{"gene":"GRB14","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":1996,"finding":"GRB14 SH2 domain binds to activated PDGF receptors in vitro; GRB14 is phosphorylated on serine residues in response to PDGF stimulation, identifying it as a target for a PDGF-regulated serine kinase.","method":"GST-SH2 domain pulldown, in vivo serine phosphorylation labeling","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — GST pulldown and phosphorylation labeling, single lab, two methods but no functional follow-up on the in vivo interaction","pmids":["8647858"],"is_preprint":false},{"year":1998,"finding":"Rat Grb14 binds the insulin receptor in an insulin-dependent manner in vivo; the PIR domain (between PH and SH2) is the primary IR-binding domain, while the SH2 domain contributes secondarily. Binding requires IR tyrosines Y1150/Y1151. Overexpression of Grb14 in CHO-IR cells decreases insulin-stimulated IRS-1 tyrosine phosphorylation and reduces glycogen and DNA synthesis, establishing Grb14 as an inhibitory regulator of insulin signaling.","method":"Yeast two-hybrid, co-immunoprecipitation, deletion mutant in vitro binding, CHO cell overexpression with metabolic readouts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, Co-IP, in vitro binding, cell-based functional assays), replicated by subsequent studies","pmids":["9748281"],"is_preprint":false},{"year":2001,"finding":"Grb14 directly inhibits insulin receptor (IR) substrate phosphorylation in vitro, acting as an uncompetitive inhibitor with respect to peptide substrate but not altering Km for ATP. The PIR/BPS domain alone is sufficient for this inhibitory effect; the SH2 domain has no effect on IR catalytic activity. In CHO-IR cells, Grb14 binding protects the IR from tyrosine phosphatases, maintaining it in a phosphorylated state, but delays Akt and ERK1/2 activation.","method":"In vitro kinase assay with deletion mutants, CHO cell co-immunoprecipitation, western blot for downstream signaling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with domain dissection and mutagenesis, replicated across multiple studies","pmids":["11726652"],"is_preprint":false},{"year":2001,"finding":"Tankyrase 2 was identified as a novel binding partner for Grb14 via yeast two-hybrid screen. The interaction is mediated by the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10–19 of tankyrase 2. Both proteins co-fractionate in the low-density microsome fraction in vivo, and their association can be detected by co-immunoprecipitation.","method":"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation, deletion analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and fractionation, single lab, two orthogonal methods","pmids":["11278563"],"is_preprint":false},{"year":2002,"finding":"ZIP (PKCζ-interacting protein) binds Grb14 through its ZZ zinc finger domain interacting with the PIR domain of Grb14, forming a PKCζ–ZIP–Grb14 heterotrimeric complex. PKCζ phosphorylates Grb14 in vitro and in vivo, and this phosphorylation is increased by insulin stimulation. Phosphorylation of Grb14 by PKCζ further enhances Grb14's inhibitory effect on IR tyrosine kinase activity. In Xenopus oocytes, ZIP potentiates Grb14's inhibitory action on insulin-induced maturation in a PKCζ- and phosphorylation-dependent manner.","method":"Co-immunoprecipitation, in vitro kinase assay, in vivo [32P] labeling, Xenopus oocyte functional assay, deletion mapping","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus in vivo labeling plus functional oocyte model, multiple orthogonal methods, single lab","pmids":["12242277"],"is_preprint":false},{"year":2003,"finding":"The isolated PIR domain of Grb14 is intrinsically unstructured (lacks secondary structure) as demonstrated by NMR, yet retains full biological activity, inhibiting insulin-induced oocyte maturation in Xenopus.","method":"NMR (15N-labeled recombinant PIR), Xenopus oocyte functional assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structural characterization with functional validation in oocytes, single lab","pmids":["14623073"],"is_preprint":false},{"year":2004,"finding":"Grb14 knockout in mice leads to improved glucose tolerance, lower circulating insulin, increased glycogen synthesis in liver and skeletal muscle, and enhanced insulin-induced glucose uptake in soleus muscle. In liver, despite lower IR autophosphorylation, IRS-1 phosphorylation and PKB activation are enhanced. This demonstrates Grb14 as a tissue-specific in vivo repressor of IR-mediated IRS-1 tyrosine phosphorylation.","method":"Gene targeting (knockout mouse), glucose/insulin tolerance tests, ex vivo tissue metabolic assays, western blot for signaling","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with multiple defined cellular and metabolic phenotypes and signaling readouts, replicated by subsequent studies","pmids":["14749734"],"is_preprint":false},{"year":2004,"finding":"The PIR domain of Grb14 is natively unstructured but contains a potentially structured short stretch (residues 399–407) that may undergo a structural transition upon binding to a partner, as evidenced by SAXS and circular dichroism with TFE.","method":"Small-angle X-ray scattering (SAXS), circular dichroism, sequence analysis","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — SAXS and CD provide structural characterization, but functional consequence of the transient structure is not directly tested, single lab","pmids":["15465854"],"is_preprint":false},{"year":2004,"finding":"Grb14 binds PDK-1 constitutively through a PDK-1 binding motif on Grb14. This interaction is required for insulin-triggered membrane translocation of PDK-1 and facilitates Akt activation. Disruption of this interaction by point mutation or SH2 domain deletion significantly decreases insulin-dependent Akt activation.","method":"Directed proteomics pulldown, co-immunoprecipitation, point mutagenesis, subcellular fractionation, Akt activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding plus mutagenesis plus functional localization assay with Akt readout, multiple orthogonal methods","pmids":["15210700"],"is_preprint":false},{"year":2004,"finding":"In adipose tissue of ob/ob mice, Goto-Kakizaki rats, and type 2 diabetic humans, Grb14 expression is increased. Insulin stimulates Grb14 expression in 3T3-F442A adipocytes, while thiazolidinediones decrease it, indicating hormonal regulation of Grb14 abundance as part of insulin resistance.","method":"Western blot, RT-PCR, 3T3-F442A cell culture treatment assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple models and human tissue, but purely expression-level regulation without direct mechanistic dissection; included because insulin-dependent transcriptional feedback is experimentally shown","pmids":["15059968"],"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, occupying the substrate peptide-binding groove of the kinase. Together with the SH2 domain crystal structure, a model is presented in which Grb14 simultaneously anchors via its SH2 domain to IR phosphotyrosines and occludes the substrate groove via BPS.","method":"X-ray crystallography (BPS–IR kinase domain complex and SH2 domain), structural modeling","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of the complex with mechanistic interpretation, foundational structural study replicated and cited widely","pmids":["16246733"],"is_preprint":false},{"year":2000,"finding":"Grb14 binds activated FGFR1 in a kinase-activity-dependent manner in mammalian cells; the SH2 domain partially mediates binding, but full-length Grb14 restricts specificity to FGFR1 (not PDGFR). Deletion of SH2 eliminates dependence on receptor activation. Overexpression of Grb14 inhibits FGF-2-induced cell proliferation, while an SH2 point mutant that cannot bind FGFR1 enhances mitogenesis. FGF-2 treatment enhances Grb14 serine/threonine phosphorylation.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, cell proliferation assay, point mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — Y2H, Co-IP, and functional proliferation assays with point mutant control, multiple orthogonal methods","pmids":["10713090"],"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 in insulin-stimulated Grb14-overexpressing cells. Estradiol blocks the insulin-induced upregulation of Grb14 expression.","method":"Overexpression in MCF-7 cells, cell cycle analysis, western blot for ERK1/2 phosphorylation","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — overexpression with defined cell cycle and signaling readouts, single lab, limited domain dissection","pmids":["15372466"],"is_preprint":false},{"year":2006,"finding":"Using real-time BRET in living cells, Grb14 is rapidly recruited to the activated IR upon insulin stimulation in a dose-dependent manner. Grb14 reduces insulin-induced IR–PTP1B interaction (BRET). Grb14 protects IR kinase-loop tyrosines (Y1158/Y1162/Y1163) from dephosphorylation by PTP1B while favoring dephosphorylation of Y972, thereby decreasing IRS-1 recruitment to the IR and reducing ERK pathway activation.","method":"BRET in living HEK cells, site-specific phosphotyrosine antibodies, co-immunoprecipitation","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — real-time BRET plus site-specific phospho-antibodies plus Co-IP, multiple orthogonal methods, mechanism precisely defined","pmids":["16582879"],"is_preprint":false},{"year":2006,"finding":"Grb14 undergoes insulin-induced dimerization as demonstrated by BRET saturation experiments, likely as a result of two Grb14 molecules binding simultaneously on the activated IR dimer. Grb14 reduces IR–PTP1B interaction in living cells, protecting kinase-loop phosphotyrosines while favoring dephosphorylation of Y972.","method":"BRET saturation experiments, co-immunoprecipitation, site-specific phospho-antibodies","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BRET saturation and Co-IP, single lab, mechanistically consistent with EMBO reports study from same period","pmids":["16934761"],"is_preprint":false},{"year":2005,"finding":"The PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. Grb14–IRS-1 complexes are detected in retinal tissue lysates. This suggests that Grb14 may sequester IRS-1 via its NPXY motif, acting as a dominant negative for IRS-1 function in the retina.","method":"Yeast two-hybrid (bovine retinal library screen), in vitro pulldown, co-immunoprecipitation from retinal lysates","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Y2H plus Co-IP from native tissue, single lab, functional consequence inferred but not directly tested","pmids":["15924411"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of Grb14 RA and PH domains reveals they form an integrated dimeric structural unit. Biochemical studies demonstrate that Grb14 binds activated Ras (GTP-loaded), suggesting a timing mechanism for downregulation of insulin signaling. The RA-PH tandem unit is structurally related to RIAM, lamellipodin, and MIG-10.","method":"X-ray crystallography (RA-PH domains of Grb10, 2.6 Å), biochemical Ras-binding assay for Grb14","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus biochemical binding assay; note structure is of Grb10 RA-PH but Grb14 Ras binding is directly demonstrated biochemically","pmids":["19648926"],"is_preprint":false},{"year":2009,"finding":"In compound Grb10/Grb14 double-knockout mice, ablation of both adaptors does not further enhance insulin-induced IRS-1 phosphorylation or Akt activation beyond single knockouts, suggesting context-dependent limiting mechanisms including IR hypophosphorylation and decreased IRS-1 expression. However, double KO mice are protected from high-fat diet-induced glucose intolerance whereas single KOs are not.","method":"Compound gene knockout mice, insulin tolerance and glucose tolerance tests, western blot for IR/IRS-1/Akt signaling","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in compound KO mice with defined signaling and metabolic phenotypes, multiple readouts","pmids":["19541746"],"is_preprint":false},{"year":2009,"finding":"Molecular determinants of Grb14–IR interaction: Grb14 L404 contacts IR L1038 (αC-helix) and Grb14 R385 contacts IR K1168 (activation loop). The L404 contact is likely important for IR-binding specificity among Grb7 family members. Phosphorylation status of Grb14-S370 controls biological activity. Insulin-induced Grb14–PDK1 interaction is required in addition to Grb14–IR binding for maximal inhibition of insulin signaling.","method":"Mutagenesis, co-immunoprecipitation, BRET, Xenopus oocyte assay, Grb14 KO MEF complementation assay","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with BRET, Co-IP, and multiple functional assays, multiple orthogonal methods in single study","pmids":["19359342"],"is_preprint":false},{"year":2010,"finding":"Grb14 is recruited to activated FGFR1 in a trimeric complex also containing PLCγ. Grb14 binding to pY766 of FGFR1 (the PLCγ binding site) alters FGF-induced PLCγ phosphorylation and activation. A molecular model proposes that Grb14 binding induces a conformational change that traps and inactivates PLCγ.","method":"BRET, Xenopus oocyte functional assay (FGF-induced maturation), site-directed mutagenesis of FGFR pY766","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BRET plus functional oocyte assay plus mutagenesis, single lab, mechanistic model supported by experiment","pmids":["20932831"],"is_preprint":false},{"year":2011,"finding":"Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for competitive inhibition of PTP1B activity. Rhodopsin-regulated Src kinase activation in retina phosphorylates Grb14. Ablation of Grb14 results in significantly elevated retinal PTP1B activity in vivo, identifying Grb14 as an endogenous, phosphorylation-regulated inhibitor of PTP1B.","method":"In vitro PTP1B activity assay, site-directed mutagenesis (Y347), retinal Grb14 KO mice, in vivo phosphorylation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzyme assay with mutagenesis plus in vivo KO validation plus identification of upstream kinase (Src), multiple orthogonal methods","pmids":["21791607"],"is_preprint":false},{"year":2011,"finding":"Grb14 promotes closure of photoreceptor-specific CNG channel alpha subunit (CNGA1) through its RA domain via electrostatic interaction. Grb14 competes with cGMP for the CNGA1 binding pocket; Glu180-182 in Grb14 are critical for electrostatic interaction with Arg559 in the cGMP-binding pocket of CNGA1. Lys140 also participates. Grb7 and Grb10 do not modulate channel activity, indicating specificity.","method":"In vitro binding assay, electrophysiology (CNG channel recordings), site-directed mutagenesis of Grb14 RA domain","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus binding assay plus channel functional readout, single lab","pmids":["22180090"],"is_preprint":false},{"year":2011,"finding":"In thyroid cancer cells, Grb14 knockdown diminishes RET phosphorylation and reduces Akt and STAT3 activation, decreasing cell proliferation and invasion. Forced Grb14 expression interrupts IR signaling but facilitates RET activation and STAT3/Akt phosphorylation, enhancing cell invasion and metastasis in xenograft models.","method":"Stable shRNA knockdown, forced overexpression, western blot, in vitro invasion assay, orthotopic mouse xenograft","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with in vitro and in vivo readouts, single lab","pmids":["22158039"],"is_preprint":false},{"year":2012,"finding":"Grb14 SH2 domain binds to the ITAM-like sequence of the phagocytic receptor CEACAM3 (identified by SH2 domain microarray). Grb14 is rapidly recruited to sites of bacteria–host cell contact. Both knockdown and overexpression of Grb14 reduce CEACAM3-mediated phagocytosis of Neisseria gonorrhoeae, establishing Grb14 as a negative regulator of CEACAM3-initiated bacterial phagocytosis.","method":"SH2 domain microarray, co-immunoprecipitation, FRET-FLIM, RNAi knockdown, overexpression, phagocytosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including FRET-FLIM for direct interaction in intact cells, functional phagocytosis assay, both KD and OE tested","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. The RA and PH domains form an integrated unit capable of simultaneously binding activated Ras and phosphoinositide lipids. Binding mode of Grb14 RA to Ras resembles RalGDS and Raf1 RA domains but with distinct features.","method":"X-ray crystallography (2.4 Å, Grb14 RA-PH + H-Ras G12V complex)","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of the complex at high resolution with structural analysis of binding interface","pmids":["23967305"],"is_preprint":false},{"year":2008,"finding":"In rat liver in vivo, insulin injection causes rapid, dose-dependent redistribution of endogenous Grb14 from cytosol to plasma membrane and then to Golgi/endosome fractions, paralleling IR phosphorylation. Co-immunoprecipitation shows Grb14 associates with phosphorylated IR beta-subunit in these fractions. Removal of endogenous Grb14 from membrane fractions by KCl treatment increases IR tyrosine kinase activity, demonstrating that endogenous Grb14 exerts negative feedback on IR activity in vivo.","method":"Rat liver subcellular fractionation, co-immunoprecipitation, in vitro IR kinase assay after KCl extraction","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo fractionation with kinase activity functional readout, multiple orthogonal methods, physiological system","pmids":["18657188"],"is_preprint":false},{"year":2008,"finding":"Grb14 knockdown in primary hepatocytes via RNAi enhances insulin-induced Akt, GSK3, and FOXO1 phosphorylation, consistent with IR inhibition by Grb14. However, Grb14 depletion completely abrogates insulin-induced SREBP-1c maturation, blunting lipogenic gene expression despite improved Akt signaling, revealing a dual role for Grb14 in liver: inhibitor of IR and positive regulator of SREBP-1c-mediated lipogenesis.","method":"RNAi in primary mouse hepatocytes, western blot, gene expression analysis","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean RNAi with multiple metabolic readouts, single lab, unexpected dual role with no further mechanistic dissection at this level","pmids":["18339716"],"is_preprint":false},{"year":2013,"finding":"In rod photoreceptors, Grb14 deletion accelerates response decay (decreased integration time and τREC) and decreases the limiting time constant τD, indicating that Grb14 modulates both cGMP-gated channel sensitivity and the rate of PDE6 inactivation. Grb14 effects are present in dark-adapted rods and enhanced after bleaching adaptation.","method":"Suction-electrode recordings from single mouse rods (Grb14 KO vs. WT), dark- and light-adapted conditions","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with electrophysiological readout, single lab, mechanism not fully resolved at molecular level","pmids":["24273167"],"is_preprint":false},{"year":2013,"finding":"In retinal neurons, phosphorylation of the BPS region of Grb14 promotes IR activation by inhibiting PTP1B, whereas unphosphorylated Grb14 acts with PTP1B to negatively regulate IR. Decreased Grb14 phosphorylation was observed in diabetic Ins2Akita mouse retinas, correlating with decreased IR activation.","method":"Co-immunoprecipitation, proximity ligation assay (PLA), phospho-specific analysis, Ins2Akita diabetic mouse retina","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — PLA and Co-IP with disease model validation, but mechanistic model relies partly on inference from prior studies","pmids":["24350791"],"is_preprint":false},{"year":2014,"finding":"GSK-3 phosphorylates serine residues (Ser358, Ser362, Ser366) in the N-terminal BPS domain of Grb14. Pharmacological inhibition or knockdown of GSK-3 facilitates Grb14-IR complex formation. Serine-to-alanine substitution at these sites also facilitates Grb14-IR binding. Phospho-Ser366 of endogenous Grb14 is regulated by insulin in HepG2 cells, establishing GSK-3-mediated phosphorylation as a negative regulator of Grb14-IR complex formation.","method":"In vitro kinase assay, proximity ligation assay, pharmacological GSK-3 inhibition, siRNA knockdown, site-directed mutagenesis","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus cell-based PLA plus mutagenesis, multiple orthogonal methods identifying the writer and target residues","pmids":["24535599"],"is_preprint":false},{"year":2015,"finding":"Chfr ubiquitin ligase is a binding partner of Grb14 (binding at T220 of Grb14 upon insulin stimulation) and potentiates Grb14's inhibitory effect on insulin-induced cell division. Chfr ligase activity and phosphorylation of its T39 residue (an Akt target) are required. In the presence of insulin, Chfr binding to Grb14 activates Chfr E3 ligase, leading to Aurora A and Polo-like kinase degradation and blocking cell division.","method":"Co-immunoprecipitation, Xenopus oocyte G2/M assay, mutagenesis (T220, Chfr T39, Chfr RING domain), mammalian cell proliferation assays","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, oocyte assay, mutagenesis of both partners), functional consequence (Aurora A/PLK degradation) defined","pmids":["25578860"],"is_preprint":false},{"year":2016,"finding":"Grb14 knockdown in mouse liver silences de novo fatty acid synthesis despite enhancing Akt signaling. Mechanistically, Grb14 binds p62/sqstm1; upon Grb14 knockdown, released p62 activates the transcription factor Nrf2, which represses the lipogenic nuclear receptor LXR, thereby suppressing lipogenesis. This identifies Grb14 as a signaling node linking the IR to the p62-Nrf2-LXR pathway.","method":"Liver-specific AAV shRNA knockdown in lean and obese mice, western blot, gene expression, de novo lipogenesis assay, co-immunoprecipitation of Grb14-p62","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo liver-specific KD with molecular mechanism dissected (Grb14–p62 Co-IP, Nrf2/LXR pathway), multiple readouts","pmids":["27215388"],"is_preprint":false},{"year":2017,"finding":"Phosphorylation of N-terminal BPS domain serines (Ser358, Ser362, Ser366) by GSK-3 negatively regulates Grb14–IR complex formation; glutamic acid substitutions at these sites reduce Kd from ~8 nM to undetectable by SPR. Protein phosphatase 1 (PP1) dephosphorylates Ser358 and Ser362, and this dephosphorylation facilitates Grb14–IR complex formation.","method":"In vitro kinase assay, surface plasmon resonance (SPR), yeast two-hybrid, co-immunoprecipitation, phosphopeptide phosphatase assay","journal":"Journal of biochemistry / Journal of peptide science","confidence":"High","confidence_rationale":"Tier 1 / Strong — SPR provides quantitative affinity, plus in vitro phosphatase assay identifies PP1, plus Co-IP and Y2H, multiple orthogonal methods","pmids":["28130417","31347216"],"is_preprint":false}],"current_model":"GRB14 is a multi-domain adaptor protein (RA–PH–BPS–SH2) that functions primarily as a tissue-specific negative regulator of insulin receptor (IR) signaling: its BPS/PIR domain acts as a pseudosubstrate inhibitor occupying the IR substrate-binding groove (crystal structure), while the SH2 domain anchors to phosphotyrosines on the activated IR; GSK-3 phosphorylates BPS-domain serines (Ser358/362/366) to prevent premature IR association, and PP1 dephosphorylates these sites to permit recruitment; once at the IR, Grb14 selectively protects kinase-loop phosphotyrosines from PTP1B while favoring dephosphorylation of Y972, thereby reducing IRS-1 recruitment and ERK/Akt signaling; PKCζ (recruited via the ZIP adaptor) phosphorylates Grb14 to further enhance its inhibitory activity; Grb14 also recruits PDK-1 to the IR to facilitate Akt activation, revealing a dual role; in liver, Grb14 sequesters p62 to suppress the Nrf2–LXR lipogenic axis; Grb14 additionally interacts with activated Ras (via RA domain), FGFR1 (blocking PLCγ), tankyrase 2, IRS-1 PTB domain, PTP1B (inhibiting phosphatase activity when Grb14-Y347 is phosphorylated by Src), CEACAM3 (inhibiting bacterial phagocytosis), the Chfr ubiquitin ligase (blocking insulin-driven cell division via Aurora A/PLK degradation), and the photoreceptor CNG channel (closing it via electrostatic RA-domain interaction with CNGA1)."},"narrative":{"mechanistic_narrative":"GRB14 is a multi-domain adaptor protein that functions principally as a tissue-specific negative regulator of insulin receptor (IR) signaling, binding the activated IR in an insulin-dependent manner and dampening downstream IRS-1 phosphorylation, glycogen synthesis, and DNA synthesis [PMID:9748281, PMID:14749734]. Its inhibitory core is the BPS/PIR domain, which acts as a pseudosubstrate inhibitor occupying the IR kinase substrate-binding groove while the SH2 domain anchors to receptor phosphotyrosines, as resolved by the BPS–IR kinase domain crystal structure [PMID:16246733]; biochemically the BPS domain alone inhibits IR substrate phosphorylation uncompetitively, and although intrinsically unstructured it retains full activity [PMID:11726652, PMID:14623073]. Once bound, Grb14 selectively protects IR kinase-loop phosphotyrosines from PTP1B while favoring dephosphorylation of Y972, reducing IRS-1 recruitment and ERK activation [PMID:16582879], and is itself recruited and dimerized on the activated receptor dimer in real time [PMID:16934761, PMID:18657188]. Recruitment is gated by phosphorylation: GSK-3 phosphorylates BPS serines Ser358/362/366 to prevent premature IR association, while PP1 dephosphorylates these sites to permit high-affinity binding [PMID:24535599, PMID:28130417, PMID:31347216], and PKCζ (via the ZIP adaptor) phosphorylates Grb14 to enhance its inhibitory activity [PMID:12242277]. Grb14 carries a dual role, additionally recruiting PDK-1 to facilitate insulin-dependent Akt activation [PMID:15210700] and, in liver, sequestering p62 to permit SREBP-1c– and LXR-driven lipogenesis [PMID:18339716, PMID:27215388]. Beyond insulin signaling, Grb14 binds activated Ras through an integrated RA–PH unit [PMID:19648926, PMID:23967305], restricts FGFR1–PLCγ signaling [PMID:10713090, PMID:20932831], inhibits PTP1B when Grb14-Y347 is phosphorylated by Src in retina [PMID:21791607], gates the photoreceptor CNG channel via its RA domain [PMID:22180090, PMID:24273167], and negatively regulates CEACAM3-mediated bacterial phagocytosis through its SH2 domain [PMID:22948154].","teleology":[{"year":1996,"claim":"Established Grb14 as a phosphotyrosine-binding adaptor coupled to receptor tyrosine kinase signaling, the first clue to its function.","evidence":"GST-SH2 domain pulldown against activated PDGF receptors and in vivo serine phosphorylation labeling","pmids":["8647858"],"confidence":"Medium","gaps":["No functional consequence of PDGFR binding tested","Identity of the PDGF-regulated serine kinase unknown"]},{"year":1998,"claim":"Defined Grb14 as an inhibitory regulator of insulin signaling that binds the IR via its PIR domain, reframing it as a negative feedback adaptor rather than a positive coupler.","evidence":"Yeast two-hybrid, Co-IP, deletion-mutant binding, and CHO-IR cell metabolic readouts","pmids":["9748281"],"confidence":"High","gaps":["Did not resolve the catalytic mechanism of inhibition","Tissue relevance not yet established"]},{"year":2001,"claim":"Showed the BPS/PIR domain alone uncompetitively inhibits IR substrate phosphorylation and protects the IR from phosphatases, separating substrate occlusion from receptor anchoring.","evidence":"In vitro kinase assays with deletion mutants and CHO-IR cell signaling readouts","pmids":["11726652"],"confidence":"High","gaps":["Structural basis of substrate-groove occlusion not yet visualized","Which phosphatase is antagonized unspecified at this stage"]},{"year":2001,"claim":"Identified tankyrase 2 as an N-terminal binding partner, hinting at additional Grb14 interactions beyond the IR.","evidence":"Yeast two-hybrid, reciprocal Co-IP, and microsomal fractionation","pmids":["11278563"],"confidence":"Medium","gaps":["Functional consequence of tankyrase 2 binding not established","No link to insulin signaling demonstrated"]},{"year":2002,"claim":"Placed Grb14 in a PKCζ–ZIP heterotrimeric complex and showed phosphorylation by PKCζ enhances its IR inhibition, adding a kinase input that tunes inhibitory strength.","evidence":"Co-IP, in vitro kinase assay, in vivo 32P labeling, and Xenopus oocyte maturation assay","pmids":["12242277"],"confidence":"High","gaps":["Phosphorylated residues on Grb14 not mapped here","Physiological relevance in metabolic tissues untested"]},{"year":2003,"claim":"Demonstrated the inhibitory PIR domain is intrinsically unstructured yet fully active, indicating function does not require a stable fold.","evidence":"NMR of 15N-labeled recombinant PIR and Xenopus oocyte assay","pmids":["14623073"],"confidence":"Medium","gaps":["Conformation when bound to IR not resolved","No partner-induced folding shown"]},{"year":2004,"claim":"Genetic ablation in mice proved Grb14 is a physiological, tissue-specific repressor of IR–IRS-1 signaling controlling glucose homeostasis.","evidence":"Knockout mouse with glucose/insulin tolerance tests, ex vivo metabolic assays, and signaling western blots","pmids":["14749734"],"confidence":"High","gaps":["Tissue-specific limiting factors not identified","Did not explain how liver IRS-1 phosphorylation rises despite lower IR autophosphorylation"]},{"year":2004,"claim":"Revealed a dual role by which Grb14 constitutively recruits PDK-1 to facilitate Akt activation, complicating its purely inhibitory image.","evidence":"Directed proteomics pulldown, Co-IP, point mutagenesis, fractionation, and Akt activity assay","pmids":["15210700"],"confidence":"High","gaps":["Balance between IR inhibition and Akt facilitation not quantified","PDK-1 binding motif structural detail limited"]},{"year":2004,"claim":"Linked Grb14 abundance to insulin resistance, showing hormonal regulation of its expression across diabetic models.","evidence":"Western blot, RT-PCR, and 3T3-F442A adipocyte treatment assays in ob/ob, GK rat, and human diabetic tissue","pmids":["15059968"],"confidence":"Medium","gaps":["Causal direction between Grb14 levels and insulin resistance not established","Transcriptional regulators of Grb14 unidentified"]},{"year":2004,"claim":"Biophysical characterization detected a transiently structured stretch (399–407) in the disordered PIR, suggesting partner-induced ordering.","evidence":"SAXS, circular dichroism with TFE, and sequence analysis","pmids":["15465854"],"confidence":"Medium","gaps":["Functional consequence of the transient structure untested","Binding partner triggering the transition unknown"]},{"year":2005,"claim":"The BPS–IR kinase domain crystal structure resolved the pseudosubstrate inhibition mechanism, providing the structural basis for substrate-groove occlusion plus SH2 anchoring.","evidence":"X-ray crystallography of the BPS–IR kinase complex and SH2 domain with modeling","pmids":["16246733"],"confidence":"High","gaps":["Full-length Grb14 conformation on the IR dimer not crystallized","Regulatory phosphorylation not captured in structure"]},{"year":2005,"claim":"Extended Grb14's inhibitory reach to estrogen- and insulin-driven proliferation in breast cancer cells, tying it to mitogenic control.","evidence":"MCF-7 overexpression, cell cycle analysis, and ERK1/2 phospho western blots","pmids":["15372466"],"confidence":"Medium","gaps":["Domain requirements for the antiproliferative effect not dissected","Overexpression-only evidence without loss-of-function"]},{"year":2005,"claim":"Identified phosphorylation-independent binding of the IRS-1 PTB domain to a Grb14 NPXY motif, proposing IRS-1 sequestration in retina.","evidence":"Yeast two-hybrid retinal screen, in vitro pulldown, and Co-IP from retinal lysates","pmids":["15924411"],"confidence":"Medium","gaps":["Functional sequestration of IRS-1 inferred but not directly tested","Retinal relevance of the complex not validated in vivo here"]},{"year":2000,"claim":"Showed Grb14 binds activated FGFR1 with receptor specificity and inhibits FGF-2-induced proliferation, broadening its RTK targets beyond the IR.","evidence":"Yeast two-hybrid, Co-IP, in vitro binding, proliferation assay, and SH2 point mutagenesis","pmids":["10713090"],"confidence":"High","gaps":["Molecular mechanism of FGFR1 inhibition not yet defined","In vivo relevance untested"]},{"year":2006,"claim":"Real-time imaging defined how Grb14 reshapes IR phosphorylation—protecting kinase-loop tyrosines from PTP1B while favoring Y972 dephosphorylation—to selectively curtail IRS-1 recruitment.","evidence":"BRET in living HEK cells, site-specific phosphotyrosine antibodies, and Co-IP","pmids":["16582879"],"confidence":"High","gaps":["Structural basis of site-selective phosphatase protection unresolved","Generality across tissues not addressed"]},{"year":2006,"claim":"Demonstrated insulin-induced Grb14 dimerization, consistent with two molecules binding the activated IR dimer.","evidence":"BRET saturation experiments, Co-IP, and site-specific phospho-antibodies","pmids":["16934761"],"confidence":"Medium","gaps":["Dimerization interface not mapped","Functional necessity of dimerization untested"]},{"year":2008,"claim":"Captured insulin-driven redistribution of endogenous Grb14 to the IR in rat liver and confirmed negative feedback by showing its removal raises IR kinase activity.","evidence":"Rat liver subcellular fractionation, Co-IP, and in vitro IR kinase assay after KCl extraction","pmids":["18657188"],"confidence":"High","gaps":["Trafficking determinants directing Grb14 to membrane/Golgi/endosome unclear","Link between localization and inhibition mechanism not resolved"]},{"year":2008,"claim":"Uncovered the liver dual role: Grb14 inhibits IR but is required for insulin-induced SREBP-1c maturation and lipogenic gene expression.","evidence":"RNAi in primary mouse hepatocytes with signaling and gene-expression readouts","pmids":["18339716"],"confidence":"Medium","gaps":["Molecular link from Grb14 to SREBP-1c not yet defined at this stage","How lipogenesis persists despite improved Akt unexplained"]},{"year":2009,"claim":"Structural and biochemical work defined the RA–PH tandem as an integrated unit that binds GTP-loaded Ras, proposing Ras-dependent timing of insulin signaling downregulation.","evidence":"X-ray crystallography of RA-PH (Grb10) and biochemical Ras-binding assay for Grb14","pmids":["19648926"],"confidence":"High","gaps":["Functional consequence of Grb14–Ras binding in cells not tested","Crystal structure was of Grb10 RA-PH, not Grb14"]},{"year":2009,"claim":"Compound Grb10/Grb14 knockout established genetic epistasis and showed combined loss protects against diet-induced glucose intolerance, revealing limiting compensatory mechanisms.","evidence":"Double-knockout mice with tolerance tests and IR/IRS-1/Akt signaling readouts","pmids":["19541746"],"confidence":"High","gaps":["Identity of the limiting factors (IR hypophosphorylation, IRS-1 levels) not mechanistically resolved","Tissue contributions to protection unseparated"]},{"year":2009,"claim":"Mutagenesis pinpointed the Grb14–IR contact residues conferring specificity and confirmed the PDK-1 interaction is required alongside IR binding for maximal inhibition.","evidence":"Mutagenesis, Co-IP, BRET, Xenopus oocyte assay, and Grb14 KO MEF complementation","pmids":["19359342"],"confidence":"High","gaps":["How S370 phosphorylation status mechanistically gates activity not fully resolved","Structural model of full assembly incomplete"]},{"year":2010,"claim":"Defined the FGFR1 inhibition mechanism: Grb14 binds FGFR1 pY766 within a PLCγ-containing trimeric complex to trap and inactivate PLCγ.","evidence":"BRET, Xenopus oocyte FGF-maturation assay, and FGFR pY766 mutagenesis","pmids":["20932831"],"confidence":"Medium","gaps":["Conformational trapping model not structurally confirmed","Physiological role in FGF biology untested"]},{"year":2011,"claim":"Identified Grb14 as a phosphorylation-regulated inhibitor of PTP1B in retina, with Tyr-347 phosphorylation by rhodopsin-activated Src enabling competitive PTP1B inhibition.","evidence":"In vitro PTP1B activity assay, Y347 mutagenesis, retinal Grb14 KO mice, and in vivo phosphorylation assays","pmids":["21791607"],"confidence":"High","gaps":["How Y347 phosphorylation reconciles with IR-protective role unclear","Retinal substrate landscape of derepressed PTP1B not mapped"]},{"year":2011,"claim":"Revealed a non-adaptor channel-gating function: Grb14's RA domain electrostatically competes with cGMP to close the photoreceptor CNGA1 channel, with isoform specificity.","evidence":"In vitro binding, CNG channel electrophysiology, and RA-domain mutagenesis","pmids":["22180090"],"confidence":"Medium","gaps":["In vivo channel regulation not yet shown here","Structural detail of the RA–CNGA1 interface limited"]},{"year":2011,"claim":"Showed Grb14 can act as a positive driver of oncogenic RET signaling in thyroid cancer, promoting Akt/STAT3 activation, invasion, and metastasis.","evidence":"Stable shRNA knockdown, overexpression, invasion assay, and orthotopic mouse xenograft","pmids":["22158039"],"confidence":"Medium","gaps":["Direct Grb14–RET binding not structurally defined","Mechanism distinguishing RET facilitation from IR inhibition unresolved"]},{"year":2012,"claim":"Defined Grb14 as a negative regulator of CEACAM3-mediated bacterial phagocytosis via SH2 binding to the receptor's ITAM-like motif.","evidence":"SH2 domain microarray, Co-IP, FRET-FLIM, RNAi, overexpression, and phagocytosis assay","pmids":["22948154"],"confidence":"High","gaps":["Downstream signaling blocked by Grb14 at CEACAM3 not detailed","Why both knockdown and overexpression reduce phagocytosis unexplained mechanistically"]},{"year":2013,"claim":"High-resolution crystal structure of Grb14 RA–PH bound to GTP-loaded H-Ras showed the integrated unit can simultaneously engage Ras and phosphoinositides.","evidence":"X-ray crystallography at 2.4 Å of the Grb14 RA-PH–H-Ras G12V complex","pmids":["23967305"],"confidence":"High","gaps":["Cellular function of simultaneous Ras/lipid engagement not tested","Coupling to IR inhibition timing not demonstrated structurally"]},{"year":2013,"claim":"Single-rod electrophysiology showed Grb14 modulates both CNG channel sensitivity and PDE6 inactivation rate, defining a physiological role in phototransduction.","evidence":"Suction-electrode recordings from single Grb14 KO vs WT mouse rods in dark- and light-adapted states","pmids":["24273167"],"confidence":"Medium","gaps":["Molecular basis of PDE6 inactivation effect unresolved","Whether channel and PDE6 effects share a mechanism unclear"]},{"year":2013,"claim":"Showed the BPS phosphorylation state switches Grb14 between IR-promoting (PTP1B-inhibiting) and IR-inhibiting modes in retinal neurons, with relevance to diabetic retinopathy.","evidence":"Co-IP, proximity ligation assay, phospho-specific analysis, and Ins2Akita diabetic mouse retina","pmids":["24350791"],"confidence":"Medium","gaps":["Switch mechanism relies partly on inference from prior studies","Kinase/phosphatase setting the retinal phospho-state not fully defined"]},{"year":2014,"claim":"Identified GSK-3 as the kinase phosphorylating BPS serines Ser358/362/366 to block premature Grb14–IR complex formation, defining an upstream gating input.","evidence":"In vitro kinase assay, proximity ligation assay, GSK-3 inhibition/knockdown, and S-to-A mutagenesis","pmids":["24535599"],"confidence":"High","gaps":["How insulin lowers GSK-3 activity to permit binding not detailed here","The opposing phosphatase not yet identified at this stage"]},{"year":2015,"claim":"Connected Grb14 to cell-cycle control by showing insulin-dependent Chfr binding activates the E3 ligase to degrade Aurora A and PLK, blocking insulin-driven division.","evidence":"Co-IP, Xenopus oocyte G2/M assay, mutagenesis of Grb14 T220 and Chfr T39/RING, and mammalian proliferation assays","pmids":["25578860"],"confidence":"High","gaps":["Physiological context of Grb14–Chfr-driven division control unclear","Tissue specificity of the effect untested"]},{"year":2016,"claim":"Resolved the liver lipogenic dual role mechanistically: Grb14 sequesters p62, and its loss releases p62 to activate Nrf2 and repress LXR, silencing de novo lipogenesis.","evidence":"Liver-specific AAV shRNA in lean and obese mice with lipogenesis assays, gene expression, and Grb14–p62 Co-IP","pmids":["27215388"],"confidence":"High","gaps":["Structural basis of Grb14–p62 binding undefined","How IR engagement is coupled to p62 release unresolved"]},{"year":2017,"claim":"Quantified the phospho-gated affinity switch and identified PP1 as the phosphatase that, by dephosphorylating Ser358/362, licenses high-affinity Grb14–IR binding.","evidence":"In vitro kinase assay, surface plasmon resonance, yeast two-hybrid, Co-IP, and phosphopeptide phosphatase assay","pmids":["28130417","31347216"],"confidence":"High","gaps":["How insulin coordinates GSK-3 inactivation with PP1 activation in vivo unclear","Spatiotemporal control of the phospho-switch at the membrane undefined"]},{"year":null,"claim":"How Grb14's many context-specific roles—IR inhibition vs Akt/RET facilitation, lipogenic regulation, channel gating, and immune phagocytosis—are coordinated within a single full-length protein in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length Grb14 structure on an intact receptor","Tissue-specific determinants selecting inhibitory vs facilitatory output unknown","Integration of RA/Ras, BPS/IR, and SH2 inputs into a unified regulatory logic not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,8,30,31]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,10,13,20]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[15,31]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,25]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[25]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[25]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,13,18]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6,26,31]},{"term_id":"R-HSA-9709957","term_label":"Sensory Perception","supporting_discovery_ids":[21,27]}],"complexes":["PKCζ–ZIP–Grb14 complex"],"partners":["INSR","PTP1B","PDPK1","FGFR1","HRAS","SQSTM1","CHFR","CEACAM3"],"other_free_text":[]}},"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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reports","url":"https://pubmed.ncbi.nlm.nih.gov/29203791","citation_count":6,"is_preprint":false},{"pmid":"24535599","id":"PMC_24535599","title":"Phosphorylation of Grb14 BPS domain by GSK-3 correlates with complex forming of Grb14 and insulin receptor.","date":"2014","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24535599","citation_count":5,"is_preprint":false},{"pmid":"28130417","id":"PMC_28130417","title":"Phosphorylation of clustered serine residues in the N-terminus of BPS domain negatively regulates formation of the complex between human Grb14 and insulin receptor.","date":"2017","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28130417","citation_count":5,"is_preprint":false},{"pmid":"39343875","id":"PMC_39343875","title":"Grb7, Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism.","date":"2024","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/39343875","citation_count":4,"is_preprint":false},{"pmid":"25578860","id":"PMC_25578860","title":"Insulin-induced cell division is controlled by the adaptor Grb14 in a Chfr-dependent manner.","date":"2015","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/25578860","citation_count":4,"is_preprint":false},{"pmid":"22180090","id":"PMC_22180090","title":"Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotidegated channel by the tyrosine kinase adapter protein Grb14.","date":"2011","source":"Protein & cell","url":"https://pubmed.ncbi.nlm.nih.gov/22180090","citation_count":4,"is_preprint":false},{"pmid":"15322292","id":"PMC_15322292","title":"Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain.","date":"2004","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/15322292","citation_count":3,"is_preprint":false},{"pmid":"12522317","id":"PMC_12522317","title":"Assignment of backbone 1H, 13C, and 15N resonances of the SH2 domain of human Grb14.","date":"2002","source":"Journal of biomolecular NMR","url":"https://pubmed.ncbi.nlm.nih.gov/12522317","citation_count":2,"is_preprint":false},{"pmid":"31347216","id":"PMC_31347216","title":"Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation.","date":"2019","source":"Journal of peptide science : an official publication of the European Peptide Society","url":"https://pubmed.ncbi.nlm.nih.gov/31347216","citation_count":1,"is_preprint":false},{"pmid":"25190572","id":"PMC_25190572","title":"[Control of insulin signalisation and action by the Grb14 protein].","date":"2014","source":"Biologie aujourd'hui","url":"https://pubmed.ncbi.nlm.nih.gov/25190572","citation_count":0,"is_preprint":false},{"pmid":"38202781","id":"PMC_38202781","title":"Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.","date":"2023","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/38202781","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26901,"output_tokens":8801,"usd":0.106359,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18689,"output_tokens":8566,"usd":0.153798,"stage2_stop_reason":"end_turn"},"total_usd":0.260157,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"GRB14 SH2 domain binds to activated PDGF receptors in vitro; GRB14 is phosphorylated on serine residues in response to PDGF stimulation, identifying it as a target for a PDGF-regulated serine kinase.\",\n      \"method\": \"GST-SH2 domain pulldown, in vivo serine phosphorylation labeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — GST pulldown and phosphorylation labeling, single lab, two methods but no functional follow-up on the in vivo interaction\",\n      \"pmids\": [\"8647858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rat Grb14 binds the insulin receptor in an insulin-dependent manner in vivo; the PIR domain (between PH and SH2) is the primary IR-binding domain, while the SH2 domain contributes secondarily. Binding requires IR tyrosines Y1150/Y1151. Overexpression of Grb14 in CHO-IR cells decreases insulin-stimulated IRS-1 tyrosine phosphorylation and reduces glycogen and DNA synthesis, establishing Grb14 as an inhibitory regulator of insulin signaling.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, deletion mutant in vitro binding, CHO cell overexpression with metabolic readouts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, Co-IP, in vitro binding, cell-based functional assays), replicated by subsequent studies\",\n      \"pmids\": [\"9748281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Grb14 directly inhibits insulin receptor (IR) substrate phosphorylation in vitro, acting as an uncompetitive inhibitor with respect to peptide substrate but not altering Km for ATP. The PIR/BPS domain alone is sufficient for this inhibitory effect; the SH2 domain has no effect on IR catalytic activity. In CHO-IR cells, Grb14 binding protects the IR from tyrosine phosphatases, maintaining it in a phosphorylated state, but delays Akt and ERK1/2 activation.\",\n      \"method\": \"In vitro kinase assay with deletion mutants, CHO cell co-immunoprecipitation, western blot for downstream signaling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with domain dissection and mutagenesis, replicated across multiple studies\",\n      \"pmids\": [\"11726652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Tankyrase 2 was identified as a novel binding partner for Grb14 via yeast two-hybrid screen. The interaction is mediated by the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10–19 of tankyrase 2. Both proteins co-fractionate in the low-density microsome fraction in vivo, and their association can be detected by co-immunoprecipitation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation, deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and fractionation, single lab, two orthogonal methods\",\n      \"pmids\": [\"11278563\"],\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 domain of Grb14, forming a PKCζ–ZIP–Grb14 heterotrimeric complex. PKCζ phosphorylates Grb14 in vitro and in vivo, and this phosphorylation is increased by insulin stimulation. Phosphorylation of Grb14 by PKCζ further enhances Grb14's inhibitory effect on IR tyrosine kinase activity. In Xenopus oocytes, ZIP potentiates Grb14's inhibitory action on insulin-induced maturation in a PKCζ- and phosphorylation-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, in vivo [32P] labeling, Xenopus oocyte functional assay, deletion mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus in vivo labeling plus functional oocyte model, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"12242277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The isolated PIR domain of Grb14 is intrinsically unstructured (lacks secondary structure) as demonstrated by NMR, yet retains full biological activity, inhibiting insulin-induced oocyte maturation in Xenopus.\",\n      \"method\": \"NMR (15N-labeled recombinant PIR), Xenopus oocyte functional assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural characterization with functional validation in oocytes, single lab\",\n      \"pmids\": [\"14623073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Grb14 knockout in mice leads to improved glucose tolerance, lower circulating insulin, increased glycogen synthesis in liver and skeletal muscle, and enhanced insulin-induced glucose uptake in soleus muscle. In liver, despite lower IR autophosphorylation, IRS-1 phosphorylation and PKB activation are enhanced. This demonstrates Grb14 as a tissue-specific in vivo repressor of IR-mediated IRS-1 tyrosine phosphorylation.\",\n      \"method\": \"Gene targeting (knockout mouse), glucose/insulin tolerance tests, ex vivo tissue metabolic assays, western blot for signaling\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with multiple defined cellular and metabolic phenotypes and signaling readouts, replicated by subsequent studies\",\n      \"pmids\": [\"14749734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The PIR domain of Grb14 is natively unstructured but contains a potentially structured short stretch (residues 399–407) that may undergo a structural transition upon binding to a partner, as evidenced by SAXS and circular dichroism with TFE.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), circular dichroism, sequence analysis\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SAXS and CD provide structural characterization, but functional consequence of the transient structure is not directly tested, 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. This interaction is required for insulin-triggered membrane translocation of PDK-1 and facilitates Akt activation. Disruption of this interaction by point mutation or SH2 domain deletion significantly decreases insulin-dependent Akt activation.\",\n      \"method\": \"Directed proteomics pulldown, co-immunoprecipitation, point mutagenesis, subcellular fractionation, Akt activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding plus mutagenesis plus functional localization assay with Akt readout, multiple orthogonal methods\",\n      \"pmids\": [\"15210700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In adipose tissue of ob/ob mice, Goto-Kakizaki rats, and type 2 diabetic humans, Grb14 expression is increased. Insulin stimulates Grb14 expression in 3T3-F442A adipocytes, while thiazolidinediones decrease it, indicating hormonal regulation of Grb14 abundance as part of insulin resistance.\",\n      \"method\": \"Western blot, RT-PCR, 3T3-F442A cell culture treatment assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple models and human tissue, but purely expression-level regulation without direct mechanistic dissection; included because insulin-dependent transcriptional feedback is experimentally shown\",\n      \"pmids\": [\"15059968\"],\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, occupying the substrate peptide-binding groove of the kinase. Together with the SH2 domain crystal structure, a model is presented in which Grb14 simultaneously anchors via its SH2 domain to IR phosphotyrosines and occludes the substrate groove via BPS.\",\n      \"method\": \"X-ray crystallography (BPS–IR kinase domain complex and SH2 domain), structural modeling\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of the complex with mechanistic interpretation, foundational structural study replicated and cited widely\",\n      \"pmids\": [\"16246733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Grb14 binds activated FGFR1 in a kinase-activity-dependent manner in mammalian cells; the SH2 domain partially mediates binding, but full-length Grb14 restricts specificity to FGFR1 (not PDGFR). Deletion of SH2 eliminates dependence on receptor activation. Overexpression of Grb14 inhibits FGF-2-induced cell proliferation, while an SH2 point mutant that cannot bind FGFR1 enhances mitogenesis. FGF-2 treatment enhances Grb14 serine/threonine phosphorylation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, cell proliferation assay, point mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Y2H, Co-IP, and functional proliferation assays with point mutant control, multiple orthogonal methods\",\n      \"pmids\": [\"10713090\"],\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 in insulin-stimulated Grb14-overexpressing cells. Estradiol blocks the insulin-induced upregulation of Grb14 expression.\",\n      \"method\": \"Overexpression in MCF-7 cells, cell cycle analysis, western blot for ERK1/2 phosphorylation\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — overexpression with defined cell cycle and signaling readouts, single lab, limited domain dissection\",\n      \"pmids\": [\"15372466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Using real-time BRET in living cells, Grb14 is rapidly recruited to the activated IR upon insulin stimulation in a dose-dependent manner. Grb14 reduces insulin-induced IR–PTP1B interaction (BRET). Grb14 protects IR kinase-loop tyrosines (Y1158/Y1162/Y1163) from dephosphorylation by PTP1B while favoring dephosphorylation of Y972, thereby decreasing IRS-1 recruitment to the IR and reducing ERK pathway activation.\",\n      \"method\": \"BRET in living HEK cells, site-specific phosphotyrosine antibodies, co-immunoprecipitation\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — real-time BRET plus site-specific phospho-antibodies plus Co-IP, multiple orthogonal methods, mechanism precisely defined\",\n      \"pmids\": [\"16582879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Grb14 undergoes insulin-induced dimerization as demonstrated by BRET saturation experiments, likely as a result of two Grb14 molecules binding simultaneously on the activated IR dimer. Grb14 reduces IR–PTP1B interaction in living cells, protecting kinase-loop phosphotyrosines while favoring dephosphorylation of Y972.\",\n      \"method\": \"BRET saturation experiments, co-immunoprecipitation, site-specific phospho-antibodies\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BRET saturation and Co-IP, single lab, mechanistically consistent with EMBO reports study from same period\",\n      \"pmids\": [\"16934761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. Grb14–IRS-1 complexes are detected in retinal tissue lysates. This suggests that Grb14 may sequester IRS-1 via its NPXY motif, acting as a dominant negative for IRS-1 function in the retina.\",\n      \"method\": \"Yeast two-hybrid (bovine retinal library screen), in vitro pulldown, co-immunoprecipitation from retinal lysates\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Y2H plus Co-IP from native tissue, single lab, functional consequence inferred but not directly tested\",\n      \"pmids\": [\"15924411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of Grb14 RA and PH domains reveals they form an integrated dimeric structural unit. Biochemical studies demonstrate that Grb14 binds activated Ras (GTP-loaded), suggesting a timing mechanism for downregulation of insulin signaling. The RA-PH tandem unit is structurally related to RIAM, lamellipodin, and MIG-10.\",\n      \"method\": \"X-ray crystallography (RA-PH domains of Grb10, 2.6 Å), biochemical Ras-binding assay for Grb14\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus biochemical binding assay; note structure is of Grb10 RA-PH but Grb14 Ras binding is directly demonstrated biochemically\",\n      \"pmids\": [\"19648926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In compound Grb10/Grb14 double-knockout mice, ablation of both adaptors does not further enhance insulin-induced IRS-1 phosphorylation or Akt activation beyond single knockouts, suggesting context-dependent limiting mechanisms including IR hypophosphorylation and decreased IRS-1 expression. However, double KO mice are protected from high-fat diet-induced glucose intolerance whereas single KOs are not.\",\n      \"method\": \"Compound gene knockout mice, insulin tolerance and glucose tolerance tests, western blot for IR/IRS-1/Akt signaling\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in compound KO mice with defined signaling and metabolic phenotypes, multiple readouts\",\n      \"pmids\": [\"19541746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Molecular determinants of Grb14–IR interaction: Grb14 L404 contacts IR L1038 (αC-helix) and Grb14 R385 contacts IR K1168 (activation loop). The L404 contact is likely important for IR-binding specificity among Grb7 family members. Phosphorylation status of Grb14-S370 controls biological activity. Insulin-induced Grb14–PDK1 interaction is required in addition to Grb14–IR binding for maximal inhibition of insulin signaling.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, BRET, Xenopus oocyte assay, Grb14 KO MEF complementation assay\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with BRET, Co-IP, and multiple functional assays, multiple orthogonal methods in single study\",\n      \"pmids\": [\"19359342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Grb14 is recruited to activated FGFR1 in a trimeric complex also containing PLCγ. Grb14 binding to pY766 of FGFR1 (the PLCγ binding site) alters FGF-induced PLCγ phosphorylation and activation. A molecular model proposes that Grb14 binding induces a conformational change that traps and inactivates PLCγ.\",\n      \"method\": \"BRET, Xenopus oocyte functional assay (FGF-induced maturation), site-directed mutagenesis of FGFR pY766\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BRET plus functional oocyte assay plus mutagenesis, single lab, mechanistic model supported by experiment\",\n      \"pmids\": [\"20932831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for competitive inhibition of PTP1B activity. Rhodopsin-regulated Src kinase activation in retina phosphorylates Grb14. Ablation of Grb14 results in significantly elevated retinal PTP1B activity in vivo, identifying Grb14 as an endogenous, phosphorylation-regulated inhibitor of PTP1B.\",\n      \"method\": \"In vitro PTP1B activity assay, site-directed mutagenesis (Y347), retinal Grb14 KO mice, in vivo phosphorylation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzyme assay with mutagenesis plus in vivo KO validation plus identification of upstream kinase (Src), multiple orthogonal methods\",\n      \"pmids\": [\"21791607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Grb14 promotes closure of photoreceptor-specific CNG channel alpha subunit (CNGA1) through its RA domain via electrostatic interaction. Grb14 competes with cGMP for the CNGA1 binding pocket; Glu180-182 in Grb14 are critical for electrostatic interaction with Arg559 in the cGMP-binding pocket of CNGA1. Lys140 also participates. Grb7 and Grb10 do not modulate channel activity, indicating specificity.\",\n      \"method\": \"In vitro binding assay, electrophysiology (CNG channel recordings), site-directed mutagenesis of Grb14 RA domain\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus binding assay plus channel functional readout, single lab\",\n      \"pmids\": [\"22180090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In thyroid cancer cells, Grb14 knockdown diminishes RET phosphorylation and reduces Akt and STAT3 activation, decreasing cell proliferation and invasion. Forced Grb14 expression interrupts IR signaling but facilitates RET activation and STAT3/Akt phosphorylation, enhancing cell invasion and metastasis in xenograft models.\",\n      \"method\": \"Stable shRNA knockdown, forced overexpression, western blot, in vitro invasion assay, orthotopic mouse xenograft\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with in vitro and in vivo readouts, single lab\",\n      \"pmids\": [\"22158039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Grb14 SH2 domain binds to the ITAM-like sequence of the phagocytic receptor CEACAM3 (identified by SH2 domain microarray). Grb14 is rapidly recruited to sites of bacteria–host cell contact. Both knockdown and overexpression of Grb14 reduce CEACAM3-mediated phagocytosis of Neisseria gonorrhoeae, establishing Grb14 as a negative regulator of CEACAM3-initiated bacterial phagocytosis.\",\n      \"method\": \"SH2 domain microarray, co-immunoprecipitation, FRET-FLIM, RNAi knockdown, overexpression, phagocytosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including FRET-FLIM for direct interaction in intact cells, functional phagocytosis assay, both KD and OE tested\",\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. The RA and PH domains form an integrated unit capable of simultaneously binding activated Ras and phosphoinositide lipids. Binding mode of Grb14 RA to Ras resembles RalGDS and Raf1 RA domains but with distinct features.\",\n      \"method\": \"X-ray crystallography (2.4 Å, Grb14 RA-PH + H-Ras G12V complex)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of the complex at high resolution with structural analysis of binding interface\",\n      \"pmids\": [\"23967305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In rat liver in vivo, insulin injection causes rapid, dose-dependent redistribution of endogenous Grb14 from cytosol to plasma membrane and then to Golgi/endosome fractions, paralleling IR phosphorylation. Co-immunoprecipitation shows Grb14 associates with phosphorylated IR beta-subunit in these fractions. Removal of endogenous Grb14 from membrane fractions by KCl treatment increases IR tyrosine kinase activity, demonstrating that endogenous Grb14 exerts negative feedback on IR activity in vivo.\",\n      \"method\": \"Rat liver subcellular fractionation, co-immunoprecipitation, in vitro IR kinase assay after KCl extraction\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo fractionation with kinase activity functional readout, multiple orthogonal methods, physiological system\",\n      \"pmids\": [\"18657188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Grb14 knockdown in primary hepatocytes via RNAi enhances insulin-induced Akt, GSK3, and FOXO1 phosphorylation, consistent with IR inhibition by Grb14. However, Grb14 depletion completely abrogates insulin-induced SREBP-1c maturation, blunting lipogenic gene expression despite improved Akt signaling, revealing a dual role for Grb14 in liver: inhibitor of IR and positive regulator of SREBP-1c-mediated lipogenesis.\",\n      \"method\": \"RNAi in primary mouse hepatocytes, western blot, gene expression analysis\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean RNAi with multiple metabolic readouts, single lab, unexpected dual role with no further mechanistic dissection at this level\",\n      \"pmids\": [\"18339716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In rod photoreceptors, Grb14 deletion accelerates response decay (decreased integration time and τREC) and decreases the limiting time constant τD, indicating that Grb14 modulates both cGMP-gated channel sensitivity and the rate of PDE6 inactivation. Grb14 effects are present in dark-adapted rods and enhanced after bleaching adaptation.\",\n      \"method\": \"Suction-electrode recordings from single mouse rods (Grb14 KO vs. WT), dark- and light-adapted conditions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with electrophysiological readout, single lab, mechanism not fully resolved at molecular level\",\n      \"pmids\": [\"24273167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In retinal neurons, phosphorylation of the BPS region of Grb14 promotes IR activation by inhibiting PTP1B, whereas unphosphorylated Grb14 acts with PTP1B to negatively regulate IR. Decreased Grb14 phosphorylation was observed in diabetic Ins2Akita mouse retinas, correlating with decreased IR activation.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay (PLA), phospho-specific analysis, Ins2Akita diabetic mouse retina\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — PLA and Co-IP with disease model validation, but mechanistic model relies partly on inference from prior studies\",\n      \"pmids\": [\"24350791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GSK-3 phosphorylates serine residues (Ser358, Ser362, Ser366) in the N-terminal BPS domain of Grb14. Pharmacological inhibition or knockdown of GSK-3 facilitates Grb14-IR complex formation. Serine-to-alanine substitution at these sites also facilitates Grb14-IR binding. Phospho-Ser366 of endogenous Grb14 is regulated by insulin in HepG2 cells, establishing GSK-3-mediated phosphorylation as a negative regulator of Grb14-IR complex formation.\",\n      \"method\": \"In vitro kinase assay, proximity ligation assay, pharmacological GSK-3 inhibition, siRNA knockdown, site-directed mutagenesis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus cell-based PLA plus mutagenesis, multiple orthogonal methods identifying the writer and target residues\",\n      \"pmids\": [\"24535599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Chfr ubiquitin ligase is a binding partner of Grb14 (binding at T220 of Grb14 upon insulin stimulation) and potentiates Grb14's inhibitory effect on insulin-induced cell division. Chfr ligase activity and phosphorylation of its T39 residue (an Akt target) are required. In the presence of insulin, Chfr binding to Grb14 activates Chfr E3 ligase, leading to Aurora A and Polo-like kinase degradation and blocking cell division.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus oocyte G2/M assay, mutagenesis (T220, Chfr T39, Chfr RING domain), mammalian cell proliferation assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, oocyte assay, mutagenesis of both partners), functional consequence (Aurora A/PLK degradation) defined\",\n      \"pmids\": [\"25578860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Grb14 knockdown in mouse liver silences de novo fatty acid synthesis despite enhancing Akt signaling. Mechanistically, Grb14 binds p62/sqstm1; upon Grb14 knockdown, released p62 activates the transcription factor Nrf2, which represses the lipogenic nuclear receptor LXR, thereby suppressing lipogenesis. This identifies Grb14 as a signaling node linking the IR to the p62-Nrf2-LXR pathway.\",\n      \"method\": \"Liver-specific AAV shRNA knockdown in lean and obese mice, western blot, gene expression, de novo lipogenesis assay, co-immunoprecipitation of Grb14-p62\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo liver-specific KD with molecular mechanism dissected (Grb14–p62 Co-IP, Nrf2/LXR pathway), multiple readouts\",\n      \"pmids\": [\"27215388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Phosphorylation of N-terminal BPS domain serines (Ser358, Ser362, Ser366) by GSK-3 negatively regulates Grb14–IR complex formation; glutamic acid substitutions at these sites reduce Kd from ~8 nM to undetectable by SPR. Protein phosphatase 1 (PP1) dephosphorylates Ser358 and Ser362, and this dephosphorylation facilitates Grb14–IR complex formation.\",\n      \"method\": \"In vitro kinase assay, surface plasmon resonance (SPR), yeast two-hybrid, co-immunoprecipitation, phosphopeptide phosphatase assay\",\n      \"journal\": \"Journal of biochemistry / Journal of peptide science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — SPR provides quantitative affinity, plus in vitro phosphatase assay identifies PP1, plus Co-IP and Y2H, multiple orthogonal methods\",\n      \"pmids\": [\"28130417\", \"31347216\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GRB14 is a multi-domain adaptor protein (RA–PH–BPS–SH2) that functions primarily as a tissue-specific negative regulator of insulin receptor (IR) signaling: its BPS/PIR domain acts as a pseudosubstrate inhibitor occupying the IR substrate-binding groove (crystal structure), while the SH2 domain anchors to phosphotyrosines on the activated IR; GSK-3 phosphorylates BPS-domain serines (Ser358/362/366) to prevent premature IR association, and PP1 dephosphorylates these sites to permit recruitment; once at the IR, Grb14 selectively protects kinase-loop phosphotyrosines from PTP1B while favoring dephosphorylation of Y972, thereby reducing IRS-1 recruitment and ERK/Akt signaling; PKCζ (recruited via the ZIP adaptor) phosphorylates Grb14 to further enhance its inhibitory activity; Grb14 also recruits PDK-1 to the IR to facilitate Akt activation, revealing a dual role; in liver, Grb14 sequesters p62 to suppress the Nrf2–LXR lipogenic axis; Grb14 additionally interacts with activated Ras (via RA domain), FGFR1 (blocking PLCγ), tankyrase 2, IRS-1 PTB domain, PTP1B (inhibiting phosphatase activity when Grb14-Y347 is phosphorylated by Src), CEACAM3 (inhibiting bacterial phagocytosis), the Chfr ubiquitin ligase (blocking insulin-driven cell division via Aurora A/PLK degradation), and the photoreceptor CNG channel (closing it via electrostatic RA-domain interaction with CNGA1).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRB14 is a multi-domain adaptor protein that functions principally as a tissue-specific negative regulator of insulin receptor (IR) signaling, binding the activated IR in an insulin-dependent manner and dampening downstream IRS-1 phosphorylation, glycogen synthesis, and DNA synthesis [#1, #6]. Its inhibitory core is the BPS/PIR domain, which acts as a pseudosubstrate inhibitor occupying the IR kinase substrate-binding groove while the SH2 domain anchors to receptor phosphotyrosines, as resolved by the BPS\\u2013IR kinase domain crystal structure [#10]; biochemically the BPS domain alone inhibits IR substrate phosphorylation uncompetitively, and although intrinsically unstructured it retains full activity [#2, #5]. Once bound, Grb14 selectively protects IR kinase-loop phosphotyrosines from PTP1B while favoring dephosphorylation of Y972, reducing IRS-1 recruitment and ERK activation [#13], and is itself recruited and dimerized on the activated receptor dimer in real time [#14, #25]. Recruitment is gated by phosphorylation: GSK-3 phosphorylates BPS serines Ser358/362/366 to prevent premature IR association, while PP1 dephosphorylates these sites to permit high-affinity binding [#29, #32], and PKC\\u03b6 (via the ZIP adaptor) phosphorylates Grb14 to enhance its inhibitory activity [#4]. Grb14 carries a dual role, additionally recruiting PDK-1 to facilitate insulin-dependent Akt activation [#8] and, in liver, sequestering p62 to permit SREBP-1c\\u2013 and LXR-driven lipogenesis [#26, #31]. Beyond insulin signaling, Grb14 binds activated Ras through an integrated RA\\u2013PH unit [#16, #24], restricts FGFR1\\u2013PLC\\u03b3 signaling [#11, #19], inhibits PTP1B when Grb14-Y347 is phosphorylated by Src in retina [#20], gates the photoreceptor CNG channel via its RA domain [#21, #27], and negatively regulates CEACAM3-mediated bacterial phagocytosis through its SH2 domain [#23].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established Grb14 as a phosphotyrosine-binding adaptor coupled to receptor tyrosine kinase signaling, the first clue to its function.\",\n      \"evidence\": \"GST-SH2 domain pulldown against activated PDGF receptors and in vivo serine phosphorylation labeling\",\n      \"pmids\": [\"8647858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence of PDGFR binding tested\", \"Identity of the PDGF-regulated serine kinase unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined Grb14 as an inhibitory regulator of insulin signaling that binds the IR via its PIR domain, reframing it as a negative feedback adaptor rather than a positive coupler.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, deletion-mutant binding, and CHO-IR cell metabolic readouts\",\n      \"pmids\": [\"9748281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the catalytic mechanism of inhibition\", \"Tissue relevance not yet established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed the BPS/PIR domain alone uncompetitively inhibits IR substrate phosphorylation and protects the IR from phosphatases, separating substrate occlusion from receptor anchoring.\",\n      \"evidence\": \"In vitro kinase assays with deletion mutants and CHO-IR cell signaling readouts\",\n      \"pmids\": [\"11726652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate-groove occlusion not yet visualized\", \"Which phosphatase is antagonized unspecified at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified tankyrase 2 as an N-terminal binding partner, hinting at additional Grb14 interactions beyond the IR.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, and microsomal fractionation\",\n      \"pmids\": [\"11278563\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of tankyrase 2 binding not established\", \"No link to insulin signaling demonstrated\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Placed Grb14 in a PKC\\u03b6\\u2013ZIP heterotrimeric complex and showed phosphorylation by PKC\\u03b6 enhances its IR inhibition, adding a kinase input that tunes inhibitory strength.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, in vivo 32P labeling, and Xenopus oocyte maturation assay\",\n      \"pmids\": [\"12242277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylated residues on Grb14 not mapped here\", \"Physiological relevance in metabolic tissues untested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated the inhibitory PIR domain is intrinsically unstructured yet fully active, indicating function does not require a stable fold.\",\n      \"evidence\": \"NMR of 15N-labeled recombinant PIR and Xenopus oocyte assay\",\n      \"pmids\": [\"14623073\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conformation when bound to IR not resolved\", \"No partner-induced folding shown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Genetic ablation in mice proved Grb14 is a physiological, tissue-specific repressor of IR\\u2013IRS-1 signaling controlling glucose homeostasis.\",\n      \"evidence\": \"Knockout mouse with glucose/insulin tolerance tests, ex vivo metabolic assays, and signaling western blots\",\n      \"pmids\": [\"14749734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific limiting factors not identified\", \"Did not explain how liver IRS-1 phosphorylation rises despite lower IR autophosphorylation\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Revealed a dual role by which Grb14 constitutively recruits PDK-1 to facilitate Akt activation, complicating its purely inhibitory image.\",\n      \"evidence\": \"Directed proteomics pulldown, Co-IP, point mutagenesis, fractionation, and Akt activity assay\",\n      \"pmids\": [\"15210700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Balance between IR inhibition and Akt facilitation not quantified\", \"PDK-1 binding motif structural detail limited\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linked Grb14 abundance to insulin resistance, showing hormonal regulation of its expression across diabetic models.\",\n      \"evidence\": \"Western blot, RT-PCR, and 3T3-F442A adipocyte treatment assays in ob/ob, GK rat, and human diabetic tissue\",\n      \"pmids\": [\"15059968\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal direction between Grb14 levels and insulin resistance not established\", \"Transcriptional regulators of Grb14 unidentified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Biophysical characterization detected a transiently structured stretch (399\\u2013407) in the disordered PIR, suggesting partner-induced ordering.\",\n      \"evidence\": \"SAXS, circular dichroism with TFE, and sequence analysis\",\n      \"pmids\": [\"15465854\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the transient structure untested\", \"Binding partner triggering the transition unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The BPS\\u2013IR kinase domain crystal structure resolved the pseudosubstrate inhibition mechanism, providing the structural basis for substrate-groove occlusion plus SH2 anchoring.\",\n      \"evidence\": \"X-ray crystallography of the BPS\\u2013IR kinase complex and SH2 domain with modeling\",\n      \"pmids\": [\"16246733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length Grb14 conformation on the IR dimer not crystallized\", \"Regulatory phosphorylation not captured in structure\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended Grb14's inhibitory reach to estrogen- and insulin-driven proliferation in breast cancer cells, tying it to mitogenic control.\",\n      \"evidence\": \"MCF-7 overexpression, cell cycle analysis, and ERK1/2 phospho western blots\",\n      \"pmids\": [\"15372466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Domain requirements for the antiproliferative effect not dissected\", \"Overexpression-only evidence without loss-of-function\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified phosphorylation-independent binding of the IRS-1 PTB domain to a Grb14 NPXY motif, proposing IRS-1 sequestration in retina.\",\n      \"evidence\": \"Yeast two-hybrid retinal screen, in vitro pulldown, and Co-IP from retinal lysates\",\n      \"pmids\": [\"15924411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional sequestration of IRS-1 inferred but not directly tested\", \"Retinal relevance of the complex not validated in vivo here\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed Grb14 binds activated FGFR1 with receptor specificity and inhibits FGF-2-induced proliferation, broadening its RTK targets beyond the IR.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, in vitro binding, proliferation assay, and SH2 point mutagenesis\",\n      \"pmids\": [\"10713090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of FGFR1 inhibition not yet defined\", \"In vivo relevance untested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Real-time imaging defined how Grb14 reshapes IR phosphorylation\\u2014protecting kinase-loop tyrosines from PTP1B while favoring Y972 dephosphorylation\\u2014to selectively curtail IRS-1 recruitment.\",\n      \"evidence\": \"BRET in living HEK cells, site-specific phosphotyrosine antibodies, and Co-IP\",\n      \"pmids\": [\"16582879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of site-selective phosphatase protection unresolved\", \"Generality across tissues not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated insulin-induced Grb14 dimerization, consistent with two molecules binding the activated IR dimer.\",\n      \"evidence\": \"BRET saturation experiments, Co-IP, and site-specific phospho-antibodies\",\n      \"pmids\": [\"16934761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dimerization interface not mapped\", \"Functional necessity of dimerization untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Captured insulin-driven redistribution of endogenous Grb14 to the IR in rat liver and confirmed negative feedback by showing its removal raises IR kinase activity.\",\n      \"evidence\": \"Rat liver subcellular fractionation, Co-IP, and in vitro IR kinase assay after KCl extraction\",\n      \"pmids\": [\"18657188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking determinants directing Grb14 to membrane/Golgi/endosome unclear\", \"Link between localization and inhibition mechanism not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Uncovered the liver dual role: Grb14 inhibits IR but is required for insulin-induced SREBP-1c maturation and lipogenic gene expression.\",\n      \"evidence\": \"RNAi in primary mouse hepatocytes with signaling and gene-expression readouts\",\n      \"pmids\": [\"18339716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link from Grb14 to SREBP-1c not yet defined at this stage\", \"How lipogenesis persists despite improved Akt unexplained\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Structural and biochemical work defined the RA\\u2013PH tandem as an integrated unit that binds GTP-loaded Ras, proposing Ras-dependent timing of insulin signaling downregulation.\",\n      \"evidence\": \"X-ray crystallography of RA-PH (Grb10) and biochemical Ras-binding assay for Grb14\",\n      \"pmids\": [\"19648926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Grb14\\u2013Ras binding in cells not tested\", \"Crystal structure was of Grb10 RA-PH, not Grb14\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Compound Grb10/Grb14 knockout established genetic epistasis and showed combined loss protects against diet-induced glucose intolerance, revealing limiting compensatory mechanisms.\",\n      \"evidence\": \"Double-knockout mice with tolerance tests and IR/IRS-1/Akt signaling readouts\",\n      \"pmids\": [\"19541746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the limiting factors (IR hypophosphorylation, IRS-1 levels) not mechanistically resolved\", \"Tissue contributions to protection unseparated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mutagenesis pinpointed the Grb14\\u2013IR contact residues conferring specificity and confirmed the PDK-1 interaction is required alongside IR binding for maximal inhibition.\",\n      \"evidence\": \"Mutagenesis, Co-IP, BRET, Xenopus oocyte assay, and Grb14 KO MEF complementation\",\n      \"pmids\": [\"19359342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How S370 phosphorylation status mechanistically gates activity not fully resolved\", \"Structural model of full assembly incomplete\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the FGFR1 inhibition mechanism: Grb14 binds FGFR1 pY766 within a PLC\\u03b3-containing trimeric complex to trap and inactivate PLC\\u03b3.\",\n      \"evidence\": \"BRET, Xenopus oocyte FGF-maturation assay, and FGFR pY766 mutagenesis\",\n      \"pmids\": [\"20932831\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conformational trapping model not structurally confirmed\", \"Physiological role in FGF biology untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified Grb14 as a phosphorylation-regulated inhibitor of PTP1B in retina, with Tyr-347 phosphorylation by rhodopsin-activated Src enabling competitive PTP1B inhibition.\",\n      \"evidence\": \"In vitro PTP1B activity assay, Y347 mutagenesis, retinal Grb14 KO mice, and in vivo phosphorylation assays\",\n      \"pmids\": [\"21791607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Y347 phosphorylation reconciles with IR-protective role unclear\", \"Retinal substrate landscape of derepressed PTP1B not mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a non-adaptor channel-gating function: Grb14's RA domain electrostatically competes with cGMP to close the photoreceptor CNGA1 channel, with isoform specificity.\",\n      \"evidence\": \"In vitro binding, CNG channel electrophysiology, and RA-domain mutagenesis\",\n      \"pmids\": [\"22180090\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo channel regulation not yet shown here\", \"Structural detail of the RA\\u2013CNGA1 interface limited\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed Grb14 can act as a positive driver of oncogenic RET signaling in thyroid cancer, promoting Akt/STAT3 activation, invasion, and metastasis.\",\n      \"evidence\": \"Stable shRNA knockdown, overexpression, invasion assay, and orthotopic mouse xenograft\",\n      \"pmids\": [\"22158039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Grb14\\u2013RET binding not structurally defined\", \"Mechanism distinguishing RET facilitation from IR inhibition unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined Grb14 as a negative regulator of CEACAM3-mediated bacterial phagocytosis via SH2 binding to the receptor's ITAM-like motif.\",\n      \"evidence\": \"SH2 domain microarray, Co-IP, FRET-FLIM, RNAi, overexpression, and phagocytosis assay\",\n      \"pmids\": [\"22948154\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling blocked by Grb14 at CEACAM3 not detailed\", \"Why both knockdown and overexpression reduce phagocytosis unexplained mechanistically\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"High-resolution crystal structure of Grb14 RA\\u2013PH bound to GTP-loaded H-Ras showed the integrated unit can simultaneously engage Ras and phosphoinositides.\",\n      \"evidence\": \"X-ray crystallography at 2.4 \\u00c5 of the Grb14 RA-PH\\u2013H-Ras G12V complex\",\n      \"pmids\": [\"23967305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular function of simultaneous Ras/lipid engagement not tested\", \"Coupling to IR inhibition timing not demonstrated structurally\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Single-rod electrophysiology showed Grb14 modulates both CNG channel sensitivity and PDE6 inactivation rate, defining a physiological role in phototransduction.\",\n      \"evidence\": \"Suction-electrode recordings from single Grb14 KO vs WT mouse rods in dark- and light-adapted states\",\n      \"pmids\": [\"24273167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of PDE6 inactivation effect unresolved\", \"Whether channel and PDE6 effects share a mechanism unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed the BPS phosphorylation state switches Grb14 between IR-promoting (PTP1B-inhibiting) and IR-inhibiting modes in retinal neurons, with relevance to diabetic retinopathy.\",\n      \"evidence\": \"Co-IP, proximity ligation assay, phospho-specific analysis, and Ins2Akita diabetic mouse retina\",\n      \"pmids\": [\"24350791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Switch mechanism relies partly on inference from prior studies\", \"Kinase/phosphatase setting the retinal phospho-state not fully defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified GSK-3 as the kinase phosphorylating BPS serines Ser358/362/366 to block premature Grb14\\u2013IR complex formation, defining an upstream gating input.\",\n      \"evidence\": \"In vitro kinase assay, proximity ligation assay, GSK-3 inhibition/knockdown, and S-to-A mutagenesis\",\n      \"pmids\": [\"24535599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How insulin lowers GSK-3 activity to permit binding not detailed here\", \"The opposing phosphatase not yet identified at this stage\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected Grb14 to cell-cycle control by showing insulin-dependent Chfr binding activates the E3 ligase to degrade Aurora A and PLK, blocking insulin-driven division.\",\n      \"evidence\": \"Co-IP, Xenopus oocyte G2/M assay, mutagenesis of Grb14 T220 and Chfr T39/RING, and mammalian proliferation assays\",\n      \"pmids\": [\"25578860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of Grb14\\u2013Chfr-driven division control unclear\", \"Tissue specificity of the effect untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the liver lipogenic dual role mechanistically: Grb14 sequesters p62, and its loss releases p62 to activate Nrf2 and repress LXR, silencing de novo lipogenesis.\",\n      \"evidence\": \"Liver-specific AAV shRNA in lean and obese mice with lipogenesis assays, gene expression, and Grb14\\u2013p62 Co-IP\",\n      \"pmids\": [\"27215388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Grb14\\u2013p62 binding undefined\", \"How IR engagement is coupled to p62 release unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Quantified the phospho-gated affinity switch and identified PP1 as the phosphatase that, by dephosphorylating Ser358/362, licenses high-affinity Grb14\\u2013IR binding.\",\n      \"evidence\": \"In vitro kinase assay, surface plasmon resonance, yeast two-hybrid, Co-IP, and phosphopeptide phosphatase assay\",\n      \"pmids\": [\"28130417\", \"31347216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How insulin coordinates GSK-3 inactivation with PP1 activation in vivo unclear\", \"Spatiotemporal control of the phospho-switch at the membrane undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Grb14's many context-specific roles\\u2014IR inhibition vs Akt/RET facilitation, lipogenic regulation, channel gating, and immune phagocytosis\\u2014are coordinated within a single full-length protein in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length Grb14 structure on an intact receptor\", \"Tissue-specific determinants selecting inhibitory vs facilitatory output unknown\", \"Integration of RA/Ras, BPS/IR, and SH2 inputs into a unified regulatory logic not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 8, 30, 31]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 10, 13, 20]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [15, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 25]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [25]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 13, 18]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6, 26, 31]},\n      {\"term_id\": \"R-HSA-9709957\", \"supporting_discovery_ids\": [21, 27]}\n    ],\n    \"complexes\": [\"PKC\\u03b6\\u2013ZIP\\u2013Grb14 complex\"],\n    \"partners\": [\"INSR\", \"PTP1B\", \"PDPK1\", \"FGFR1\", \"HRAS\", \"SQSTM1\", \"CHFR\", \"CEACAM3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}