","citation_count":142,"is_preprint":false},{"pmid":"24699409","id":"PMC_24699409","title":"A central role for GRB10 in regulation of islet function in man.","date":"2014","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24699409","citation_count":140,"is_preprint":false},{"pmid":"15901248","id":"PMC_15901248","title":"Grb10 and Grb14: enigmatic regulators of insulin action--and more?","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15901248","citation_count":138,"is_preprint":false},{"pmid":"7731717","id":"PMC_7731717","title":"The cloning of Grb10 reveals a new family of SH2 domain proteins.","date":"1995","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/7731717","citation_count":138,"is_preprint":false},{"pmid":"8798570","id":"PMC_8798570","title":"Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8798570","citation_count":137,"is_preprint":false},{"pmid":"9448292","id":"PMC_9448292","title":"Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver-Russell syndrome gene.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9448292","citation_count":134,"is_preprint":false},{"pmid":"8798417","id":"PMC_8798417","title":"Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors. 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this was the first receptor described to use GRB10 as a signaling intermediate.\",\n      \"method\": \"Yeast two-hybrid, GST fusion protein pulldown, in vivo co-immunoprecipitation with EGFR/Ret chimera\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by GST pulldown and in vivo co-IP, single lab\",\n      \"pmids\": [\"7665556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"GRB10 binds to the insulin receptor and IGF-I receptor via its SH2 domain in a kinase-dependent manner; GRB10 SH2 domain fusion protein microinjection inhibits insulin- and IGF-I-stimulated mitogenesis but not EGF-induced mitogenesis, indicating a positive role in insulin/IGF-I signaling.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown with purified insulin receptor, microinjection of SH2 domain fusion protein\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro binding plus functional microinjection assay, single lab\",\n      \"pmids\": [\"8798417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Ligand-activated ELK (Eph-related) receptor recruits GRB10 via its SH2 domain in a phosphorylation-dependent manner; Tyr-929 of ELK was identified as required for GRB10 (but not GRB2) interaction.\",\n      \"method\": \"Yeast two-hybrid, GST-ELKcy pulldown from endothelial cell extracts, site-directed mutagenesis, co-immunoprecipitation after ligand stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including mutagenesis and in vivo co-IP, single lab\",\n      \"pmids\": [\"8798570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"GRB10 SH2 domain interacts with the insulin receptor carboxyl terminus (specifically phospho-Tyr-1322) in an insulin-dependent and kinase-dependent manner; GRB10 does not associate with IRS-1, indicating an IRS-1-independent function.\",\n      \"method\": \"Yeast two-hybrid, GST fusion protein pulldown from cell lysates, co-precipitation with purified insulin receptor, phosphopeptide binding\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal in vitro and cell-based methods, phosphopeptide specificity mapped\",\n      \"pmids\": [\"8621530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"GRB10 was identified as a direct binding partner of the IGF-I receptor intracellular domain via yeast two-hybrid; binding requires a catalytically active receptor and maps to residues 1229–1245 of the IGF-IR; GRB10 co-precipitates with IGF-IR in cell lysates.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, IGF-IR mutant analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by co-IP with mutational mapping, single lab\",\n      \"pmids\": [\"8764099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"GRB10 interacts with the IGF-I receptor via its SH2 domain in a kinase-dependent manner; yeast two-hybrid also identified GRB10 as binding to IRS-1 and Shc binding partners of the IGF-IR, and GRB10 interaction does not require the juxtamembrane Tyr-950.\",\n      \"method\": \"Yeast two-hybrid interaction trap, reporter gene activation assay\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid with kinase-dead receptor controls, single lab\",\n      \"pmids\": [\"8776723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"GRB10 protein translocates from cytosol to membrane upon insulin stimulation; its SH2 domain binds at least two sites in the insulin receptor (kinase activation loop > juxtamembrane); c-Abl SH3 domain (but not Fyn, PI3K p85, or Grb2 SH3) binds GRB10; GRB10 also binds PDGF and EGF receptors.\",\n      \"method\": \"Cell fractionation/localization, GST pulldown, phosphopeptide binding, co-immunoprecipitation, synthetic mutant receptor analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including localization, binding site mapping, and interactor specificity\",\n      \"pmids\": [\"9006901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"GRB10 associates preferentially with the insulin receptor compared to the IGF-I receptor in intact mouse fibroblasts; association is hormone-activated and sustained 5–10 min after insulin stimulation.\",\n      \"method\": \"Co-immunoprecipitation from R- cells (IGF-IR knockout) and transfected R-IR or R+ cells\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean receptor-specific co-IP system using knockout cell lines, single lab\",\n      \"pmids\": [\"9062339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRB10 contains a second novel receptor-binding domain (BPS domain, ~50 amino acids between the PH and SH2 domains) that interacts with the insulin receptor and IGF-I receptor in a kinase-dependent manner requiring the activation loop tyrosines (Y1150/Y1151); the SH2 and BPS domains cooperate to determine receptor binding specificity.\",\n      \"method\": \"Domain deletion/mutagenesis analysis, GST pulldown, yeast two-hybrid with mutant receptors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis of both receptor and adaptor combined with in vitro binding, single lab but rigorous\",\n      \"pmids\": [\"9506989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRB10 SH2 domain interacts in a phosphotyrosine-independent manner with Raf-1 and MEK1 kinases; interaction with MEK1 requires insulin treatment and follows MAP kinase activation; overexpression of GRB10 SH2 domain mutants promotes apoptosis, reversed by co-expression of wild-type GRB10.\",\n      \"method\": \"Yeast two-hybrid (MEK1 as bait), random mutagenesis of SH2 domain, co-expression in HTC-IR and COS-7 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — two-hybrid plus mutagenesis plus cell-based apoptosis assay, single lab\",\n      \"pmids\": [\"9553107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRB10 was identified as a regulator of growth hormone (GH) signaling: GRB10 associates with the GH receptor and Jak2 under GH stimulation, and inhibits transcription of SRE- and GH-response element-containing reporter genes but not STAT5-dependent reporters.\",\n      \"method\": \"Modified receptor target cloning procedure, co-transfection co-immunoprecipitation in 293 cells, transcriptional reporter assays in Huh-7 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — binding confirmed by co-IP with functional reporter assays, single lab\",\n      \"pmids\": [\"9632636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRB10 was identified as a binding partner of BCR-ABL via its SH2 domain at a Bcr autophosphorylation site distinct from the Grb2 binding site; interaction is kinase-activation-dependent; a BCR-ABL mutant lacking the GRB10 binding site has reduced capacity to induce IL-3 independence and focus formation.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation in CML cells, temperature-sensitive BCR-ABL system, transformation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods plus functional consequence via mutant BCR-ABL, single lab\",\n      \"pmids\": [\"9747873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRB10/GrbIR is phosphorylated by the non-receptor tyrosine kinase Tec (but not by Syk, Jak2, or insulin receptor) in a transient expression system; GRB10 expression suppresses Tec-driven activation of the c-fos promoter, acting as a downstream effector/suppressor of Tec signaling.\",\n      \"method\": \"Yeast two-hybrid, transient transfection in HEK293 cells, tyrosine phosphorylation assay, transcriptional reporter assay\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — two-hybrid plus in-cell phosphorylation and functional reporter, single lab\",\n      \"pmids\": [\"9753425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Endogenous GRB10 is predominantly localized to mitochondria (by immunofluorescence and subcellular fractionation); small pools relocate to plasma membrane and actin-rich ruffles after IGF-I or serum treatment; endogenous GRB10 and Raf-1 co-immunoprecipitate from mitochondrial fractions, with interaction enhanced by UV-activated Raf-1, suggesting GRB10 regulates mitochondrial Raf-1 anti-apoptotic activity.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, co-immunoprecipitation from mitochondrial extract, yeast two-hybrid mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by two independent methods plus co-IP from fractionated mitochondria, single lab\",\n      \"pmids\": [\"10585452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"GRB10 functions as a positive stimulatory signaling adapter in PDGF-BB-, IGF-I-, and insulin-mediated mitogenesis; Tyr-771 of PDGFRβ mediates GRB10 SH2 domain association; microinjection and cell-permeable peptide mimetics of the GRB10 SH2 domain inhibit DNA synthesis; overexpression increases cell proliferation.\",\n      \"method\": \"Ecdysone-inducible expression, microinjection, cell-permeable peptide mimetics (antennapedia-fused), DNA synthesis assay, cell number counts\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — four independent experimental strategies consistently supporting same conclusion, single lab\",\n      \"pmids\": [\"10454568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"GRB10 is phosphorylated on tyrosine (specifically Tyr-67) by Src family kinases (Src and Fyn) but not by the insulin receptor kinase itself; this Src/Fyn-mediated phosphorylation negatively regulates GRB10 binding to the insulin receptor.\",\n      \"method\": \"Pharmacological inhibition (herbimycin A), dominant-negative and constitutively active Src/Fyn expression, purified kinase in vitro assay, site-directed mutagenesis (Y67G), co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis plus multiple cell-based approaches, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"10871840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The BPS domain of GRB10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and IGF-1 receptor in vitro; inhibition depends on activation-loop phosphorylation but the BPS domain does not bind directly to phosphotyrosine.\",\n      \"method\": \"In vitro kinase assay with purified recombinant proteins, peptide competition experiments\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro kinase inhibition assay with purified components and peptide competition, mechanistically defined\",\n      \"pmids\": [\"11287005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GRB10 is a positive regulator of VEGF-R2 (KDR) signaling: overexpression of GRB10 increases KDR protein levels and tyrosine phosphorylation and activates MAP kinase; GRB10 undergoes VEGF-induced tyrosine phosphorylation partly through Src, requiring an intact SH2 domain; GRB10's positive effect on KDR is SH2-domain-independent.\",\n      \"method\": \"Transfection in HUVEC and 293 cells, co-immunoprecipitation, mutant expression, immunoblotting\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP and mutational analysis in relevant cell types, single lab\",\n      \"pmids\": [\"11494124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GRB10 inhibits the IRS/PI3K/Akt signaling pathway by physically blocking IRS-1/IRS-2 access to the insulin receptor; overexpression reduces insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation and Akt phosphorylation; yeast tri-hybrid studies show GRB10 SH2 domain is required for blocking IRS-IR association; GRB10 does not reduce IR catalytic activity toward activation loop and juxtamembrane tyrosines.\",\n      \"method\": \"Stable overexpression in CHO/IR cells and adipocytes, yeast tri-hybrid, RNAi knockdown, immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell systems plus yeast tri-hybrid, mechanism defined at SH2-domain level, replicated with RNAi\",\n      \"pmids\": [\"12493740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GRB10 forms a constitutive complex with Akt; overexpression of GRB10 and c-kit synergistically activates Akt in a wortmannin-sensitive, PI3K-activity-independent manner; both PH and SH2 domains of GRB10 are required for Akt activation; GRB10 can rescue deficient Akt activation by a c-kit mutant lacking the PI3K binding site.\",\n      \"method\": \"Yeast two-hybrid (c-kit as bait), co-immunoprecipitation, overexpression in Ba/F3 cells, kinase assay, IL-3-independence growth assay, domain deletion mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — constitutive complex by co-IP plus functional rescue experiment with domain mutants, single lab\",\n      \"pmids\": [\"11809791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"GRB10 forms a complex with the E3 ubiquitin ligase Nedd4 and the IGF-IR; GRB10 acts as adapter bringing Nedd4 to the IGF-IR, promoting ligand-dependent IGF-IR ubiquitination, increased internalization, and shortened receptor half-life via both proteasomal and lysosomal pathways; the GRB10 SH2 domain is required for this effect.\",\n      \"method\": \"Co-immunoprecipitation (triple complex), overexpression of catalytically inactive Nedd4 mutant, SH2 domain deletion mutant, pulse-chase half-life assay, proteasome/lysosome inhibitors, dansylcadaverine treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — mechanistic dissection with catalytically-dead enzyme mutant, domain deletion, and multiple degradation pathway inhibitors, replicated by later studies\",\n      \"pmids\": [\"12697834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"GRB10 N-terminus interacts with two novel proteins GIGYF1 and GIGYF2 via their GYF domains binding to tandem proline-rich regions in GRB10; IGF-I stimulation increases GIGYF1 binding to GRB10 and transient binding of both to IGF-IR; overexpression of GIGYF1-GRB10-binding fragment increases IGF-I-stimulated receptor tyrosine phosphorylation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, mutation analysis, overexpression in R+ fibroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by co-IP in cells, functional consequence shown, single lab\",\n      \"pmids\": [\"12771153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"GRB10 negatively regulates insulin-stimulated MAPK signaling by blocking Shc tyrosine phosphorylation; GRB10 overexpression reduces MAPK and Shc phosphorylation; the inhibitory effect requires the GRB10 SH2 domain; RNAi knockdown of GRB10 enhances MAPK, Shc, and Akt phosphorylation.\",\n      \"method\": \"Overexpression in CHO/IR cells and 3T3-L1 adipocytes, SH2 domain deletion, RNAi knockdown in HeLa/IR cells, immunoblotting\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — overexpression and RNAi with SH2 domain mutant converge on same mechanism, single lab\",\n      \"pmids\": [\"14615605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mouse GRB10 links the insulin receptor to p85 PI3-kinase directly (without involving IRS proteins), regulating PI3K activity and downstream metabolic insulin responses (glycogen synthesis, glucose/amino acid transport, lipogenesis, Akt/PKB, GSK, and glycogen synthase); dominant-negative GRB10 SH2 domain eliminates metabolic insulin responses in 3T3-L1 adipocytes.\",\n      \"method\": \"Co-immunoprecipitation of GRB10-p85 complex, dominant-negative domain peptides, metabolic assays in differentiated adipocytes and L6 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated plus multiple metabolic readouts with dominant-negative peptides, single lab\",\n      \"pmids\": [\"12783867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Crystal structure of the GRB10 SH2 domain at 1.65 Å reveals a non-covalent homodimer under physiologic conditions; the dimer interface involves residues flanking the C-terminal alpha helix conserved in the Grb7/10/14 family; structural features (Val-522, Asp-500) favor binding to dimeric phosphotyrosine sequences such as the insulin and IGF-1R activation loops.\",\n      \"method\": \"X-ray crystallography, analytical ultracentrifugation (solution dimerization), structural analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure at high resolution with biochemical validation of dimerization\",\n      \"pmids\": [\"12551896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"GRB10 disruption in mice (maternal allele) results in ~30% overgrowth of embryo and placenta by an IGF-2-independent mechanism; genetic epistasis with Igf2 mutation shows GRB10 acts on a distinct fetal growth axis.\",\n      \"method\": \"Gene-trap knockout, genetic cross with Igf2 mutant mice (epistasis), body weight measurements\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in defined knockout/double-mutant animals, replicated across multiple studies\",\n      \"pmids\": [\"12829789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"GRB10 constitutively associates with Nedd4 and prevents Nedd4-mediated degradation of VEGF-R2; Nedd4 overexpression causes VEGF-R2 disappearance, but co-expression with GRB10 restores VEGF-R2 levels; VEGF-R2 is ubiquitinated but Nedd4 is not the direct E3 ligase for VEGF-R2 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of Nedd4 and GRB10 in cells, ubiquitination assay, Nedd4 catalytic mutant (C854S), MG132 treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus catalytic mutant plus rescue experiment, single lab\",\n      \"pmids\": [\"15060076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RNAi knockdown of endogenous GRB10 enhances IGF-I-stimulated IRS phosphorylation, Akt/PKB, and ERK1/2, and increases DNA synthesis; GRB10 knockdown paradoxically decreases IGF-IR autophosphorylation, an effect partially reversed by phosphatase inhibitor pervanadate, suggesting GRB10 protects the activated receptor from phosphatases.\",\n      \"method\": \"siRNA knockdown, immunoblotting of downstream signaling, DNA synthesis assay, pervanadate pretreatment\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA with multiple downstream readouts and pharmacological test of mechanism, single lab\",\n      \"pmids\": [\"16037382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Transgenic overexpression of Meg1/Grb10 in mice causes postnatal growth retardation and hyperinsulinemic insulin resistance in vivo, confirming that GRB10 negatively regulates both IGF1R- and IR-dependent signaling pathways in vivo.\",\n      \"method\": \"Transgenic mouse lines (4 independent lines), glucose tolerance test, insulin tolerance test, body weight measurement\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — four independent transgenic lines with consistent phenotype, in vivo metabolic testing\",\n      \"pmids\": [\"15752742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Phosphorylation of GRB10 (at Ser-428) by Akt creates a binding site for 14-3-3 proteins; Akt directly binds GRB10 and phosphorylates it in vitro; only the phosphorylated form of GRB10 co-immunoprecipitates with endogenous 14-3-3.\",\n      \"method\": \"Yeast two-hybrid (14-3-3 as binding partner), co-immunoprecipitation, in vitro Akt kinase assay, site-directed mutagenesis (S428A)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis plus co-IP, identifies writer (Akt) and reader (14-3-3)\",\n      \"pmids\": [\"15722337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GRB10 mediates insulin-stimulated proteasomal degradation of the insulin receptor; suppression of GRB10 by RNAi leads to increased IR protein levels and reduced insulin-stimulated IR ubiquitination; overexpression reduces IR levels without affecting IR mRNA; IR reduction is blocked by MG132 but not chloroquine.\",\n      \"method\": \"RNAi knockdown, stable overexpression, qRT-PCR (mRNA unchanged), ubiquitination assay, proteasome/lysosome inhibitors\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional genetic manipulation plus pharmacological inhibition with ubiquitination assay, single lab\",\n      \"pmids\": [\"16434550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Peripheral (maternal allele) knockout of Grb10 in mice leads to enhanced insulin-stimulated Akt and MAPK phosphorylation in skeletal muscle and fat, and increased whole-body insulin sensitivity by hyperinsulinemic-euglycemic clamp, establishing GRB10 as an in vivo negative regulator of insulin signaling.\",\n      \"method\": \"Gene-trap knockout mice (maternal inheritance), hyperinsulinemic-euglycemic clamp, insulin-stimulated kinase phosphorylation in tissues\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with gold-standard in vivo clamp measurements, independently confirmed by other groups\",\n      \"pmids\": [\"17620412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Grb10-deficient (Grb10Δ2-4) mice show improved glucose tolerance and insulin sensitivity, and tissue-specific changes in IR tyrosine phosphorylation consistent with GRB10 blocking phosphatase access to IR activation loop; IRS-1 tyrosine phosphorylation is also enhanced, supporting attenuation of IR→IRS-1 signal transmission.\",\n      \"method\": \"Knockout mouse (Grb10Δ2-4), glucose and insulin tolerance tests, tissue IR phosphorylation analysis, IRS-1 phosphorylation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with mechanistic tissue-level signaling analysis, replicated across labs\",\n      \"pmids\": [\"17562854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"GRB10 and active Raf-1 promote Bad-dependent cell survival; both Grb10 and Raf-1 knockout cells show enhanced apoptosis in response to Bad; GRB10 requires its SH2, proline-rich, and PH domains plus Akt phosphorylation site (and consequent 14-3-3 binding) for anti-apoptotic function; Raf-1 requires its kinase activity and Ras-associated domain binding to GRB10 SH2.\",\n      \"method\": \"Knockout MEFs (Grb10 and Raf-1 deficient), siRNA, mutagenesis of GRB10 domains, signaling inhibitors, kinase assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple domain mutants in KO cells with convergent results, single lab\",\n      \"pmids\": [\"17535812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"GRB10 interacts with the Wnt co-receptor LRP6 intracellular domain and negatively regulates canonical Wnt signaling; GRB10 overexpression attenuates Wnt3a-induced β-catenin accumulation and TCF reporter activity; GRB10 interferes with Axin binding to LRP6; RNAi knockdown of GRB10 stimulates Wnt signaling.\",\n      \"method\": \"Co-immunoprecipitation of GRB10-LRP6, TCF reporter assays, RNAi knockdown, β-catenin accumulation assay, Axin binding competition assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus RNAi plus reporter assay and mechanistic Axin competition, single lab\",\n      \"pmids\": [\"17376403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The GRB10/Nedd4 complex mediates multiubiquitination (not polyubiquitination) of the IGF-IR upon ligand stimulation, which is required for receptor internalization; GRB10 and Nedd4 associate with IGF-IR in early endosomes and caveosomes but are not degraded and are directed to recycling endosomes.\",\n      \"method\": \"Ubiquitin chain analysis, clathrin-dependent and -independent internalization assays, confocal microscopy, subcellular fractionation, co-immunoprecipitation from endosomal fractions\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple trafficking assays plus subcellular fractionation and localization, single lab\",\n      \"pmids\": [\"18286479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of the Grb10 RA-PH tandem domain at 2.6 Å reveals an integrated dimeric structural unit (RA+PH+linker); biochemical studies show Grb14 (family member) binds activated Ras via its RA domain; these domains illuminate membrane-recruitment mechanisms shared with MIG-10, RIAM, lamellipodin, and Pico.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays for Ras interaction\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical validation, mechanistically defines domain architecture\",\n      \"pmids\": [\"19648926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the NEDD4 C2 domain – GRB10 SH2 domain complex at 2.0 Å shows three interaction interfaces, with the main interface being an antiparallel β-sheet; NEDD4 C2 binds at non-classical sites on the SH2 surface far from the phosphotyrosine pocket (phosphotyrosine-independent); GRB10 SH2 can simultaneously bind NEDD4 C2 and IGF-1R kinase domain.\",\n      \"method\": \"X-ray crystallography, structural analysis, binding interface characterization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure mechanistically explaining how GRB10 bridges NEDD4 and IGF-1R\",\n      \"pmids\": [\"20980250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"mTORC1-mediated phosphorylation of GRB10 stabilizes GRB10 protein, leading to feedback inhibition of both the PI3K and ERK-MAPK pathways; identified by large-scale quantitative phosphoproteomics as a direct mTORC1 substrate.\",\n      \"method\": \"Quantitative phosphoproteomics (large-scale), mTORC1 substrate characterization, rapamycin treatment, loss-of-function experiments\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — large-scale phosphoproteomics plus functional characterization, highly cited, replicated by others\",\n      \"pmids\": [\"21659605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Within the brain, Grb10 is expressed from the paternal allele; ablation of paternal Grb10 specifically increases social dominance behavior in mice; maternal allele ablation causes peripheral overgrowth—demonstrating tissue-specific allelic function controlling distinct physiological processes.\",\n      \"method\": \"Allele-specific knockout mice, behavioral testing (social dominance, allogrooming), body weight measurement\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — allele-specific knockout with specific behavioral and growth phenotypes, replicated across multiple behavioral paradigms\",\n      \"pmids\": [\"21270893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"GRB10 deletion in mice increases myofiber number (not fiber size) in skeletal muscle; the hypermuscular phenotype originates during embryonic development; Grb10-deficient neonatal muscle shows upregulated functional gene signatures for myogenic signaling and proliferation.\",\n      \"method\": \"Grb10 knockout mice, histomorphometry (fiber number and cross-sectional area), neonatal limb measurements, gene expression profiling\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockout with specific morphological and transcriptional phenotypic readouts, single lab\",\n      \"pmids\": [\"22623587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"GRB10 physically associates with FLT3 at phospho-tyrosines 572 and 793 in a ligand-dependent manner, and constitutively with oncogenic FLT3-ITD; GRB10 enhances FLT3-induced Akt phosphorylation by directly interacting with p85 PI3K subunit; GRB10 depletion reduces Akt phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, receptor phosphorylation site mapping (Y572F/Y793F mutants), p85 pulldown, siRNA knockdown, cell proliferation and colony formation assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with mutational mapping of binding site plus functional signaling and cell growth assays, single lab\",\n      \"pmids\": [\"23246379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"mTOR-mediated phosphorylation of GRB10 at Ser501/503 switches its binding preference from the insulin receptor to raptor (mTOR complex component), leading to raptor-mTOR dissociation and mTORC1 downregulation; fat-specific GRB10 disruption increases mTORC1 signaling, suppresses lipolysis, and reduces thermogenesis; these effects are abolished by rapamycin.\",\n      \"method\": \"mTORC1 substrate phosphorylation assay (Ser501/503), co-immunoprecipitation of GRB10-raptor complex, fat-specific Grb10 knockout mice, in vivo rapamycin treatment, lipolysis assay, thermogenic gene expression\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — phospho-site switching mechanism with co-IP, tissue-specific KO, and in vivo rescue with rapamycin; multiple orthogonal methods\",\n      \"pmids\": [\"24746805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GRB10 associates with IRS-2, NEDD4.2, IL-4Rα, and γc after IL-4 stimulation in macrophages; GRB10 knockdown enhances Tyr(P)-IRS-2 and promotes M2 macrophage gene expression (CD200R, CCL22, MMP12, TGM2); IL-4Rα and γc are ubiquitinated after IL-4 treatment, suggesting GRB10 regulates IL-4 receptor complex degradation through NEDD4.2.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, M2 gene expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP of multi-protein complex plus siRNA with gene expression readout, single lab\",\n      \"pmids\": [\"27742835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of the maternal allele of Grb10 in mice substantially increases hematopoietic stem cell (HSC) long-term repopulating capacity and accelerates HSC regeneration after irradiation; enhanced HSC regeneration is dependent on activation of the Akt/mTORC1 pathway; Grb10-deficient HSCs show increased proliferation with upregulation of CDK4 and Cyclin E.\",\n      \"method\": \"Maternal allele-specific Grb10 knockout mice, competitive transplantation, total body irradiation, flow cytometry, pathway inhibitor studies (Akt/mTORC1), cell cycle marker analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — allele-specific KO with competitive transplantation and pharmacological pathway validation, single lab\",\n      \"pmids\": [\"27806297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Ablation of Grb10 specifically in muscle (muscle-specific KO using α-skeletal actin-Cre) is sufficient to enlarge muscle fibers and increase insulin-stimulated glucose uptake and phospho-Akt in muscle, confirming a cell-autonomous role of GRB10 as modulator of proximal insulin receptor signaling in muscle.\",\n      \"method\": \"Muscle-specific conditional knockout (Cre-lox), hyperinsulinemic-euglycemic clamp, immunoblotting of insulin signaling\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific conditional KO with in vivo clamp measurement, cell-autonomous function established\",\n      \"pmids\": [\"29370381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRB10 expression is silenced in adult mouse liver but can be reactivated by acute ER stress (tunicamycin or short-term high-fat diet) via ATF4-mediated transcriptional induction; liver-specific GRB10 KO suppresses lipogenic gene expression and ER-stress-induced hepatosteatosis, establishing GRB10 as a mediator of acute ER stress-induced hepatic lipid dysregulation.\",\n      \"method\": \"Liver-specific GRB10 knockout, ER stress induction (tunicamycin, HFD), ATF4 ChIP/transcription analysis, lipogenic gene expression, hepatic lipid assays\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with mechanistic ATF4 transcriptional link, single lab\",\n      \"pmids\": [\"29555819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"mTORC1 phosphorylates GRB10 at Ser476 in human skeletal muscle cells; insulin and amino acids independently and additively stimulate this phosphorylation; mTORC1 controls PI3K/Akt signaling through GRB10-mediated modulation of insulin receptor protein abundance; rapamycin blocks Grb10 Ser476 phosphorylation and reduces GRB10 protein levels with corresponding increase in IR.\",\n      \"method\": \"Grb10 knockdown in primary human myotubes, rapamycin treatment, phospho-Ser476 immunoblotting, glucose uptake assay, IR protein quantification\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human primary cell knockdown with rapamycin pharmacology and defined phosphorylation site, single lab\",\n      \"pmids\": [\"31794259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GRB10 in hypothalamic AgRP and POMC neurons interacts with the leptin receptor and enhances leptin signaling; GRB10 ablation in AgRP neurons promotes weight gain while overexpression reduces body weight; GRB10 in AgRP neurons enhances leptin inhibition via ATP-sensitive K+ channels and in POMC neurons via transient receptor potential channels.\",\n      \"method\": \"Neuron-specific conditional knockout and overexpression (AAV-mediated), electrophysiology (patch-clamp), co-immunoprecipitation of GRB10-leptin receptor, body weight and food intake measurements\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — neuron-specific bidirectional manipulation plus electrophysiology plus co-IP, multiple cell-type specific phenotypes defined\",\n      \"pmids\": [\"36593271\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GRB10 is a multi-domain adapter protein (containing RA, PH, BPS, and SH2 domains) that functions primarily as a negative regulator of insulin and IGF-1 receptor signaling: its BPS domain acts as a pseudosubstrate inhibitor of the IR/IGF-1R kinase, its SH2 domain physically blocks IRS protein access to the receptor, and it recruits the E3 ubiquitin ligase NEDD4 to promote receptor ubiquitination and degradation; additionally, mTORC1 phosphorylates GRB10 (at Ser501/503 in mice, Ser476 in humans) to stabilize it and mediate feedback inhibition of both PI3K and MAPK pathways, while in adipose tissue this phosphorylation switches GRB10 binding from the insulin receptor to raptor to downregulate mTORC1, and in hypothalamic neurons GRB10 instead enhances leptin receptor signaling to promote energy homeostasis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GRB10 is a multi-domain adaptor protein that functions as a central negative regulator of receptor tyrosine kinase signaling, particularly insulin receptor (IR) and IGF-1 receptor (IGF-1R), integrating growth factor inputs with nutrient-sensing pathways to control growth, metabolism, and cell survival. Its BPS domain acts as a pseudosubstrate inhibitor of IR/IGF-1R kinase activity [PMID:11287005], while its SH2 domain physically blocks IRS protein access to activated receptors, thereby attenuating PI3K/Akt and MAPK signaling [PMID:12493740, PMID:14615605]; simultaneously, GRB10 recruits the E3 ubiquitin ligase NEDD4 to promote receptor multiubiquitination, internalization, and degradation [PMID:12697834, PMID:18286479]. mTORC1 phosphorylates GRB10 to stabilize it, creating a feedback loop that restrains PI3K and MAPK pathways; in adipose tissue, this phosphorylation switches GRB10 binding from IR to raptor, thereby downregulating mTORC1 itself [PMID:21659605, PMID:24746805]. In vivo, maternal-allele GRB10 loss causes fetal overgrowth and enhanced insulin sensitivity, while paternal-allele loss in brain increases social dominance behavior, and in hypothalamic neurons GRB10 enhances leptin receptor signaling to regulate energy homeostasis [PMID:12829789, PMID:21270893, PMID:36593271].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of GRB10 as a new SH2-containing adaptor protein that binds activated receptor tyrosine kinases established it as a candidate signaling intermediate downstream of growth factor receptors.\",\n      \"evidence\": \"Expression library screening with phosphorylated EGF receptor; yeast two-hybrid and GST pulldowns with RET receptor\",\n      \"pmids\": [\"7731717\", \"7665556\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence of GRB10–RTK interaction determined\", \"Signaling role (positive vs. negative) unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Mapping GRB10 SH2 domain binding to specific phosphotyrosines on the insulin receptor and IGF-1R defined the molecular basis of its receptor specificity and raised the question of whether GRB10 promotes or inhibits insulin/IGF signaling.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldowns, phosphopeptide binding (IR pY1322), microinjection of SH2 domain fusion proteins; multiple independent labs\",\n      \"pmids\": [\"8621530\", \"8798417\", \"8764099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Positive vs. negative signaling role debated\", \"BPS domain not yet discovered\", \"In vivo relevance unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Discovery of the BPS domain as a second receptor-binding module that cooperates with the SH2 domain revealed a bipartite receptor engagement mechanism unique to the Grb7/10/14 family.\",\n      \"evidence\": \"Domain deletion/mutagenesis with GST pulldown and yeast two-hybrid using mutant IR/IGF-1R (activation loop tyrosine mutants)\",\n      \"pmids\": [\"9506989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"BPS domain mechanism of action (pseudosubstrate inhibition) not yet demonstrated\", \"Structural basis undefined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstration that the BPS domain directly inhibits IR/IGF-1R kinase activity toward substrates in a reconstituted system established GRB10 as a bona fide kinase inhibitor, not merely a docking partner.\",\n      \"evidence\": \"In vitro kinase assay with purified recombinant BPS domain and IR/IGF-1R kinase domains, peptide competition\",\n      \"pmids\": [\"11287005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of BPS-mediated inhibition not yet shown\", \"Relative contribution of BPS vs. SH2 in intact cells unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Resolving the positive-vs-negative debate: GRB10 SH2 domain physically blocks IRS-1/IRS-2 access to the insulin receptor, attenuating PI3K/Akt signaling, while RNAi depletion enhances insulin signaling—establishing GRB10 as a negative regulator.\",\n      \"evidence\": \"Stable overexpression and RNAi knockdown in CHO/IR cells and adipocytes, yeast tri-hybrid showing competitive exclusion of IRS\",\n      \"pmids\": [\"12493740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation in whole animals pending\", \"Contribution to MAPK pathway inhibition not yet addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"GRB10 was shown to recruit the E3 ligase NEDD4 to IGF-1R, promoting receptor ubiquitination, internalization, and degradation—revealing a second inhibitory mechanism beyond kinase/substrate blockade.\",\n      \"evidence\": \"Triple-complex co-IP (GRB10–NEDD4–IGF-1R), catalytically inactive NEDD4 mutant, pulse-chase receptor half-life, proteasome/lysosome inhibitors\",\n      \"pmids\": [\"12697834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain topology on IGF-1R not characterized\", \"Whether NEDD4 recruitment also targets IR in vivo uncertain\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Crystal structures of the SH2 domain (homodimer at 1.65 Å) revealed how the Grb7/10/14 family recognizes dimeric phosphotyrosine motifs on the IR/IGF-1R activation loop, providing the first structural framework for GRB10-receptor interaction.\",\n      \"evidence\": \"X-ray crystallography, analytical ultracentrifugation\",\n      \"pmids\": [\"12551896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length GRB10 structure unavailable\", \"BPS domain structure not yet solved for GRB10\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Maternal-allele Grb10 knockout mice exhibited ~30% embryonic overgrowth, genetically establishing GRB10 as a maternally imprinted growth suppressor acting on a pathway distinct from IGF-2.\",\n      \"evidence\": \"Gene-trap knockout crossed with Igf2 mutant mice (epistasis analysis), body/placenta weight measurements\",\n      \"pmids\": [\"12829789\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific contributions to overgrowth not dissected\", \"Receptor target in placenta not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Whole-body insulin sensitivity measurements in Grb10-deficient mice using hyperinsulinemic-euglycemic clamps confirmed that GRB10 constrains insulin signaling in vivo, with enhanced Akt and MAPK phosphorylation in muscle and fat.\",\n      \"evidence\": \"Gene-trap knockout mice, hyperinsulinemic-euglycemic clamp, tissue-level signaling immunoblots\",\n      \"pmids\": [\"17620412\", \"17562854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-autonomous vs. systemic effects not separated\", \"Brain-specific functions of paternal allele not yet known\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The crystal structure of the NEDD4 C2–GRB10 SH2 complex showed that NEDD4 binds GRB10 at sites distinct from the phosphotyrosine pocket, explaining how GRB10 can simultaneously engage both NEDD4 and the receptor kinase domain to bridge them.\",\n      \"evidence\": \"X-ray crystallography at 2.0 Å resolution, structural interface analysis\",\n      \"pmids\": [\"20980250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full ternary complex structure (GRB10–NEDD4–receptor)\", \"Stoichiometry and dynamics in vivo not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of GRB10 as a direct mTORC1 substrate whose phosphorylation-dependent stabilization mediates feedback inhibition of both PI3K and MAPK pathways placed GRB10 at a critical node linking nutrient sensing to growth factor signaling.\",\n      \"evidence\": \"Large-scale quantitative phosphoproteomics, rapamycin treatment, loss-of-function experiments\",\n      \"pmids\": [\"21659605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylation sites in human GRB10 partially mapped\", \"Phosphatase(s) that dephosphorylate GRB10 unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that the paternal Grb10 allele is selectively expressed in brain and controls social dominance behavior revealed an unexpected imprinting-based functional partition: maternal allele governs peripheral growth, paternal allele governs behavior.\",\n      \"evidence\": \"Allele-specific knockout mice, behavioral testing panels (social dominance, allogrooming)\",\n      \"pmids\": [\"21270893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of GRB10 in social behavior unknown\", \"Receptor partners in brain neurons not identified at this time\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that mTOR-mediated phosphorylation switches GRB10 binding from insulin receptor to raptor, causing mTORC1 dissociation and downregulation in adipose tissue, revealed a phosphorylation-dependent partner-switching mechanism integrating GRB10 into bidirectional mTORC1 feedback.\",\n      \"evidence\": \"Phospho-site mutant analysis, co-IP of GRB10–raptor complex, fat-specific Grb10 KO mice, rapamycin rescue in vivo\",\n      \"pmids\": [\"24746805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether raptor-switching occurs in all tissues or is fat-specific\", \"Structural basis of phospho-dependent partner switch unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Muscle-specific GRB10 deletion confirmed cell-autonomous control of insulin-stimulated glucose uptake and fiber size, establishing that the metabolic and growth phenotypes of global knockouts originate in muscle itself.\",\n      \"evidence\": \"Muscle-specific conditional knockout (α-skeletal actin-Cre), hyperinsulinemic-euglycemic clamp, insulin signaling immunoblots\",\n      \"pmids\": [\"29370381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Developmental vs. postnatal contribution to muscle hypertrophy not fully resolved\", \"Whether BPS or SH2 domain is dominant in muscle unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"GRB10 was found to interact with the leptin receptor in hypothalamic neurons and enhance leptin signaling, broadening its role from a growth factor signaling inhibitor to a positive modulator of energy homeostasis in the CNS.\",\n      \"evidence\": \"Neuron-specific conditional KO and AAV-mediated overexpression in AgRP/POMC neurons, patch-clamp electrophysiology, GRB10–leptin receptor co-IP, body weight/food intake measurements\",\n      \"pmids\": [\"36593271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GRB10 enhances (rather than inhibits) leptin receptor signaling not fully defined\", \"Whether NEDD4 or BPS domain mechanisms are involved in leptin signaling unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structural model of GRB10 in complex with activated receptor and NEDD4 is lacking, and the molecular basis for context-dependent switching between positive and negative signaling roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length GRB10 structure\", \"Mechanism determining positive (leptin/VEGFR/c-Kit) vs. negative (IR/IGF-1R) signaling roles unknown\", \"Upstream signals controlling allele-specific expression in brain vs. periphery not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 4, 9, 19, 21, 38, 44]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [17, 19, 23, 31, 39, 43]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 4, 19, 23, 30, 39, 43, 49]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [17, 21, 31, 38]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [26, 40, 41]}\n    ],\n    \"complexes\": [\n      \"GRB10–NEDD4 complex\",\n      \"mTORC1 (via raptor interaction)\"\n    ],\n    \"partners\": [\n      \"NEDD4\",\n      \"INSR\",\n      \"IGF1R\",\n      \"RPTOR\",\n      \"LEPR\",\n      \"RAF1\",\n      \"GIGYF1\",\n      \"GIGYF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}