{"gene":"GRB14","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1996,"finding":"GRB14 was identified as a novel member of the GRB7 gene family containing a PH domain, a BPS/PIR region, and a C-terminal SH2 domain. The GST-Grb14 SH2 domain fusion protein exhibited strong binding to activated PDGF receptors in vitro, and Grb14 was phosphorylated on serine residues in a PDGF-regulated manner.","method":"cDNA library screening, GST pulldown, co-immunoprecipitation, Western blot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — original cloning paper with in vitro binding assays and phosphorylation studies, single lab","pmids":["8647858"],"is_preprint":false},{"year":1998,"finding":"Rat Grb14 binds to the insulin receptor (IR) in an insulin-dependent manner via two domains: the SH2 domain and a region between PH and SH2 domains (PIR/BPS), with PIR being the major binding domain. The interaction requires IR tyrosines Y1150/Y1151. Grb14 overexpression decreases insulin-stimulated IRS-1 tyrosine phosphorylation, DNA synthesis, and glycogen synthesis without altering IR autophosphorylation.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding with deletion mutants, cell-based overexpression assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods identifying binding domains and functional consequences, replicated in subsequent studies","pmids":["9748281"],"is_preprint":false},{"year":2000,"finding":"Grb14 binds activated FGFR1 in a kinase-activity-dependent manner, requiring the FGFR1 C-tail tyrosines and juxtamembrane domain. The SH2 domain of Grb14 mediates binding but regions upstream of SH2 confer specificity for FGFR1 over PDGFR. Grb14 overexpression inhibits FGF-2-induced cell proliferation, whereas an SH2-binding-deficient mutant enhances it.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, cell proliferation assay, domain deletion analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, in vitro binding, functional gain/loss-of-function with mutant rescue, single lab but multiple orthogonal methods","pmids":["10713090"],"is_preprint":false},{"year":2001,"finding":"Tankyrase 2, a poly(ADP-ribose) polymerase, was identified as a binding partner of Grb14 via yeast two-hybrid screening. The interaction is mediated by the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10-19 of tankyrase 2. Both proteins co-localize in the low-density microsome fraction and co-immunoprecipitate in vivo.","method":"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation, deletion mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP and fractionation with domain mapping, single lab","pmids":["11278563"],"is_preprint":false},{"year":2001,"finding":"Grb14 directly inhibits IR substrate phosphorylation in vitro, acting as an uncompetitive inhibitor for IR substrate without altering Km for ATP. The PIR/BPS domain alone mediates this inhibitory effect. Grb14 binding protects IR kinase-loop phosphotyrosines from phosphatases but delays downstream Akt and ERK1/2 activation.","method":"In vitro kinase assay, domain deletion analysis, CHO cell overexpression, Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with domain mapping and mechanistic follow-up in cells, replicated across labs","pmids":["11726652"],"is_preprint":false},{"year":2002,"finding":"ZIP (PKCζ interacting protein) binds Grb14 through its ZZ zinc finger domain interacting with the PIR region of Grb14, forming a PKCζ-ZIP-Grb14 heterotrimeric complex. PKCζ phosphorylates Grb14 in vitro and in vivo, and this phosphorylation increases Grb14's inhibitory effect on IR tyrosine kinase activity. In Xenopus oocytes, ZIP potentiates Grb14-mediated inhibition of insulin-induced maturation in a PKCζ- and phosphorylation-dependent manner.","method":"Co-immunoprecipitation, in vitro kinase assay, in vivo radiolabeling, Xenopus oocyte functional assay, domain deletion mapping","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay, in vivo phosphorylation, functional epistasis in Xenopus model, multiple orthogonal methods","pmids":["12242277"],"is_preprint":false},{"year":2003,"finding":"The PIR domain of Grb14 is an intrinsically unstructured protein by NMR analysis, yet retains biological activity as demonstrated by inhibition of insulin-induced meiosis reinitiation in Xenopus oocytes.","method":"NMR spectroscopy, Xenopus oocyte functional assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 — NMR structure determination with functional validation in oocyte model, single lab","pmids":["14623073"],"is_preprint":false},{"year":2003,"finding":"Grb14 (but not Grb10) inhibits FGF receptor-induced maturation in Xenopus oocytes by binding to the receptor and inhibiting the Ras-dependent pathway. The PIR and SH2 domains of Grb14 have different roles in FGFR signaling inhibition compared to its paralogs.","method":"Xenopus oocyte maturation assay, co-immunoprecipitation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — functional oocyte assay with biochemical co-IP, single lab","pmids":["12885405"],"is_preprint":false},{"year":2004,"finding":"Grb14 knockout mice display improved glucose tolerance, lower circulating insulin, enhanced glycogen synthesis in liver and skeletal muscle, and increased insulin-induced IRS-1 and PKB phosphorylation in a tissue-specific manner (liver and skeletal muscle, but not adipose), demonstrating that Grb14 is a tissue-specific negative regulator of insulin signaling in vivo.","method":"Gene knockout mouse model, glucose tolerance test, insulin signaling assays (phosphorylation of IR, IRS-1, PKB), ex vivo glucose uptake","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with multiple defined molecular and metabolic phenotypes, replicated in subsequent studies","pmids":["14749734"],"is_preprint":false},{"year":2004,"finding":"The PIR domain of Grb14 is natively unfolded in solution (SAXS and CD analyses), with a potentially structured short stretch around residues 399-407 that may undergo structural transition upon binding to a partner.","method":"Small-angle X-ray scattering (SAXS), circular dichroism, NMR","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1 — multiple biophysical methods, single lab","pmids":["15465854"],"is_preprint":false},{"year":2004,"finding":"Grb14 binds PDK-1 constitutively through a PDK-1 binding motif on Grb14 (distinct from SH2 domain). This interaction facilitates insulin-triggered membrane translocation of PDK-1 to activated IR and promotes Akt phosphorylation. Disruption of the Grb14-PDK-1 interaction significantly decreases insulin-dependent Akt activation.","method":"Directed proteomics, co-immunoprecipitation, membrane translocation assay, dominant-negative overexpression, Akt activation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, point mutant disruption, functional consequence on Akt, moderate evidence","pmids":["15210700"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of the Grb14 BPS region in complex with the insulin receptor tyrosine kinase domain reveals that the N-terminal portion of BPS acts as a pseudosubstrate inhibitor binding in the substrate peptide-binding groove of the kinase. Together with the SH2 domain crystal structure, a model for dual-domain engagement of IR by Grb14 is presented.","method":"X-ray crystallography (crystal structure of BPS-IR kinase domain complex and SH2 domain)","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional mechanistic model, highly cited foundational structural study","pmids":["16246733"],"is_preprint":false},{"year":2005,"finding":"Grb14 overexpression inhibits insulin- and estrogen-induced cell cycle progression in MCF-7 breast cancer cells, associated with decreased ERK1/2 activation. Estradiol represses insulin-induced Grb14 expression, while anti-estrogen ICI 182780 increases Grb14 levels.","method":"Cell overexpression, cell cycle analysis, ERK1/2 phosphorylation assay, hormonal treatment","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — overexpression with defined signaling and cell cycle readouts, single lab","pmids":["15372466"],"is_preprint":false},{"year":2005,"finding":"Grb14 contains an FXNPXY motif, and the PTB domain of IRS-1 binds to this NPXY motif of Grb14 in a phosphorylation-independent manner. Grb14-IRS-1 complexes are detected in retinal tissue lysates, suggesting Grb14 acts as a dominant negative for IRS-1 functions in the retina.","method":"Yeast two-hybrid (retinal cDNA library screen), GST pulldown, co-immunoprecipitation from retinal tissue","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vitro binding assay and tissue co-IP, single lab","pmids":["15924411"],"is_preprint":false},{"year":2006,"finding":"Real-time BRET analysis in living HEK cells shows that Grb14 is rapidly and dose-dependently recruited to the activated IR upon insulin stimulation. Grb14 reduces IR-PTP1B interaction (BRET), protects kinase-loop tyrosines from PTP1B dephosphorylation, while promoting dephosphorylation of Y972, thereby decreasing IRS-1 binding to IR and attenuating ERK pathway activation. Insulin also induces Grb14 dimerization.","method":"BRET (bioluminescence resonance energy transfer) in living cells, co-immunoprecipitation, site-specific phosphotyrosine antibodies","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — real-time BRET, co-IP, and site-specific phospho-antibodies providing mechanistic detail on PTP1B regulation, multiple orthogonal methods","pmids":["16582879","16934761"],"is_preprint":false},{"year":2008,"finding":"In rat liver, Grb14 localizes predominantly to microsomal and cytosolic fractions at baseline and translocates to the plasma membrane and Golgi/endosome fractions upon insulin stimulation, co-localizing with phosphorylated IR. KCl removal of Grb14 from fractions increases IR tyrosine kinase activity, demonstrating endogenous Grb14 exerts negative feedback on liver IR activity in vivo.","method":"Subcellular fractionation, co-immunoprecipitation, in vitro phosphatase assay, KCl extraction, in vivo rat liver model","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 — subcellular fractionation with functional consequence (KCl stripping assay), in vivo rat model, multiple methods","pmids":["18657188"],"is_preprint":false},{"year":2008,"finding":"In primary hepatocytes with Grb14 knockdown, insulin-induced Akt phosphorylation and inhibition of gluconeogenesis are increased, but insulin-stimulated SREBP-1c maturation is completely blunted, blocking lipogenic gene expression and glycogen synthesis. This reveals a dual role for Grb14 in liver: inhibiting IR kinase activity and promoting a distal step (SREBP-1c maturation) required for lipogenic insulin action.","method":"RNAi knockdown in primary mouse hepatocytes, insulin signaling assays (Akt, GSK3, FOXO1 phosphorylation), gene expression analysis, glucose production measurement","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 — clean RNAi KD with multiple molecular readouts revealing dual mechanistic role, single lab but multiple orthogonal assays","pmids":["18339716"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of the Grb14 RA and PH domains at 2.4-Å resolution shows these domains form an integrated structural unit. Biochemical studies demonstrate that Grb14 binds to activated Ras (GTP-loaded), which may serve as a timing mechanism for downregulation of insulin signaling. The RA-PH unit can simultaneously engage small GTPases and phosphoinositide lipids.","method":"X-ray crystallography (RA-PH structure in complex with H-Ras G12V at 2.4-Å), biochemical binding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical validation, published in high-impact journal","pmids":["19648926"],"is_preprint":false},{"year":2009,"finding":"Dual knockout of Grb10 and Grb14 in mice does not further increase IRS-1 phosphorylation and Akt activation beyond single knockouts, suggesting context-dependent limiting mechanisms including IR hypophosphorylation and decreased IRS-1 expression. Double KO mice are protected from high-fat diet-induced glucose intolerance whereas single KOs are not, suggesting additional glucose homeostasis regulatory mechanisms beyond IRS-1/Akt.","method":"Compound gene knockout mice, glucose tolerance test, insulin signaling assays, high-fat diet challenge","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis using double KO mice with defined molecular phenotypes, clean experimental design","pmids":["19541746"],"is_preprint":false},{"year":2009,"finding":"Molecular determinants of Grb14-IR interaction include Grb14-L404 interacting with IR-L1038 (IR αC-helix) and Grb14-R385 interacting with IR-K1168 (activation loop), with L404 likely contributing to specificity for IR over other Grb7 family members. Grb14 S370 phosphorylation status controls its biological activity, and insulin-induced Grb14-PDK1 interaction is required for maximal inhibition of insulin signaling.","method":"Site-directed mutagenesis, co-immunoprecipitation, BRET, Xenopus oocyte assay, MEF functional complementation assay","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis with multiple functional readouts including structural contacts and signaling outcomes, multiple orthogonal methods","pmids":["19359342"],"is_preprint":false},{"year":2010,"finding":"Grb14 inhibits FGFR signaling by being recruited to FGFR1 in a trimeric complex containing PLCγ. Grb14 binds to FGFR1 pY766 (the PLCγ docking site) via its SH2 domain, competitively trapping and inactivating PLCγ, and altering FGF-induced PLCγ phosphorylation and activation.","method":"BRET in living cells, Xenopus oocyte maturation assay, co-immunoprecipitation, Western blot for PLCγ phosphorylation","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — BRET, co-IP, and functional oocyte assay, with mechanistic model involving competitive binding at Y766, multiple methods","pmids":["20932831"],"is_preprint":false},{"year":2010,"finding":"Tie2 receptor tyrosine kinase phosphorylates Grb14 on tyrosine residues in a manner requiring kinase-competent Tie2 and intact Tie2 tyrosines Y1100 and Y1106. A complete Grb14 SH2 domain is required for this phosphorylation. Grb14 tyrosine phosphorylation also occurs in primary endothelial cells treated with COMP-Ang1.","method":"Co-immunoprecipitation, Western blot with phosphotyrosine antibodies, kinase assay, dominant-negative Tie2 mutant, primary endothelial cells","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP with kinase-dead controls and primary cell validation, single lab","pmids":["20973951"],"is_preprint":false},{"year":2011,"finding":"Phosphorylation of Tyr-347 in the BPS domain of Grb14 (by Src kinase, activated downstream of rhodopsin) enables Grb14 to competitively inhibit PTP1B activity. Ablation of Grb14 in mouse retina results in significantly elevated PTP1B activity in vivo, demonstrating that phosphorylated Grb14 is an endogenous inhibitor of retinal PTP1B.","method":"In vitro PTP1B activity assay, site-directed mutagenesis (Y347), Grb14 KO mouse retina assay, Src kinase activity measurement","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro enzyme assay, mutagenesis of critical tyrosine, in vivo KO validation, multiple methods","pmids":["21791607"],"is_preprint":false},{"year":2011,"finding":"In thyroid cancer cells, Grb14 knockdown reduces RET receptor phosphorylation and downstream Akt and STAT3 activation while facilitating IR signaling, whereas Grb14 overexpression facilitates RET activation and STAT3/Akt phosphorylation. This reveals a novel role for Grb14 in promoting RET-mediated thyroid cancer progression.","method":"Stable shRNA knockdown, overexpression, in vitro proliferation/invasion assays, in vivo xenograft mouse model, phosphorylation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function with defined molecular readouts in vitro and in vivo, single lab","pmids":["22158039"],"is_preprint":false},{"year":2011,"finding":"Grb14 RA domain mediates closure of the photoreceptor-specific CNG channel (CNGA1) through electrostatic interactions; three Glu residues (180-182) in Grb14 interact electrostatically with Arg559 in the cGMP binding pocket of CNGA1, and Lys140 of Grb14 also contributes. Grb14 competes with cGMP for the CNGA1 binding pocket. This activity is specific to Grb14 and not shared by Grb7 or Grb10.","method":"In vitro binding assay, site-directed mutagenesis of electrostatic residues, channel activity assay","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 1-2 — mutagenesis identifying specific electrostatic contacts with functional channel assay, single lab","pmids":["22180090"],"is_preprint":false},{"year":2012,"finding":"Grb14 SH2 domain binds the ITAM-like sequence in the cytoplasmic domain of CEACAM3 (identified by SH2 domain microarray). Grb14 is rapidly recruited to sites of bacteria-host cell contact, and both Grb14 knockdown and overexpression indicate an inhibitory role for Grb14 in CEACAM3-mediated bacterial phagocytosis.","method":"SH2 domain microarray, co-immunoprecipitation, FRET-FLIM in intact cells, RNAi knockdown, overexpression, phagocytosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including FRET-FLIM, reciprocal gain/loss-of-function, SH2 microarray identification","pmids":["22948154"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of Grb14 RA-PH domains in complex with GTP-loaded H-Ras(G12V) at 2.4-Å resolution reveals the integrated RA-PH structural unit binds simultaneously to activated Ras and phosphoinositide lipids. Binding mode of Grb14 RA domain to H-Ras is similar to RalGDS and Raf1 RA domains but with distinct features.","method":"X-ray crystallography (RA-PH domains in complex with H-Ras G12V)","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure providing detailed molecular basis for Ras binding","pmids":["23967305"],"is_preprint":false},{"year":2014,"finding":"GSK-3 phosphorylates Ser358, Ser362, and Ser366 in the BPS domain of Grb14 (requiring a priming phospho-Ser370), and this phosphorylation suppresses Grb14-IR complex formation. Pharmacological inhibition or knockdown of GSK-3 facilitates Grb14-IR binding, and Ala substitutions at these serines enhance Grb14-IR association.","method":"In vitro kinase assay with phosphopeptides, proximity ligation assay, pharmacological GSK-3 inhibition, siRNA knockdown, co-immunoprecipitation","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro kinase assay with mutagenesis and cell-based co-IP validation, single lab","pmids":["24535599"],"is_preprint":false},{"year":2015,"finding":"The ubiquitin ligase Chfr binds to the T220 residue of Grb14 in an insulin-stimulated manner and potentiates Grb14's inhibitory effect on insulin-induced cell division. Chfr ligase activity and phosphorylation of its T39 residue (an Akt substrate) are required for this function. Insulin-stimulated Chfr binding to Grb14 activates Chfr ligase activity, leading to Aurora A and Polo-like kinase degradation, blocking cell division.","method":"Co-immunoprecipitation, Xenopus oocyte G2/M transition assay, targeted mutagenesis, mammalian cell line overexpression/knockdown","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 — co-IP, mutagenesis, functional oocyte assay, multiple orthogonal methods establishing pathway","pmids":["25578860"],"is_preprint":false},{"year":2016,"finding":"Liver-specific Grb14 knockdown in mice enhances insulin signaling but also releases p62/SQSTM1 (a Grb14 binding partner), which activates the Nrf2 transcription factor, which in turn represses LXR and thereby inhibits de novo lipogenesis. This reveals Grb14 as a signaling node at the crossroads of the IR and p62-Nrf2-LXR pathways regulating both glucose homeostasis and hepatic lipid synthesis.","method":"In vivo siRNA/shRNA liver knockdown, mouse metabolic phenotyping, co-immunoprecipitation (Grb14-p62), gene expression analysis, Nrf2/LXR reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo knockdown, co-IP establishing binding, multiple pathway readouts in insulin-resistant mouse models","pmids":["27215388"],"is_preprint":false},{"year":2017,"finding":"GSK-3-mediated phosphorylation of N-terminal BPS domain serines (Ser358, Ser362, Ser366) in human Grb14 negatively regulates Grb14-IR complex formation; surface plasmon resonance shows Kd of 8 nM for Grb14-IR, which is abolished by Glu substitutions at these serines. The BPS domain alone does not show this differential, indicating additional domain contributions.","method":"In vitro kinase assay, co-immunoprecipitation, yeast two-hybrid, surface plasmon resonance","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 — SPR quantitative binding, yeast two-hybrid, in vitro kinase, single lab confirming prior findings","pmids":["28130417"],"is_preprint":false},{"year":2019,"finding":"Protein phosphatase 1 (PP1) dephosphorylates Ser358 and Ser362 in the N-terminal BPS domain of human Grb14, and this dephosphorylation facilitates Grb14-IR complex formation as demonstrated by co-immunoprecipitation with phosphomimetic substitutions.","method":"In vitro phosphatase assay with synthetic phosphopeptides, co-immunoprecipitation","journal":"Journal of peptide science","confidence":"Medium","confidence_rationale":"Tier 1-3 — in vitro phosphatase assay with cell-based co-IP, single lab extending prior GSK-3 phosphorylation findings","pmids":["31347216"],"is_preprint":false}],"current_model":"GRB14 is a multidomain adaptor protein (containing RA, PH, BPS/PIR, and SH2 domains) that functions primarily as a tissue-specific negative regulator of insulin receptor (IR) signaling: the BPS/PIR region acts as a pseudosubstrate inhibitor in the IR kinase substrate-binding groove, the SH2 domain docks to IR phosphotyrosines, and together these interactions inhibit IR-mediated IRS-1 phosphorylation and downstream Akt/ERK activation; GRB14 also recruits PDK-1 to the IR to modulate Akt signaling, interacts with p62/SQSTM1 to regulate the Nrf2-LXR lipogenic pathway, is phosphorylated by PKCζ (via ZIP adaptor) and Src kinase (enabling PTP1B inhibition) and regulated by GSK-3/PP1 at BPS domain serines, binds activated Ras through its RA domain, inhibits FGFR signaling by competing with PLCγ at pY766, inhibits CEACAM3-mediated phagocytosis via its SH2 domain, and collaborates with Chfr ubiquitin ligase to suppress insulin-induced cell division."},"narrative":{"teleology":[{"year":1996,"claim":"Identification of GRB14 as a new GRB7-family adaptor with PH, BPS, and SH2 domains that binds activated PDGF receptors established it as a candidate RTK-signaling adaptor.","evidence":"cDNA library cloning, GST-SH2 pulldown with PDGFR, serine phosphorylation analysis","pmids":["8647858"],"confidence":"Medium","gaps":["No endogenous function demonstrated","PDGFR binding not confirmed by reciprocal IP","In vivo relevance unknown"]},{"year":1998,"claim":"Demonstration that Grb14 binds the insulin receptor in an insulin-dependent manner via two domains (PIR/BPS and SH2) and that overexpression suppresses IRS-1 phosphorylation, DNA synthesis, and glycogen synthesis established GRB14 as a negative regulator of insulin signaling.","evidence":"Yeast two-hybrid, co-IP with IR deletion/point mutants (Y1150/Y1151), overexpression in CHO cells","pmids":["9748281"],"confidence":"High","gaps":["Mechanism of inhibition (competitive vs. pseudosubstrate) unresolved","In vivo relevance not tested","Contribution of individual domains not fully dissected"]},{"year":2000,"claim":"Extending the adaptor's scope beyond IR, binding to activated FGFR1 and inhibition of FGF-induced proliferation revealed GRB14 as a broader RTK-signaling inhibitor.","evidence":"Yeast two-hybrid, co-IP, domain deletion analysis, cell proliferation assay in FGFR1-expressing cells","pmids":["10713090"],"confidence":"High","gaps":["Mechanism of FGFR inhibition not identified","Relative contribution of SH2 vs. upstream regions unclear"]},{"year":2001,"claim":"In vitro kinase assays showed the BPS/PIR domain alone inhibits IR kinase activity toward substrates without altering ATP Km, establishing an uncompetitive pseudosubstrate inhibition mechanism and linking this to delayed Akt/ERK activation in cells.","evidence":"In vitro IR kinase assay with purified BPS domain, CHO cell overexpression, phospho-specific Western blots","pmids":["11726652"],"confidence":"High","gaps":["Structural basis of pseudosubstrate mechanism not yet solved","In vivo significance not demonstrated"]},{"year":2002,"claim":"Discovery of the PKCζ–ZIP–Grb14 trimeric complex and PKCζ-dependent phosphorylation of Grb14 that enhances its IR-inhibitory activity identified a regulatory input controlling GRB14 potency.","evidence":"Co-IP, in vitro/in vivo kinase assay, Xenopus oocyte insulin-maturation assay with epistasis analysis","pmids":["12242277"],"confidence":"High","gaps":["Phosphorylation sites on Grb14 not mapped","Relevance in mammalian metabolic tissues not tested"]},{"year":2004,"claim":"Grb14 knockout mice showed improved glucose tolerance, lower insulin levels, and enhanced IRS-1/Akt signaling specifically in liver and muscle but not adipose, providing the first in vivo genetic evidence that GRB14 is a tissue-specific physiological brake on insulin action.","evidence":"Grb14−/− mouse model with glucose tolerance tests, insulin signaling assays, ex vivo glucose uptake","pmids":["14749734"],"confidence":"High","gaps":["Molecular basis for tissue specificity unclear","Compensation by Grb10 not addressed","Lipogenic consequences not explored"]},{"year":2004,"claim":"Identification of constitutive Grb14–PDK-1 binding through a distinct motif, which recruits PDK-1 to the IR and is required for full insulin-stimulated Akt activation, revealed a paradoxical positive-signaling role for an otherwise inhibitory adaptor.","evidence":"Co-IP, membrane translocation assay, dominant-negative disruption, Akt phosphorylation readout","pmids":["15210700"],"confidence":"High","gaps":["Relative contribution of positive (PDK-1 recruitment) vs. negative (pseudosubstrate) roles not quantified","PDK-1 binding site not structurally characterized"]},{"year":2005,"claim":"The crystal structure of the BPS region bound to the IR kinase domain at atomic resolution confirmed the pseudosubstrate model: the BPS N-terminus occupies the substrate-binding groove, providing the structural basis for GRB14's inhibitory mechanism.","evidence":"X-ray crystallography of BPS–IR kinase complex and SH2 domain","pmids":["16246733"],"confidence":"High","gaps":["Full-length Grb14–IR complex structure not available","Dynamics of engagement/disengagement not addressed"]},{"year":2006,"claim":"Real-time BRET in living cells showed that Grb14 recruitment to IR is rapid and dose-dependent, that Grb14 reduces IR–PTP1B interaction while selectively promoting dephosphorylation of Y972 (the IRS-1 docking site) and protecting activation-loop phosphotyrosines, mechanistically explaining site-selective signal attenuation.","evidence":"BRET donor/acceptor pairs in HEK cells, site-specific phospho-antibodies, co-IP","pmids":["16582879","16934761"],"confidence":"High","gaps":["Insulin-induced Grb14 dimerization function unclear","Whether PTP1B modulation occurs in all IR-expressing tissues unknown"]},{"year":2008,"claim":"Hepatocyte Grb14 knockdown revealed a dual role: while enhancing proximal IR signaling, it abolished SREBP-1c maturation and lipogenic gene expression, showing GRB14 promotes a distal lipogenic step independently of its kinase-inhibitory function.","evidence":"siRNA knockdown in primary mouse hepatocytes with Akt/FOXO1 phosphorylation, SREBP-1c processing, and gluconeogenesis assays","pmids":["18339716"],"confidence":"High","gaps":["Mechanism linking Grb14 to SREBP-1c maturation not identified","Whether this is direct or via intermediary pathway unknown"]},{"year":2009,"claim":"Crystal structure of the RA-PH integrated unit bound to GTP-loaded H-Ras established that GRB14 directly engages activated Ras, potentially serving as a timing mechanism linking Ras activity to insulin-signaling downregulation.","evidence":"X-ray crystallography at 2.4 Å, biochemical Ras-binding assays","pmids":["19648926"],"confidence":"High","gaps":["Functional consequence of Ras binding on IR inhibition not tested in cells","Whether Ras binding recruits Grb14 to specific membrane microdomains unknown"]},{"year":2010,"claim":"Demonstration that Grb14 SH2 domain binds FGFR1 pY766 (the PLCγ docking site) in a trimeric complex, competitively trapping and inactivating PLCγ, provided the mechanism for GRB14's inhibition of FGFR signaling.","evidence":"BRET, co-IP, PLCγ phosphorylation Western blot, Xenopus oocyte maturation assay","pmids":["20932831"],"confidence":"High","gaps":["In vivo physiological relevance for FGFR regulation not demonstrated","Whether GRB14 inhibits other FGFR family members unknown"]},{"year":2011,"claim":"Src-mediated phosphorylation of BPS Tyr-347 converts GRB14 into an endogenous PTP1B inhibitor, as shown by elevated retinal PTP1B activity in Grb14 KO mice; this added a gain-of-function dimension to the adaptor.","evidence":"In vitro PTP1B assay, Y347 mutagenesis, Grb14 KO mouse retina, Src kinase activity measurement","pmids":["21791607"],"confidence":"High","gaps":["Whether pY347-mediated PTP1B inhibition occurs outside the retina not tested","Structural basis of pY347–PTP1B interaction unknown"]},{"year":2012,"claim":"SH2 domain microarray and functional assays identified CEACAM3 as a GRB14 binding partner, with Grb14 rapidly recruited to bacteria-host contact sites to inhibit phagocytosis, extending GRB14 function to innate immunity.","evidence":"SH2 microarray, FRET-FLIM, RNAi knockdown and overexpression, phagocytosis assay","pmids":["22948154"],"confidence":"High","gaps":["Mechanism of phagocytosis inhibition beyond SH2 binding not resolved","In vivo infection model not tested"]},{"year":2014,"claim":"Identification of GSK-3 as a kinase that phosphorylates BPS serines 358/362/366 to disrupt Grb14–IR complex formation, with PP1 serving as the opposing phosphatase, established a phosphorylation toggle controlling GRB14's access to its primary target.","evidence":"In vitro kinase/phosphatase assays, Ala/Glu substitutions, co-IP, SPR (Kd ~8 nM abolished by phosphomimetics)","pmids":["24535599","28130417","31347216"],"confidence":"Medium","gaps":["Physiological contexts triggering GSK-3/PP1 regulation of Grb14 not defined","Whether priming at S370 is constitutive or regulated unknown","Single-lab findings"]},{"year":2015,"claim":"Identification of the Chfr ubiquitin ligase as an insulin-stimulated Grb14 partner that potentiates cell-cycle arrest by degrading Aurora A and Plk established a pathway by which GRB14 links insulin signaling to mitotic checkpoint control.","evidence":"Co-IP, Xenopus G2/M oocyte assay, mutagenesis of Chfr T39 and Grb14 T220, mammalian cell knockdown/overexpression","pmids":["25578860"],"confidence":"High","gaps":["In vivo relevance for tumor suppression not shown","Whether Chfr–Grb14 interaction is direct or bridged unclear"]},{"year":2016,"claim":"Liver-specific Grb14 knockdown revealed that GRB14 sequesters p62/SQSTM1, and its loss releases p62 to activate Nrf2, which represses LXR-driven lipogenesis, resolving the molecular link between GRB14 and hepatic de novo lipogenesis.","evidence":"In vivo hepatic siRNA/shRNA, co-IP of Grb14–p62, Nrf2/LXR reporter assays, metabolic phenotyping in insulin-resistant mice","pmids":["27215388"],"confidence":"High","gaps":["Direct binding interface between Grb14 and p62 not mapped","Interaction of this pathway with SREBP-1c maturation defect from 2008 study not reconciled"]},{"year":null,"claim":"A full-length structural model of GRB14 bound to the intact IR, the precise mechanism linking GRB14 to SREBP-1c maturation, the physiological significance of Ras binding via the RA domain in metabolic tissues, and the in vivo relevance of GRB14 in innate immune phagocytosis remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length Grb14–IR structure","SREBP-1c connection mechanistically unexplained","Ras–RA domain function not tested in metabolic models","CEACAM3 inhibition not validated in infection models"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,4,10,11,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,11,22,20]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[17,26]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[15]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[15]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,8,10,11,14,16,22,23]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8,16,29]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[12,28]}],"complexes":[],"partners":["INSR","FGFR1","PDPK1","SQSTM1","PLCG1","CHFR","PRKCZ","TNKS2"],"other_free_text":[]},"mechanistic_narrative":"GRB14 is a multidomain adaptor protein that functions as a tissue-specific negative regulator of receptor tyrosine kinase signaling, most prominently insulin receptor (IR) signaling. Its BPS/PIR domain acts as a pseudosubstrate inhibitor occupying the IR kinase substrate-binding groove, while its SH2 domain docks to IR activation-loop phosphotyrosines; together these interactions suppress IRS-1 phosphorylation and downstream Akt/ERK activation, as demonstrated by crystal structures, in vitro kinase assays, and improved glucose homeostasis in Grb14 knockout mice [PMID:16246733, PMID:11726652, PMID:14749734]. Beyond IR, GRB14 inhibits FGFR1 signaling by competitively displacing PLCγ from pY766 via its SH2 domain [PMID:20932831], inhibits CEACAM3-mediated phagocytosis [PMID:22948154], and when phosphorylated on Tyr-347 by Src acts as an endogenous inhibitor of PTP1B in the retina [PMID:21791607]. GRB14 activity is regulated by PKCζ-mediated phosphorylation (enhanced IR inhibition) [PMID:12242277], GSK-3 phosphorylation of BPS-domain serines (which opposes IR binding and is reversed by PP1) [PMID:24535599, PMID:31347216], binding to activated Ras through its integrated RA-PH structural unit [PMID:19648926], and interaction with p62/SQSTM1 to modulate the Nrf2–LXR lipogenic pathway in liver [PMID:27215388]."},"prefetch_data":{"uniprot":{"accession":"Q14449","full_name":"Growth factor receptor-bound protein 14","aliases":["GRB14 adapter protein"],"length_aa":540,"mass_kda":61.0,"function":"Adapter protein which modulates coupling of cell surface receptor kinases with specific signaling pathways. Binds to, and suppresses signals from, the activated insulin receptor (INSR). Potent inhibitor of insulin-stimulated MAPK3 phosphorylation. Plays a critical role regulating PDPK1 membrane translocation in response to insulin stimulation and serves as an adapter protein to recruit PDPK1 to activated insulin receptor, thus promoting PKB/AKT1 phosphorylation and transduction of the insulin signal","subcellular_location":"Cytoplasm; Endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q14449/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GRB14","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GRB14","total_profiled":1310},"omim":[{"mim_id":"609036","title":"APBB1-INTERACTING PROTEIN; APBB1IP","url":"https://www.omim.org/entry/609036"},{"mim_id":"607128","title":"TANKYRASE 2; TNKS2","url":"https://www.omim.org/entry/607128"},{"mim_id":"601524","title":"GROWTH FACTOR RECEPTOR-BOUND PROTEIN 14; GRB14","url":"https://www.omim.org/entry/601524"},{"mim_id":"601523","title":"GROWTH FACTOR RECEPTOR-BOUND PROTEIN 10; GRB10","url":"https://www.omim.org/entry/601523"},{"mim_id":"601522","title":"GROWTH FACTOR RECEPTOR-BOUND PROTEIN 7; GRB7","url":"https://www.omim.org/entry/601522"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"epididymis","ntpm":23.8},{"tissue":"liver","ntpm":29.1},{"tissue":"tongue","ntpm":43.3}],"url":"https://www.proteinatlas.org/search/GRB14"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q14449","domains":[{"cath_id":"3.10.20.90","chopping":"106-188","consensus_level":"high","plddt":93.7395,"start":106,"end":188},{"cath_id":"2.30.29.30","chopping":"194-212_220-350","consensus_level":"high","plddt":92.037,"start":194,"end":350},{"cath_id":"3.30.505.10","chopping":"439-533","consensus_level":"high","plddt":94.004,"start":439,"end":533}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14449","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14449-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14449-F1-predicted_aligned_error_v6.png","plddt_mean":76.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GRB14","jax_strain_url":"https://www.jax.org/strain/search?query=GRB14"},"sequence":{"accession":"Q14449","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14449.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14449/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14449"}},"corpus_meta":[{"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":"8647858","id":"PMC_8647858","title":"Cloning and characterization of GRB14, a novel member of the GRB7 gene family.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8647858","citation_count":117,"is_preprint":false},{"pmid":"14749734","id":"PMC_14749734","title":"Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice.","date":"2004","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/14749734","citation_count":112,"is_preprint":false},{"pmid":"16849634","id":"PMC_16849634","title":"GRB14, GPD1, and GDF8 as potential network collaborators in weight loss-induced improvements in insulin action in human skeletal muscle.","date":"2006","source":"Physiological genomics","url":"https://pubmed.ncbi.nlm.nih.gov/16849634","citation_count":112,"is_preprint":false},{"pmid":"11278563","id":"PMC_11278563","title":"Identification of a novel human tankyrase through its interaction with the adaptor protein Grb14.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11278563","citation_count":110,"is_preprint":false},{"pmid":"16246733","id":"PMC_16246733","title":"Structural basis for inhibition of the insulin receptor by the adaptor protein Grb14.","date":"2005","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/16246733","citation_count":98,"is_preprint":false},{"pmid":"11726652","id":"PMC_11726652","title":"Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11726652","citation_count":97,"is_preprint":false},{"pmid":"9748281","id":"PMC_9748281","title":"Identification of the rat adapter Grb14 as an inhibitor of insulin actions.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9748281","citation_count":93,"is_preprint":false},{"pmid":"19648926","id":"PMC_19648926","title":"Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14.","date":"2009","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19648926","citation_count":64,"is_preprint":false},{"pmid":"10713090","id":"PMC_10713090","title":"Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. 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a C-terminal SH2 domain. The GST-Grb14 SH2 domain fusion protein exhibited strong binding to activated PDGF receptors in vitro, and Grb14 was phosphorylated on serine residues in a PDGF-regulated manner.\",\n      \"method\": \"cDNA library screening, GST pulldown, co-immunoprecipitation, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — original cloning paper with in vitro binding assays and phosphorylation studies, single lab\",\n      \"pmids\": [\"8647858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rat Grb14 binds to the insulin receptor (IR) in an insulin-dependent manner via two domains: the SH2 domain and a region between PH and SH2 domains (PIR/BPS), with PIR being the major binding domain. The interaction requires IR tyrosines Y1150/Y1151. Grb14 overexpression decreases insulin-stimulated IRS-1 tyrosine phosphorylation, DNA synthesis, and glycogen synthesis without altering IR autophosphorylation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding with deletion mutants, cell-based overexpression assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods identifying binding domains and functional consequences, replicated in subsequent studies\",\n      \"pmids\": [\"9748281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Grb14 binds activated FGFR1 in a kinase-activity-dependent manner, requiring the FGFR1 C-tail tyrosines and juxtamembrane domain. The SH2 domain of Grb14 mediates binding but regions upstream of SH2 confer specificity for FGFR1 over PDGFR. Grb14 overexpression inhibits FGF-2-induced cell proliferation, whereas an SH2-binding-deficient mutant enhances it.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, cell proliferation assay, domain deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, in vitro binding, functional gain/loss-of-function with mutant rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"10713090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Tankyrase 2, a poly(ADP-ribose) polymerase, was identified as a binding partner of Grb14 via yeast two-hybrid screening. The interaction is mediated by the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10-19 of tankyrase 2. Both proteins co-localize in the low-density microsome fraction and co-immunoprecipitate in vivo.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation, deletion mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and fractionation with domain mapping, single lab\",\n      \"pmids\": [\"11278563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Grb14 directly inhibits IR substrate phosphorylation in vitro, acting as an uncompetitive inhibitor for IR substrate without altering Km for ATP. The PIR/BPS domain alone mediates this inhibitory effect. Grb14 binding protects IR kinase-loop phosphotyrosines from phosphatases but delays downstream Akt and ERK1/2 activation.\",\n      \"method\": \"In vitro kinase assay, domain deletion analysis, CHO cell overexpression, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with domain mapping and mechanistic follow-up in cells, replicated across labs\",\n      \"pmids\": [\"11726652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ZIP (PKCζ interacting protein) binds Grb14 through its ZZ zinc finger domain interacting with the PIR region of Grb14, forming a PKCζ-ZIP-Grb14 heterotrimeric complex. PKCζ phosphorylates Grb14 in vitro and in vivo, and this phosphorylation increases Grb14's inhibitory effect on IR tyrosine kinase activity. In Xenopus oocytes, ZIP potentiates Grb14-mediated inhibition of insulin-induced maturation in a PKCζ- and phosphorylation-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, in vivo radiolabeling, Xenopus oocyte functional assay, domain deletion mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay, in vivo phosphorylation, functional epistasis in Xenopus model, multiple orthogonal methods\",\n      \"pmids\": [\"12242277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The PIR domain of Grb14 is an intrinsically unstructured protein by NMR analysis, yet retains biological activity as demonstrated by inhibition of insulin-induced meiosis reinitiation in Xenopus oocytes.\",\n      \"method\": \"NMR spectroscopy, Xenopus oocyte functional assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure determination with functional validation in oocyte model, single lab\",\n      \"pmids\": [\"14623073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Grb14 (but not Grb10) inhibits FGF receptor-induced maturation in Xenopus oocytes by binding to the receptor and inhibiting the Ras-dependent pathway. The PIR and SH2 domains of Grb14 have different roles in FGFR signaling inhibition compared to its paralogs.\",\n      \"method\": \"Xenopus oocyte maturation assay, co-immunoprecipitation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional oocyte assay with biochemical co-IP, single lab\",\n      \"pmids\": [\"12885405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Grb14 knockout mice display improved glucose tolerance, lower circulating insulin, enhanced glycogen synthesis in liver and skeletal muscle, and increased insulin-induced IRS-1 and PKB phosphorylation in a tissue-specific manner (liver and skeletal muscle, but not adipose), demonstrating that Grb14 is a tissue-specific negative regulator of insulin signaling in vivo.\",\n      \"method\": \"Gene knockout mouse model, glucose tolerance test, insulin signaling assays (phosphorylation of IR, IRS-1, PKB), ex vivo glucose uptake\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with multiple defined molecular and metabolic phenotypes, replicated in subsequent studies\",\n      \"pmids\": [\"14749734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The PIR domain of Grb14 is natively unfolded in solution (SAXS and CD analyses), with a potentially structured short stretch around residues 399-407 that may undergo structural transition upon binding to a partner.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), circular dichroism, NMR\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — multiple biophysical methods, single lab\",\n      \"pmids\": [\"15465854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Grb14 binds PDK-1 constitutively through a PDK-1 binding motif on Grb14 (distinct from SH2 domain). This interaction facilitates insulin-triggered membrane translocation of PDK-1 to activated IR and promotes Akt phosphorylation. Disruption of the Grb14-PDK-1 interaction significantly decreases insulin-dependent Akt activation.\",\n      \"method\": \"Directed proteomics, co-immunoprecipitation, membrane translocation assay, dominant-negative overexpression, Akt activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, point mutant disruption, functional consequence on Akt, moderate evidence\",\n      \"pmids\": [\"15210700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of the Grb14 BPS region in complex with the insulin receptor tyrosine kinase domain reveals that the N-terminal portion of BPS acts as a pseudosubstrate inhibitor binding in the substrate peptide-binding groove of the kinase. Together with the SH2 domain crystal structure, a model for dual-domain engagement of IR by Grb14 is presented.\",\n      \"method\": \"X-ray crystallography (crystal structure of BPS-IR kinase domain complex and SH2 domain)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional mechanistic model, highly cited foundational structural study\",\n      \"pmids\": [\"16246733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Grb14 overexpression inhibits insulin- and estrogen-induced cell cycle progression in MCF-7 breast cancer cells, associated with decreased ERK1/2 activation. Estradiol represses insulin-induced Grb14 expression, while anti-estrogen ICI 182780 increases Grb14 levels.\",\n      \"method\": \"Cell overexpression, cell cycle analysis, ERK1/2 phosphorylation assay, hormonal treatment\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — overexpression with defined signaling and cell cycle readouts, single lab\",\n      \"pmids\": [\"15372466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Grb14 contains an FXNPXY motif, and the PTB domain of IRS-1 binds to this NPXY motif of Grb14 in a phosphorylation-independent manner. Grb14-IRS-1 complexes are detected in retinal tissue lysates, suggesting Grb14 acts as a dominant negative for IRS-1 functions in the retina.\",\n      \"method\": \"Yeast two-hybrid (retinal cDNA library screen), GST pulldown, co-immunoprecipitation from retinal tissue\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vitro binding assay and tissue co-IP, single lab\",\n      \"pmids\": [\"15924411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Real-time BRET analysis in living HEK cells shows that Grb14 is rapidly and dose-dependently recruited to the activated IR upon insulin stimulation. Grb14 reduces IR-PTP1B interaction (BRET), protects kinase-loop tyrosines from PTP1B dephosphorylation, while promoting dephosphorylation of Y972, thereby decreasing IRS-1 binding to IR and attenuating ERK pathway activation. Insulin also induces Grb14 dimerization.\",\n      \"method\": \"BRET (bioluminescence resonance energy transfer) in living cells, co-immunoprecipitation, site-specific phosphotyrosine antibodies\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — real-time BRET, co-IP, and site-specific phospho-antibodies providing mechanistic detail on PTP1B regulation, multiple orthogonal methods\",\n      \"pmids\": [\"16582879\", \"16934761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In rat liver, Grb14 localizes predominantly to microsomal and cytosolic fractions at baseline and translocates to the plasma membrane and Golgi/endosome fractions upon insulin stimulation, co-localizing with phosphorylated IR. KCl removal of Grb14 from fractions increases IR tyrosine kinase activity, demonstrating endogenous Grb14 exerts negative feedback on liver IR activity in vivo.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, in vitro phosphatase assay, KCl extraction, in vivo rat liver model\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — subcellular fractionation with functional consequence (KCl stripping assay), in vivo rat model, multiple methods\",\n      \"pmids\": [\"18657188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In primary hepatocytes with Grb14 knockdown, insulin-induced Akt phosphorylation and inhibition of gluconeogenesis are increased, but insulin-stimulated SREBP-1c maturation is completely blunted, blocking lipogenic gene expression and glycogen synthesis. This reveals a dual role for Grb14 in liver: inhibiting IR kinase activity and promoting a distal step (SREBP-1c maturation) required for lipogenic insulin action.\",\n      \"method\": \"RNAi knockdown in primary mouse hepatocytes, insulin signaling assays (Akt, GSK3, FOXO1 phosphorylation), gene expression analysis, glucose production measurement\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean RNAi KD with multiple molecular readouts revealing dual mechanistic role, single lab but multiple orthogonal assays\",\n      \"pmids\": [\"18339716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of the Grb14 RA and PH domains at 2.4-Å resolution shows these domains form an integrated structural unit. Biochemical studies demonstrate that Grb14 binds to activated Ras (GTP-loaded), which may serve as a timing mechanism for downregulation of insulin signaling. The RA-PH unit can simultaneously engage small GTPases and phosphoinositide lipids.\",\n      \"method\": \"X-ray crystallography (RA-PH structure in complex with H-Ras G12V at 2.4-Å), biochemical binding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical validation, published in high-impact journal\",\n      \"pmids\": [\"19648926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Dual knockout of Grb10 and Grb14 in mice does not further increase IRS-1 phosphorylation and Akt activation beyond single knockouts, suggesting context-dependent limiting mechanisms including IR hypophosphorylation and decreased IRS-1 expression. Double KO mice are protected from high-fat diet-induced glucose intolerance whereas single KOs are not, suggesting additional glucose homeostasis regulatory mechanisms beyond IRS-1/Akt.\",\n      \"method\": \"Compound gene knockout mice, glucose tolerance test, insulin signaling assays, high-fat diet challenge\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis using double KO mice with defined molecular phenotypes, clean experimental design\",\n      \"pmids\": [\"19541746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Molecular determinants of Grb14-IR interaction include Grb14-L404 interacting with IR-L1038 (IR αC-helix) and Grb14-R385 interacting with IR-K1168 (activation loop), with L404 likely contributing to specificity for IR over other Grb7 family members. Grb14 S370 phosphorylation status controls its biological activity, and insulin-induced Grb14-PDK1 interaction is required for maximal inhibition of insulin signaling.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, BRET, Xenopus oocyte assay, MEF functional complementation assay\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis with multiple functional readouts including structural contacts and signaling outcomes, multiple orthogonal methods\",\n      \"pmids\": [\"19359342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Grb14 inhibits FGFR signaling by being recruited to FGFR1 in a trimeric complex containing PLCγ. Grb14 binds to FGFR1 pY766 (the PLCγ docking site) via its SH2 domain, competitively trapping and inactivating PLCγ, and altering FGF-induced PLCγ phosphorylation and activation.\",\n      \"method\": \"BRET in living cells, Xenopus oocyte maturation assay, co-immunoprecipitation, Western blot for PLCγ phosphorylation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — BRET, co-IP, and functional oocyte assay, with mechanistic model involving competitive binding at Y766, multiple methods\",\n      \"pmids\": [\"20932831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Tie2 receptor tyrosine kinase phosphorylates Grb14 on tyrosine residues in a manner requiring kinase-competent Tie2 and intact Tie2 tyrosines Y1100 and Y1106. A complete Grb14 SH2 domain is required for this phosphorylation. Grb14 tyrosine phosphorylation also occurs in primary endothelial cells treated with COMP-Ang1.\",\n      \"method\": \"Co-immunoprecipitation, Western blot with phosphotyrosine antibodies, kinase assay, dominant-negative Tie2 mutant, primary endothelial cells\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP with kinase-dead controls and primary cell validation, single lab\",\n      \"pmids\": [\"20973951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Phosphorylation of Tyr-347 in the BPS domain of Grb14 (by Src kinase, activated downstream of rhodopsin) enables Grb14 to competitively inhibit PTP1B activity. Ablation of Grb14 in mouse retina results in significantly elevated PTP1B activity in vivo, demonstrating that phosphorylated Grb14 is an endogenous inhibitor of retinal PTP1B.\",\n      \"method\": \"In vitro PTP1B activity assay, site-directed mutagenesis (Y347), Grb14 KO mouse retina assay, Src kinase activity measurement\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzyme assay, mutagenesis of critical tyrosine, in vivo KO validation, multiple methods\",\n      \"pmids\": [\"21791607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In thyroid cancer cells, Grb14 knockdown reduces RET receptor phosphorylation and downstream Akt and STAT3 activation while facilitating IR signaling, whereas Grb14 overexpression facilitates RET activation and STAT3/Akt phosphorylation. This reveals a novel role for Grb14 in promoting RET-mediated thyroid cancer progression.\",\n      \"method\": \"Stable shRNA knockdown, overexpression, in vitro proliferation/invasion assays, in vivo xenograft mouse model, phosphorylation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function with defined molecular readouts in vitro and in vivo, single lab\",\n      \"pmids\": [\"22158039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Grb14 RA domain mediates closure of the photoreceptor-specific CNG channel (CNGA1) through electrostatic interactions; three Glu residues (180-182) in Grb14 interact electrostatically with Arg559 in the cGMP binding pocket of CNGA1, and Lys140 of Grb14 also contributes. Grb14 competes with cGMP for the CNGA1 binding pocket. This activity is specific to Grb14 and not shared by Grb7 or Grb10.\",\n      \"method\": \"In vitro binding assay, site-directed mutagenesis of electrostatic residues, channel activity assay\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis identifying specific electrostatic contacts with functional channel assay, single lab\",\n      \"pmids\": [\"22180090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Grb14 SH2 domain binds the ITAM-like sequence in the cytoplasmic domain of CEACAM3 (identified by SH2 domain microarray). Grb14 is rapidly recruited to sites of bacteria-host cell contact, and both Grb14 knockdown and overexpression indicate an inhibitory role for Grb14 in CEACAM3-mediated bacterial phagocytosis.\",\n      \"method\": \"SH2 domain microarray, co-immunoprecipitation, FRET-FLIM in intact cells, RNAi knockdown, overexpression, phagocytosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including FRET-FLIM, reciprocal gain/loss-of-function, SH2 microarray identification\",\n      \"pmids\": [\"22948154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of Grb14 RA-PH domains in complex with GTP-loaded H-Ras(G12V) at 2.4-Å resolution reveals the integrated RA-PH structural unit binds simultaneously to activated Ras and phosphoinositide lipids. Binding mode of Grb14 RA domain to H-Ras is similar to RalGDS and Raf1 RA domains but with distinct features.\",\n      \"method\": \"X-ray crystallography (RA-PH domains in complex with H-Ras G12V)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure providing detailed molecular basis for Ras binding\",\n      \"pmids\": [\"23967305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GSK-3 phosphorylates Ser358, Ser362, and Ser366 in the BPS domain of Grb14 (requiring a priming phospho-Ser370), and this phosphorylation suppresses Grb14-IR complex formation. Pharmacological inhibition or knockdown of GSK-3 facilitates Grb14-IR binding, and Ala substitutions at these serines enhance Grb14-IR association.\",\n      \"method\": \"In vitro kinase assay with phosphopeptides, proximity ligation assay, pharmacological GSK-3 inhibition, siRNA knockdown, co-immunoprecipitation\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay with mutagenesis and cell-based co-IP validation, single lab\",\n      \"pmids\": [\"24535599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ubiquitin ligase Chfr binds to the T220 residue of Grb14 in an insulin-stimulated manner and potentiates Grb14's inhibitory effect on insulin-induced cell division. Chfr ligase activity and phosphorylation of its T39 residue (an Akt substrate) are required for this function. Insulin-stimulated Chfr binding to Grb14 activates Chfr ligase activity, leading to Aurora A and Polo-like kinase degradation, blocking cell division.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus oocyte G2/M transition assay, targeted mutagenesis, mammalian cell line overexpression/knockdown\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP, mutagenesis, functional oocyte assay, multiple orthogonal methods establishing pathway\",\n      \"pmids\": [\"25578860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Liver-specific Grb14 knockdown in mice enhances insulin signaling but also releases p62/SQSTM1 (a Grb14 binding partner), which activates the Nrf2 transcription factor, which in turn represses LXR and thereby inhibits de novo lipogenesis. This reveals Grb14 as a signaling node at the crossroads of the IR and p62-Nrf2-LXR pathways regulating both glucose homeostasis and hepatic lipid synthesis.\",\n      \"method\": \"In vivo siRNA/shRNA liver knockdown, mouse metabolic phenotyping, co-immunoprecipitation (Grb14-p62), gene expression analysis, Nrf2/LXR reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo knockdown, co-IP establishing binding, multiple pathway readouts in insulin-resistant mouse models\",\n      \"pmids\": [\"27215388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GSK-3-mediated phosphorylation of N-terminal BPS domain serines (Ser358, Ser362, Ser366) in human Grb14 negatively regulates Grb14-IR complex formation; surface plasmon resonance shows Kd of 8 nM for Grb14-IR, which is abolished by Glu substitutions at these serines. The BPS domain alone does not show this differential, indicating additional domain contributions.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, yeast two-hybrid, surface plasmon resonance\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — SPR quantitative binding, yeast two-hybrid, in vitro kinase, single lab confirming prior findings\",\n      \"pmids\": [\"28130417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Protein phosphatase 1 (PP1) dephosphorylates Ser358 and Ser362 in the N-terminal BPS domain of human Grb14, and this dephosphorylation facilitates Grb14-IR complex formation as demonstrated by co-immunoprecipitation with phosphomimetic substitutions.\",\n      \"method\": \"In vitro phosphatase assay with synthetic phosphopeptides, co-immunoprecipitation\",\n      \"journal\": \"Journal of peptide science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-3 — in vitro phosphatase assay with cell-based co-IP, single lab extending prior GSK-3 phosphorylation findings\",\n      \"pmids\": [\"31347216\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GRB14 is a multidomain adaptor protein (containing RA, PH, BPS/PIR, and SH2 domains) that functions primarily as a tissue-specific negative regulator of insulin receptor (IR) signaling: the BPS/PIR region acts as a pseudosubstrate inhibitor in the IR kinase substrate-binding groove, the SH2 domain docks to IR phosphotyrosines, and together these interactions inhibit IR-mediated IRS-1 phosphorylation and downstream Akt/ERK activation; GRB14 also recruits PDK-1 to the IR to modulate Akt signaling, interacts with p62/SQSTM1 to regulate the Nrf2-LXR lipogenic pathway, is phosphorylated by PKCζ (via ZIP adaptor) and Src kinase (enabling PTP1B inhibition) and regulated by GSK-3/PP1 at BPS domain serines, binds activated Ras through its RA domain, inhibits FGFR signaling by competing with PLCγ at pY766, inhibits CEACAM3-mediated phagocytosis via its SH2 domain, and collaborates with Chfr ubiquitin ligase to suppress insulin-induced cell division.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GRB14 is a multidomain adaptor protein that functions as a tissue-specific negative regulator of receptor tyrosine kinase signaling, most prominently insulin receptor (IR) signaling. Its BPS/PIR domain acts as a pseudosubstrate inhibitor occupying the IR kinase substrate-binding groove, while its SH2 domain docks to IR activation-loop phosphotyrosines; together these interactions suppress IRS-1 phosphorylation and downstream Akt/ERK activation, as demonstrated by crystal structures, in vitro kinase assays, and improved glucose homeostasis in Grb14 knockout mice [PMID:16246733, PMID:11726652, PMID:14749734]. Beyond IR, GRB14 inhibits FGFR1 signaling by competitively displacing PLCγ from pY766 via its SH2 domain [PMID:20932831], inhibits CEACAM3-mediated phagocytosis [PMID:22948154], and when phosphorylated on Tyr-347 by Src acts as an endogenous inhibitor of PTP1B in the retina [PMID:21791607]. GRB14 activity is regulated by PKCζ-mediated phosphorylation (enhanced IR inhibition) [PMID:12242277], GSK-3 phosphorylation of BPS-domain serines (which opposes IR binding and is reversed by PP1) [PMID:24535599, PMID:31347216], binding to activated Ras through its integrated RA-PH structural unit [PMID:19648926], and interaction with p62/SQSTM1 to modulate the Nrf2–LXR lipogenic pathway in liver [PMID:27215388].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Identification of GRB14 as a new GRB7-family adaptor with PH, BPS, and SH2 domains that binds activated PDGF receptors established it as a candidate RTK-signaling adaptor.\",\n      \"evidence\": \"cDNA library cloning, GST-SH2 pulldown with PDGFR, serine phosphorylation analysis\",\n      \"pmids\": [\"8647858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No endogenous function demonstrated\", \"PDGFR binding not confirmed by reciprocal IP\", \"In vivo relevance unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstration that Grb14 binds the insulin receptor in an insulin-dependent manner via two domains (PIR/BPS and SH2) and that overexpression suppresses IRS-1 phosphorylation, DNA synthesis, and glycogen synthesis established GRB14 as a negative regulator of insulin signaling.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP with IR deletion/point mutants (Y1150/Y1151), overexpression in CHO cells\",\n      \"pmids\": [\"9748281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of inhibition (competitive vs. pseudosubstrate) unresolved\", \"In vivo relevance not tested\", \"Contribution of individual domains not fully dissected\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Extending the adaptor's scope beyond IR, binding to activated FGFR1 and inhibition of FGF-induced proliferation revealed GRB14 as a broader RTK-signaling inhibitor.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, domain deletion analysis, cell proliferation assay in FGFR1-expressing cells\",\n      \"pmids\": [\"10713090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of FGFR inhibition not identified\", \"Relative contribution of SH2 vs. upstream regions unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"In vitro kinase assays showed the BPS/PIR domain alone inhibits IR kinase activity toward substrates without altering ATP Km, establishing an uncompetitive pseudosubstrate inhibition mechanism and linking this to delayed Akt/ERK activation in cells.\",\n      \"evidence\": \"In vitro IR kinase assay with purified BPS domain, CHO cell overexpression, phospho-specific Western blots\",\n      \"pmids\": [\"11726652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of pseudosubstrate mechanism not yet solved\", \"In vivo significance not demonstrated\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery of the PKCζ–ZIP–Grb14 trimeric complex and PKCζ-dependent phosphorylation of Grb14 that enhances its IR-inhibitory activity identified a regulatory input controlling GRB14 potency.\",\n      \"evidence\": \"Co-IP, in vitro/in vivo kinase assay, Xenopus oocyte insulin-maturation assay with epistasis analysis\",\n      \"pmids\": [\"12242277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation sites on Grb14 not mapped\", \"Relevance in mammalian metabolic tissues not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Grb14 knockout mice showed improved glucose tolerance, lower insulin levels, and enhanced IRS-1/Akt signaling specifically in liver and muscle but not adipose, providing the first in vivo genetic evidence that GRB14 is a tissue-specific physiological brake on insulin action.\",\n      \"evidence\": \"Grb14−/− mouse model with glucose tolerance tests, insulin signaling assays, ex vivo glucose uptake\",\n      \"pmids\": [\"14749734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for tissue specificity unclear\", \"Compensation by Grb10 not addressed\", \"Lipogenic consequences not explored\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of constitutive Grb14–PDK-1 binding through a distinct motif, which recruits PDK-1 to the IR and is required for full insulin-stimulated Akt activation, revealed a paradoxical positive-signaling role for an otherwise inhibitory adaptor.\",\n      \"evidence\": \"Co-IP, membrane translocation assay, dominant-negative disruption, Akt phosphorylation readout\",\n      \"pmids\": [\"15210700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of positive (PDK-1 recruitment) vs. negative (pseudosubstrate) roles not quantified\", \"PDK-1 binding site not structurally characterized\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The crystal structure of the BPS region bound to the IR kinase domain at atomic resolution confirmed the pseudosubstrate model: the BPS N-terminus occupies the substrate-binding groove, providing the structural basis for GRB14's inhibitory mechanism.\",\n      \"evidence\": \"X-ray crystallography of BPS–IR kinase complex and SH2 domain\",\n      \"pmids\": [\"16246733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length Grb14–IR complex structure not available\", \"Dynamics of engagement/disengagement not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Real-time BRET in living cells showed that Grb14 recruitment to IR is rapid and dose-dependent, that Grb14 reduces IR–PTP1B interaction while selectively promoting dephosphorylation of Y972 (the IRS-1 docking site) and protecting activation-loop phosphotyrosines, mechanistically explaining site-selective signal attenuation.\",\n      \"evidence\": \"BRET donor/acceptor pairs in HEK cells, site-specific phospho-antibodies, co-IP\",\n      \"pmids\": [\"16582879\", \"16934761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Insulin-induced Grb14 dimerization function unclear\", \"Whether PTP1B modulation occurs in all IR-expressing tissues unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Hepatocyte Grb14 knockdown revealed a dual role: while enhancing proximal IR signaling, it abolished SREBP-1c maturation and lipogenic gene expression, showing GRB14 promotes a distal lipogenic step independently of its kinase-inhibitory function.\",\n      \"evidence\": \"siRNA knockdown in primary mouse hepatocytes with Akt/FOXO1 phosphorylation, SREBP-1c processing, and gluconeogenesis assays\",\n      \"pmids\": [\"18339716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking Grb14 to SREBP-1c maturation not identified\", \"Whether this is direct or via intermediary pathway unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structure of the RA-PH integrated unit bound to GTP-loaded H-Ras established that GRB14 directly engages activated Ras, potentially serving as a timing mechanism linking Ras activity to insulin-signaling downregulation.\",\n      \"evidence\": \"X-ray crystallography at 2.4 Å, biochemical Ras-binding assays\",\n      \"pmids\": [\"19648926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ras binding on IR inhibition not tested in cells\", \"Whether Ras binding recruits Grb14 to specific membrane microdomains unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstration that Grb14 SH2 domain binds FGFR1 pY766 (the PLCγ docking site) in a trimeric complex, competitively trapping and inactivating PLCγ, provided the mechanism for GRB14's inhibition of FGFR signaling.\",\n      \"evidence\": \"BRET, co-IP, PLCγ phosphorylation Western blot, Xenopus oocyte maturation assay\",\n      \"pmids\": [\"20932831\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological relevance for FGFR regulation not demonstrated\", \"Whether GRB14 inhibits other FGFR family members unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Src-mediated phosphorylation of BPS Tyr-347 converts GRB14 into an endogenous PTP1B inhibitor, as shown by elevated retinal PTP1B activity in Grb14 KO mice; this added a gain-of-function dimension to the adaptor.\",\n      \"evidence\": \"In vitro PTP1B assay, Y347 mutagenesis, Grb14 KO mouse retina, Src kinase activity measurement\",\n      \"pmids\": [\"21791607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether pY347-mediated PTP1B inhibition occurs outside the retina not tested\", \"Structural basis of pY347–PTP1B interaction unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"SH2 domain microarray and functional assays identified CEACAM3 as a GRB14 binding partner, with Grb14 rapidly recruited to bacteria-host contact sites to inhibit phagocytosis, extending GRB14 function to innate immunity.\",\n      \"evidence\": \"SH2 microarray, FRET-FLIM, RNAi knockdown and overexpression, phagocytosis assay\",\n      \"pmids\": [\"22948154\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of phagocytosis inhibition beyond SH2 binding not resolved\", \"In vivo infection model not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of GSK-3 as a kinase that phosphorylates BPS serines 358/362/366 to disrupt Grb14–IR complex formation, with PP1 serving as the opposing phosphatase, established a phosphorylation toggle controlling GRB14's access to its primary target.\",\n      \"evidence\": \"In vitro kinase/phosphatase assays, Ala/Glu substitutions, co-IP, SPR (Kd ~8 nM abolished by phosphomimetics)\",\n      \"pmids\": [\"24535599\", \"28130417\", \"31347216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts triggering GSK-3/PP1 regulation of Grb14 not defined\", \"Whether priming at S370 is constitutive or regulated unknown\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of the Chfr ubiquitin ligase as an insulin-stimulated Grb14 partner that potentiates cell-cycle arrest by degrading Aurora A and Plk established a pathway by which GRB14 links insulin signaling to mitotic checkpoint control.\",\n      \"evidence\": \"Co-IP, Xenopus G2/M oocyte assay, mutagenesis of Chfr T39 and Grb14 T220, mammalian cell knockdown/overexpression\",\n      \"pmids\": [\"25578860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance for tumor suppression not shown\", \"Whether Chfr–Grb14 interaction is direct or bridged unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Liver-specific Grb14 knockdown revealed that GRB14 sequesters p62/SQSTM1, and its loss releases p62 to activate Nrf2, which represses LXR-driven lipogenesis, resolving the molecular link between GRB14 and hepatic de novo lipogenesis.\",\n      \"evidence\": \"In vivo hepatic siRNA/shRNA, co-IP of Grb14–p62, Nrf2/LXR reporter assays, metabolic phenotyping in insulin-resistant mice\",\n      \"pmids\": [\"27215388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface between Grb14 and p62 not mapped\", \"Interaction of this pathway with SREBP-1c maturation defect from 2008 study not reconciled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structural model of GRB14 bound to the intact IR, the precise mechanism linking GRB14 to SREBP-1c maturation, the physiological significance of Ras binding via the RA domain in metabolic tissues, and the in vivo relevance of GRB14 in innate immune phagocytosis remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length Grb14–IR structure\", \"SREBP-1c connection mechanistically unexplained\", \"Ras–RA domain function not tested in metabolic models\", \"CEACAM3 inhibition not validated in infection models\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 4, 10, 11, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 11, 22, 20]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [17, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 8, 10, 11, 14, 16, 22, 23]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8, 16, 29]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [12, 28]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"INSR\",\n      \"FGFR1\",\n      \"PDPK1\",\n      \"SQSTM1\",\n      \"PLCG1\",\n      \"CHFR\",\n      \"PRKCZ\",\n      \"TNKS2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}