{"gene":"NHERF4","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2002,"finding":"IKEPP (NHERF4) was identified as a novel binding partner of guanylyl cyclase C (GCC) via its COOH terminus, demonstrated by yeast two-hybrid, biochemical assays, and co-immunoprecipitation. The GCC-IKEPP interaction significantly inhibits heat-stable enterotoxin-mediated activation of GCC, but is not required for apical targeting of GCC.","method":"Yeast two-hybrid screen, co-immunoprecipitation, biochemical binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, yeast two-hybrid, and functional assay (GCC activity inhibition) in a single focused study; replicated and extended by later studies","pmids":["11950846"],"is_preprint":false},{"year":2003,"finding":"IKEPP (NHERF4) binds to the C-terminal PDZ binding motif of MRP2, and this binding is significantly increased when the MRP2 PDZ binding motif is phosphorylated (phosphorylation-mimicking mutant or phosphorylated C-terminal peptide), demonstrating that PKC-mediated phosphorylation of MRP2 modulates its interaction with IKEPP.","method":"GST pull-down assay with phosphorylation-mimicking mutants and phosphorylated peptides","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with phospho-mimetic mutants, single lab, two orthogonal approaches (whole protein and peptide)","pmids":["12615054"],"is_preprint":false},{"year":2006,"finding":"NHERF4 (IKEPP/NaPi-Cap2/PDZK2) was identified as a binding partner of the epithelial Ca2+ channels TRPV5 and TRPV6 via their carboxyl termini. The fourth PDZ domain of NHERF4 is sufficient for interaction, with PDZ domain 1 also contributing. The binding site on TRPV5/6 is conserved and distinct from the NHERF2 binding site. NHERF4 localizes predominantly at the plasma membrane independently of TRPV5.","method":"Yeast two-hybrid screen, GST pull-down (in vitro translated NHERF4 and Xenopus oocyte lysates), co-immunoprecipitation (HEK293 cells), immunolocalization","journal":"Pflugers Archiv : European journal of physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, GST pulldown, co-IP in two expression systems) in one focused study confirming the interaction and domain mapping","pmids":["16565876"],"is_preprint":false},{"year":2006,"finding":"MAP17 interacts with NHERF4 (and NHERF3), and coexpression of MAP17 with NHERF4 (or NHERF3) induces internalization of the renal Na/Pi IIa transporter (NaPiIIa) and MAP17 to the trans-Golgi network (TGN). This effect is not observed with NHERF1/2. PKC inhibition prevents TGN accumulation, and PKC activation causes NaPiIIa degradation unless lysosomal degradation is blocked. Coexpression of MAP17 and NHERF3/4 prevents the adaptive upregulation of phosphate transport in response to low phosphate.","method":"Bacterial and mammalian two-hybrid, coexpression in opossum kidney (OK) cells, confocal immunofluorescence, phosphate transport assays, PKC inhibition/activation pharmacology","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods (two-hybrid, coexpression, localization, functional transport assay) in a focused study with clear mechanistic readout","pmids":["16926447"],"is_preprint":false},{"year":2006,"finding":"PDZK2 (NHERF4) increases the transport capacity and cell surface expression of OCTN2 (organic cation/carnitine transporter) approximately 2-fold in HEK293 cells. This effect depends on physical interaction via the last four amino acids of OCTN2 (PDZ binding motif), as deletion of these residues abolishes both interaction and stimulation. In mouse kidney, PDZK2 co-localizes with OCTN2 in a subapical compartment, suggesting an intracellular pool relevant to stabilization of cell surface expression. No stimulatory effect was seen for OCT3 or OCTN1.","method":"Uptake assays in HEK293 cells, cell surface expression assay, deletion mutagenesis, co-immunolocalization in mouse kidney","journal":"Drug metabolism and disposition","confidence":"High","confidence_rationale":"Tier 2 / Moderate — functional transport assay with mutagenesis confirming PDZ-dependent mechanism, plus in vivo co-localization; single lab but multiple orthogonal approaches","pmids":["16896066"],"is_preprint":false},{"year":2007,"finding":"PDZK2 (NHERF4) interacts with TRPV6 through its fourth PDZ domain, requiring the last four amino acids (EYQI) of TRPV6 C-terminus. A TRPV6 PDZ-binding motif deletion mutant (Δ4) showed decreased peak current amplitude. Intracellular introduction of the PDZ-binding motif peptide (EYQI) or siRNA knockdown of endogenous PDZK2 significantly reduced TRPV6 divalent-free current density in HEK293 cells, establishing PDZK2 as an essential physiological modulator of TRPV6 channel activity.","method":"Yeast two-hybrid screen, GST pull-down, deletion mutagenesis, patch-clamp electrophysiology, siRNA knockdown in HEK293 cells","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct binding (GST pulldown + mutagenesis) plus functional electrophysiology and RNAi in one focused study with multiple orthogonal methods","pmids":["17645868"],"is_preprint":false},{"year":2008,"finding":"IKEPP (NHERF4) binds to the F2 region (aa 590–667) of NHE3 C-terminus in overlay assays and directly associates with NHE3 in vivo (demonstrated by FRET). In PS120 cells stably expressing both NHE3 and IKEPP, elevation of intracellular Ca2+ stimulates NHE3 Vmax activity (~40%) and increases plasma membrane expression of NHE3 by a similar amount. Elevated Ca2+ decreases the intracellular IKEPP–NHE3 association and shifts both NHE3 and IKEPP to smaller complexes. In contrast, NHERF2 mediates Ca2+-dependent inhibition and NHERF1 has no effect, demonstrating NHERF-specific regulation of NHE3.","method":"In vitro overlay assay, FRET on fixed cells, sucrose density gradient centrifugation, NHE3 activity assay (SNARF fluorescence), cell surface biotinylation, Ca2+ ionophore treatment in PS120 cells","journal":"Cellular physiology and biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (overlay, FRET, density gradient, functional assay, surface expression) in a focused study establishing both binding and functional consequence","pmids":["19088451"],"is_preprint":false},{"year":2010,"finding":"DRA (SLC26A3) interacts with IKEPP (NHERF4) within lipid rafts (LR) in intestinal Caco-2/BBE cells. The localization of IKEPP within lipid rafts is independent of DRA. Disruption of LR integrity decreases DRA surface expression and activity. This effect in HEK cells is entirely dependent on the PDZ interaction motif of DRA, establishing that NHERF4 participates in lipid-raft-dependent regulation of DRA.","method":"Detergent-resistant membrane fractionation (Triton X-100), cell surface expression assay, transport activity assay (Caco-2/BBE and HEK cells), PDZ binding motif deletion mutagenesis","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lipid raft fractionation plus functional assay with mutagenesis; single lab, IKEPP/NHERF4 finding embedded within broader DRA study","pmids":["20634435"],"is_preprint":false},{"year":2012,"finding":"NHERF4 interacts with SLC26A3 (DRA) via the third PDZ domain of NHERF4 and the C-terminal PDZ binding motif of SLC26A3. This interaction decreases SLC26A3 plasma membrane expression and induces rapid internalization, reducing anion exchange activity. The SLC26A3–NHERF4 interaction is modulated by phosphorylation; serine 329 of NHERF4-PDZ3 plays a critical role in binding selectivity.","method":"Co-immunoprecipitation, GST pull-down, cell surface expression assay, internalization assay, anion exchange activity assay, phosphorylation site mutagenesis","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-IP, pulldown, functional assay, mutagenesis) in one focused study establishing mechanism of PDZ-phosphorylation-dependent regulation","pmids":["22627094"],"is_preprint":false},{"year":2012,"finding":"IKEPP (NHERF4) interacts with the human prostacyclin receptor (hIP) via PDZ domain 1 (and to a lesser extent PDZ domain 2) binding to a C-terminal Class I PDZ ligand in the hIP. The interaction is constitutive, but agonist activation of hIP leads to PKA- and PKC-phosphorylation of IKEPP and increased IKEPP–hIP interaction. Ectopic IKEPP expression increases functional hIP expression, enhancing ligand binding and agonist-induced cAMP generation. IKEPP is expressed in vascular endothelial cells where it co-localizes with hIP, and siRNA disruption of IKEPP impairs hIP-induced endothelial cell migration and in vitro angiogenesis.","method":"Co-immunoprecipitation, co-localization, siRNA knockdown, ligand binding assay, cAMP assay, endothelial migration and angiogenesis assays","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-IP, functional assays, siRNA with defined cellular phenotypes) in one focused study","pmids":["22884631"],"is_preprint":false},{"year":2016,"finding":"USP2-45 (a ubiquitin-specific protease) interacts with NHERF4 in vitro and negatively regulates NHERF4 protein abundance in a rhythmic (circadian) manner in mouse small intestinal membrane fractions. Usp2-KO mice show strong overexpression of NHERF4 and hyperabsorption of dietary Ca2+, likely via elevated NHERF4-dependent regulation of TRPV6. USP2-45 also interacts with endogenous Clathrin Heavy Chain, suggesting a membrane protein turnover mechanism.","method":"In vitro interaction assay, immunoblot of membrane fractions, Usp2-KO mouse phenotype, co-immunoprecipitation with Clathrin Heavy Chain","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype with mechanistic link to NHERF4 protein level regulation; in vitro interaction confirmed; single lab","pmids":["26756164"],"is_preprint":false},{"year":2021,"finding":"NHERF4 physically interacts with the Mas receptor (identified by SPR-MS and confirmed by GST pull-down and co-immunoprecipitation). NHERF4 overexpression inhibits Mas-induced migration, invasion, and in vivo metastasis of clear cell renal cell carcinoma (ccRCC) cells. Mechanistically, NHERF4 suppresses Mas-stimulated AKT phosphorylation and the PLC/Ca2+ response, establishing NHERF4 as a negative regulator of the PLC/AKT signaling axis downstream of Mas.","method":"Surface plasmon resonance coupled to mass spectrometry, GST pull-down, co-immunoprecipitation, NHERF4 overexpression and knockdown, migration/invasion assays, in vivo metastasis assay, AKT phosphorylation and Ca2+ signaling assays","journal":"Cancer letters","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (SPR-MS, co-IP, GST pulldown, functional cellular and in vivo assays, signaling readouts) in one focused study","pmids":["34216689"],"is_preprint":false},{"year":2025,"finding":"The PDZ1 domain of NHERF4 directly inhibits GCC (guanylate cyclase 2C) catalytic activity, while NHERF1–3 binary binding had no impact. Two peptides (N4-110 and N4-195) within the PDZ1 domain act synergistically to mimic this inhibition; structural modeling places them at the GCC dimer interface. FRET analysis showed that NHERF4-PDZ1 domain binding interferes with GCC oligomerization. In mouse and human enteroid models, NHERF4 peptides dose-dependently reduced GCC-mediated fluid secretion. NHERF4–GCC interaction is enhanced upon heat-stable enterotoxin (ST) stimulation, positioning NHERF4 as a negative feedback regulator of aberrant GCC activity during enterotoxin-induced diarrhea.","method":"In vitro GCC activity assay, peptide competition assay, 3D structural modeling, FRET, mouse and human enteroid fluid secretion assay, ST stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro catalytic assay, mutagenesis-equivalent peptide mapping, FRET for mechanism, and organoid functional validation; single lab but multiple rigorous orthogonal methods","pmids":["40759370"],"is_preprint":false}],"current_model":"NHERF4 (IKEPP/PDZK2/NaPi-Cap2) is an epithelial-enriched, four-PDZ-domain scaffold protein that regulates ion transport, receptor signaling, and channel activity at apical membranes: its PDZ domains (particularly PDZ1, PDZ3, and PDZ4) bind the C-terminal PDZ ligands of GCC (inhibiting its catalytic activity and oligomerization), NHE3 (mediating Ca2+-dependent stimulation of NHE3 surface expression and activity), TRPV5/TRPV6 (modulating Ca2+ channel activity), SLC26A3/DRA (inducing internalization and reducing anion exchange), OCTN2 (increasing transporter cell surface expression), the prostacyclin receptor hIP (enhancing its functional expression and signaling), and the Mas receptor (suppressing PLC/AKT signaling); additionally, NHERF4 protein abundance is post-translationally regulated by the circadian deubiquitinase USP2-45, and its interaction with MAP17 routes NaPiIIa to the trans-Golgi network in a PKC-dependent manner."},"narrative":{"mechanistic_narrative":"NHERF4 (IKEPP/PDZK2/NaPi-Cap2) is an epithelial-enriched, multi-PDZ-domain scaffold protein that uses its PDZ domains to bind the C-terminal PDZ ligands of apical-membrane transporters, channels, and receptors and thereby tune their surface expression and activity [PMID:11950846, PMID:16565876, PMID:19088451]. Through distinct PDZ domains it engages a broad set of partners: PDZ1 binds and directly inhibits the catalytic activity and oligomerization of guanylyl cyclase C (GCC), positioning NHERF4 as a negative-feedback regulator of heat-stable enterotoxin-induced fluid secretion in enteroids [PMID:11950846, PMID:40759370]; the fourth PDZ domain (with a contribution from PDZ1) binds the epithelial Ca2+ channels TRPV5/TRPV6 and is an essential modulator of channel current [PMID:16565876, PMID:17645868]; and PDZ3 (via serine 329) binds SLC26A3/DRA to drive its internalization and reduce anion exchange [PMID:22627094]. NHERF4 produces partner-specific functional outcomes — it mediates Ca2+-dependent stimulation of NHE3 surface expression and activity [PMID:19088451], increases cell-surface expression of the carnitine transporter OCTN2 [PMID:16896066], and enhances functional expression and cAMP signaling of the prostacyclin receptor hIP to support endothelial migration and angiogenesis [PMID:22884631]. Several of these interactions are governed by phosphorylation of either the partner's PDZ ligand or of NHERF4 itself, including PKC-dependent regulation of MRP2 binding [PMID:12615054] and PKA/PKC phosphorylation upon hIP activation [PMID:22884631]. Beyond ion transport, NHERF4 binds the Mas receptor and suppresses its PLC/Ca2+ and AKT signaling, inhibiting renal carcinoma cell migration, invasion, and metastasis [PMID:34216689]. NHERF4 protein abundance is itself controlled post-translationally by the deubiquitinase USP2-45 in a circadian manner, linking NHERF4 levels to intestinal Ca2+ absorption [PMID:26756164].","teleology":[{"year":2002,"claim":"Established the first molecular role of NHERF4 by showing it binds guanylyl cyclase C and dampens enterotoxin-stimulated cyclase activity without affecting apical targeting, defining it as a regulatory scaffold rather than a trafficking determinant for GCC.","evidence":"Yeast two-hybrid, co-IP, and GCC activity assays","pmids":["11950846"],"confidence":"High","gaps":["Domain mediating GCC binding not yet mapped","Mechanism of catalytic inhibition unresolved at this stage"]},{"year":2003,"claim":"Showed that NHERF4's interaction with a partner (MRP2) is phosphorylation-dependent, introducing the principle that PKC-driven modification of a PDZ ligand controls NHERF4 binding.","evidence":"GST pull-down with phospho-mimetic mutants and phosphorylated peptides","pmids":["12615054"],"confidence":"Medium","gaps":["No functional consequence for MRP2 activity or trafficking demonstrated","Single-lab in vitro binding without cellular validation"]},{"year":2006,"claim":"Identified NHERF4 as a binder and modulator of epithelial Ca2+ channels TRPV5/TRPV6 and of the renal phosphate transport machinery, mapping the fourth PDZ domain as the principal interaction module and revealing partner-specific routing via MAP17 to the TGN.","evidence":"Yeast two-hybrid, GST pull-down, co-IP, immunolocalization, OK-cell coexpression with phosphate transport assays","pmids":["16565876","16926447","16896066"],"confidence":"High","gaps":["Whether NHERF4 directly gates channel activity vs. trafficking unresolved at this stage","In vivo relevance of MAP17-NHERF4 TGN routing not established"]},{"year":2007,"claim":"Demonstrated that NHERF4 is a physiological modulator of TRPV6 channel current, moving from binding to a functional electrophysiological readout.","evidence":"GST pull-down, deletion mutagenesis, patch-clamp, and siRNA knockdown in HEK293 cells","pmids":["17645868"],"confidence":"High","gaps":["Molecular mechanism by which PDZ binding alters channel gating unknown","In vivo TRPV6 regulation not tested"]},{"year":2008,"claim":"Defined NHERF4 as a NHERF-isoform-specific mediator of Ca2+-dependent stimulation of NHE3, showing that a Ca2+-induced decrease in NHERF4-NHE3 association increases NHE3 surface expression and activity.","evidence":"Overlay assay, FRET, sucrose gradient, NHE3 activity and surface biotinylation in PS120 cells","pmids":["19088451"],"confidence":"High","gaps":["Signaling link between Ca2+ rise and complex remodeling not defined","Physiological context in intact epithelium not addressed"]},{"year":2012,"claim":"Extended NHERF4's regulatory repertoire to anion exchange and GPCR signaling, mapping PDZ3 (Ser329) to SLC26A3/DRA internalization and PDZ1/2 to prostacyclin receptor potentiation with defined endothelial phenotypes.","evidence":"Co-IP, GST pull-down, surface/internalization and transport assays, phospho-site mutagenesis; siRNA, ligand binding, cAMP, migration and angiogenesis assays","pmids":["22627094","22884631","20634435"],"confidence":"High","gaps":["How a single scaffold drives opposite outcomes (internalization vs. surface stabilization) for different partners not mechanistically reconciled","Lipid-raft dependence of DRA regulation correlative"]},{"year":2016,"claim":"Revealed that NHERF4 abundance is itself regulated post-translationally by the circadian deubiquitinase USP2-45, linking scaffold levels to rhythmic intestinal Ca2+ absorption via TRPV6.","evidence":"In vitro interaction, membrane-fraction immunoblot, Usp2-KO mouse phenotype, co-IP with Clathrin Heavy Chain","pmids":["26756164"],"confidence":"Medium","gaps":["Direct ubiquitination/deubiquitination of NHERF4 not demonstrated","TRPV6 link to the calcium phenotype inferred rather than proven"]},{"year":2021,"claim":"Identified the Mas receptor as a NHERF4 partner and established NHERF4 as a negative regulator of PLC/AKT signaling that suppresses renal carcinoma metastasis, broadening NHERF4's role into oncogenic signaling.","evidence":"SPR-MS, GST pull-down, co-IP, overexpression/knockdown, migration/invasion and in vivo metastasis assays, AKT and Ca2+ readouts","pmids":["34216689"],"confidence":"High","gaps":["PDZ domain responsible for Mas binding not mapped","Whether suppression occurs via receptor sequestration or trafficking unresolved"]},{"year":2025,"claim":"Resolved the molecular basis of GCC inhibition first seen in 2002, showing PDZ1 peptides interfere with GCC oligomerization at the dimer interface and reduce GCC-mediated fluid secretion in enteroids, defining NHERF4 as a negative-feedback brake on enterotoxin-driven secretion.","evidence":"In vitro GCC activity assay, peptide competition, structural modeling, FRET, mouse/human enteroid fluid secretion with ST stimulation","pmids":["40759370"],"confidence":"High","gaps":["High-resolution structure of the NHERF4-PDZ1/GCC complex lacking","Therapeutic translation of inhibitory peptides untested in vivo"]},{"year":null,"claim":"How NHERF4's distinct PDZ domains are selected and coordinated to produce partner-specific, sometimes opposite, regulatory outcomes — and how its phosphorylation state integrates these decisions — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length NHERF4 engaging multiple partners","Logic of activation vs. internalization choice undefined","In vivo loss-of-function phenotypes largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,6,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,6,8,9,11,12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,6,7]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[4,6,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,11,12]}],"complexes":[],"partners":["GUCY2C","TRPV5","TRPV6","SLC9A3","SLC26A3","SLC22A5","PTGIR","MAS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86UT5","full_name":"Na(+)/H(+) exchange regulatory cofactor NHE-RF4","aliases":["Intestinal and kidney-enriched PDZ protein","Natrium-phosphate cotransporter IIa C-terminal-associated protein 2","Na/Pi cotransporter C-terminal-associated protein 2","NaPi-Cap2","PDZ domain-containing protein 2","PDZ domain-containing protein 3","Sodium-hydrogen exchanger regulatory factor 4"],"length_aa":571,"mass_kda":61.0,"function":"Acts as a regulatory protein that associates with GUCY2C and negatively modulates its heat-stable enterotoxin-mediated activation (PubMed:11950846). Stimulates SLC9A3 activity in the presence of elevated calcium ions (PubMed:19088451)","subcellular_location":"Cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q86UT5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NHERF4","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NHERF4","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"PDZD3","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"intestine","ntpm":18.2},{"tissue":"kidney","ntpm":17.8}],"url":"https://www.proteinatlas.org/search/PDZD3"},"hgnc":{"alias_symbol":["FLJ22756","IKEPP","NaPi-Cap2"],"prev_symbol":["PDZK2","PDZD3"]},"alphafold":{"accession":"Q86UT5","domains":[{"cath_id":"2.30.42.10","chopping":"115-206","consensus_level":"high","plddt":86.9486,"start":115,"end":206},{"cath_id":"2.30.42.10","chopping":"223-322","consensus_level":"high","plddt":89.9222,"start":223,"end":322},{"cath_id":"2.30.42.10","chopping":"329-427","consensus_level":"high","plddt":89.3216,"start":329,"end":427},{"cath_id":"2.30.42.10","chopping":"466-546","consensus_level":"high","plddt":72.6747,"start":466,"end":546}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UT5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UT5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UT5-F1-predicted_aligned_error_v6.png","plddt_mean":69.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NHERF4","jax_strain_url":"https://www.jax.org/strain/search?query=NHERF4"},"sequence":{"accession":"Q86UT5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UT5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UT5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UT5"}},"corpus_meta":[{"pmid":"16798722","id":"PMC_16798722","title":"The emerging role of PDZ adapter proteins for regulation of intestinal ion transport.","date":"2006","source":"American journal of physiology. Gastrointestinal and liver physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16798722","citation_count":116,"is_preprint":false},{"pmid":"12615054","id":"PMC_12615054","title":"C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12615054","citation_count":76,"is_preprint":false},{"pmid":"11950846","id":"PMC_11950846","title":"A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11950846","citation_count":66,"is_preprint":false},{"pmid":"24763552","id":"PMC_24763552","title":"RNA sequencing shows transcriptomic changes in rectosigmoid mucosa in patients with irritable bowel syndrome-diarrhea: a pilot case-control study.","date":"2014","source":"American journal of physiology. 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The GCC-IKEPP interaction significantly inhibits heat-stable enterotoxin-mediated activation of GCC, but is not required for apical targeting of GCC.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, biochemical binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, yeast two-hybrid, and functional assay (GCC activity inhibition) in a single focused study; replicated and extended by later studies\",\n      \"pmids\": [\"11950846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"IKEPP (NHERF4) binds to the C-terminal PDZ binding motif of MRP2, and this binding is significantly increased when the MRP2 PDZ binding motif is phosphorylated (phosphorylation-mimicking mutant or phosphorylated C-terminal peptide), demonstrating that PKC-mediated phosphorylation of MRP2 modulates its interaction with IKEPP.\",\n      \"method\": \"GST pull-down assay with phosphorylation-mimicking mutants and phosphorylated peptides\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with phospho-mimetic mutants, single lab, two orthogonal approaches (whole protein and peptide)\",\n      \"pmids\": [\"12615054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NHERF4 (IKEPP/NaPi-Cap2/PDZK2) was identified as a binding partner of the epithelial Ca2+ channels TRPV5 and TRPV6 via their carboxyl termini. The fourth PDZ domain of NHERF4 is sufficient for interaction, with PDZ domain 1 also contributing. The binding site on TRPV5/6 is conserved and distinct from the NHERF2 binding site. NHERF4 localizes predominantly at the plasma membrane independently of TRPV5.\",\n      \"method\": \"Yeast two-hybrid screen, GST pull-down (in vitro translated NHERF4 and Xenopus oocyte lysates), co-immunoprecipitation (HEK293 cells), immunolocalization\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, GST pulldown, co-IP in two expression systems) in one focused study confirming the interaction and domain mapping\",\n      \"pmids\": [\"16565876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAP17 interacts with NHERF4 (and NHERF3), and coexpression of MAP17 with NHERF4 (or NHERF3) induces internalization of the renal Na/Pi IIa transporter (NaPiIIa) and MAP17 to the trans-Golgi network (TGN). This effect is not observed with NHERF1/2. PKC inhibition prevents TGN accumulation, and PKC activation causes NaPiIIa degradation unless lysosomal degradation is blocked. Coexpression of MAP17 and NHERF3/4 prevents the adaptive upregulation of phosphate transport in response to low phosphate.\",\n      \"method\": \"Bacterial and mammalian two-hybrid, coexpression in opossum kidney (OK) cells, confocal immunofluorescence, phosphate transport assays, PKC inhibition/activation pharmacology\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods (two-hybrid, coexpression, localization, functional transport assay) in a focused study with clear mechanistic readout\",\n      \"pmids\": [\"16926447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PDZK2 (NHERF4) increases the transport capacity and cell surface expression of OCTN2 (organic cation/carnitine transporter) approximately 2-fold in HEK293 cells. This effect depends on physical interaction via the last four amino acids of OCTN2 (PDZ binding motif), as deletion of these residues abolishes both interaction and stimulation. In mouse kidney, PDZK2 co-localizes with OCTN2 in a subapical compartment, suggesting an intracellular pool relevant to stabilization of cell surface expression. No stimulatory effect was seen for OCT3 or OCTN1.\",\n      \"method\": \"Uptake assays in HEK293 cells, cell surface expression assay, deletion mutagenesis, co-immunolocalization in mouse kidney\",\n      \"journal\": \"Drug metabolism and disposition\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional transport assay with mutagenesis confirming PDZ-dependent mechanism, plus in vivo co-localization; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"16896066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PDZK2 (NHERF4) interacts with TRPV6 through its fourth PDZ domain, requiring the last four amino acids (EYQI) of TRPV6 C-terminus. A TRPV6 PDZ-binding motif deletion mutant (Δ4) showed decreased peak current amplitude. Intracellular introduction of the PDZ-binding motif peptide (EYQI) or siRNA knockdown of endogenous PDZK2 significantly reduced TRPV6 divalent-free current density in HEK293 cells, establishing PDZK2 as an essential physiological modulator of TRPV6 channel activity.\",\n      \"method\": \"Yeast two-hybrid screen, GST pull-down, deletion mutagenesis, patch-clamp electrophysiology, siRNA knockdown in HEK293 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct binding (GST pulldown + mutagenesis) plus functional electrophysiology and RNAi in one focused study with multiple orthogonal methods\",\n      \"pmids\": [\"17645868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IKEPP (NHERF4) binds to the F2 region (aa 590–667) of NHE3 C-terminus in overlay assays and directly associates with NHE3 in vivo (demonstrated by FRET). In PS120 cells stably expressing both NHE3 and IKEPP, elevation of intracellular Ca2+ stimulates NHE3 Vmax activity (~40%) and increases plasma membrane expression of NHE3 by a similar amount. Elevated Ca2+ decreases the intracellular IKEPP–NHE3 association and shifts both NHE3 and IKEPP to smaller complexes. In contrast, NHERF2 mediates Ca2+-dependent inhibition and NHERF1 has no effect, demonstrating NHERF-specific regulation of NHE3.\",\n      \"method\": \"In vitro overlay assay, FRET on fixed cells, sucrose density gradient centrifugation, NHE3 activity assay (SNARF fluorescence), cell surface biotinylation, Ca2+ ionophore treatment in PS120 cells\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (overlay, FRET, density gradient, functional assay, surface expression) in a focused study establishing both binding and functional consequence\",\n      \"pmids\": [\"19088451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"DRA (SLC26A3) interacts with IKEPP (NHERF4) within lipid rafts (LR) in intestinal Caco-2/BBE cells. The localization of IKEPP within lipid rafts is independent of DRA. Disruption of LR integrity decreases DRA surface expression and activity. This effect in HEK cells is entirely dependent on the PDZ interaction motif of DRA, establishing that NHERF4 participates in lipid-raft-dependent regulation of DRA.\",\n      \"method\": \"Detergent-resistant membrane fractionation (Triton X-100), cell surface expression assay, transport activity assay (Caco-2/BBE and HEK cells), PDZ binding motif deletion mutagenesis\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lipid raft fractionation plus functional assay with mutagenesis; single lab, IKEPP/NHERF4 finding embedded within broader DRA study\",\n      \"pmids\": [\"20634435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NHERF4 interacts with SLC26A3 (DRA) via the third PDZ domain of NHERF4 and the C-terminal PDZ binding motif of SLC26A3. This interaction decreases SLC26A3 plasma membrane expression and induces rapid internalization, reducing anion exchange activity. The SLC26A3–NHERF4 interaction is modulated by phosphorylation; serine 329 of NHERF4-PDZ3 plays a critical role in binding selectivity.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, cell surface expression assay, internalization assay, anion exchange activity assay, phosphorylation site mutagenesis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-IP, pulldown, functional assay, mutagenesis) in one focused study establishing mechanism of PDZ-phosphorylation-dependent regulation\",\n      \"pmids\": [\"22627094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"IKEPP (NHERF4) interacts with the human prostacyclin receptor (hIP) via PDZ domain 1 (and to a lesser extent PDZ domain 2) binding to a C-terminal Class I PDZ ligand in the hIP. The interaction is constitutive, but agonist activation of hIP leads to PKA- and PKC-phosphorylation of IKEPP and increased IKEPP–hIP interaction. Ectopic IKEPP expression increases functional hIP expression, enhancing ligand binding and agonist-induced cAMP generation. IKEPP is expressed in vascular endothelial cells where it co-localizes with hIP, and siRNA disruption of IKEPP impairs hIP-induced endothelial cell migration and in vitro angiogenesis.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, siRNA knockdown, ligand binding assay, cAMP assay, endothelial migration and angiogenesis assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-IP, functional assays, siRNA with defined cellular phenotypes) in one focused study\",\n      \"pmids\": [\"22884631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP2-45 (a ubiquitin-specific protease) interacts with NHERF4 in vitro and negatively regulates NHERF4 protein abundance in a rhythmic (circadian) manner in mouse small intestinal membrane fractions. Usp2-KO mice show strong overexpression of NHERF4 and hyperabsorption of dietary Ca2+, likely via elevated NHERF4-dependent regulation of TRPV6. USP2-45 also interacts with endogenous Clathrin Heavy Chain, suggesting a membrane protein turnover mechanism.\",\n      \"method\": \"In vitro interaction assay, immunoblot of membrane fractions, Usp2-KO mouse phenotype, co-immunoprecipitation with Clathrin Heavy Chain\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype with mechanistic link to NHERF4 protein level regulation; in vitro interaction confirmed; single lab\",\n      \"pmids\": [\"26756164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NHERF4 physically interacts with the Mas receptor (identified by SPR-MS and confirmed by GST pull-down and co-immunoprecipitation). NHERF4 overexpression inhibits Mas-induced migration, invasion, and in vivo metastasis of clear cell renal cell carcinoma (ccRCC) cells. Mechanistically, NHERF4 suppresses Mas-stimulated AKT phosphorylation and the PLC/Ca2+ response, establishing NHERF4 as a negative regulator of the PLC/AKT signaling axis downstream of Mas.\",\n      \"method\": \"Surface plasmon resonance coupled to mass spectrometry, GST pull-down, co-immunoprecipitation, NHERF4 overexpression and knockdown, migration/invasion assays, in vivo metastasis assay, AKT phosphorylation and Ca2+ signaling assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (SPR-MS, co-IP, GST pulldown, functional cellular and in vivo assays, signaling readouts) in one focused study\",\n      \"pmids\": [\"34216689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The PDZ1 domain of NHERF4 directly inhibits GCC (guanylate cyclase 2C) catalytic activity, while NHERF1–3 binary binding had no impact. Two peptides (N4-110 and N4-195) within the PDZ1 domain act synergistically to mimic this inhibition; structural modeling places them at the GCC dimer interface. FRET analysis showed that NHERF4-PDZ1 domain binding interferes with GCC oligomerization. In mouse and human enteroid models, NHERF4 peptides dose-dependently reduced GCC-mediated fluid secretion. NHERF4–GCC interaction is enhanced upon heat-stable enterotoxin (ST) stimulation, positioning NHERF4 as a negative feedback regulator of aberrant GCC activity during enterotoxin-induced diarrhea.\",\n      \"method\": \"In vitro GCC activity assay, peptide competition assay, 3D structural modeling, FRET, mouse and human enteroid fluid secretion assay, ST stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro catalytic assay, mutagenesis-equivalent peptide mapping, FRET for mechanism, and organoid functional validation; single lab but multiple rigorous orthogonal methods\",\n      \"pmids\": [\"40759370\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NHERF4 (IKEPP/PDZK2/NaPi-Cap2) is an epithelial-enriched, four-PDZ-domain scaffold protein that regulates ion transport, receptor signaling, and channel activity at apical membranes: its PDZ domains (particularly PDZ1, PDZ3, and PDZ4) bind the C-terminal PDZ ligands of GCC (inhibiting its catalytic activity and oligomerization), NHE3 (mediating Ca2+-dependent stimulation of NHE3 surface expression and activity), TRPV5/TRPV6 (modulating Ca2+ channel activity), SLC26A3/DRA (inducing internalization and reducing anion exchange), OCTN2 (increasing transporter cell surface expression), the prostacyclin receptor hIP (enhancing its functional expression and signaling), and the Mas receptor (suppressing PLC/AKT signaling); additionally, NHERF4 protein abundance is post-translationally regulated by the circadian deubiquitinase USP2-45, and its interaction with MAP17 routes NaPiIIa to the trans-Golgi network in a PKC-dependent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NHERF4 (IKEPP/PDZK2/NaPi-Cap2) is an epithelial-enriched, multi-PDZ-domain scaffold protein that uses its PDZ domains to bind the C-terminal PDZ ligands of apical-membrane transporters, channels, and receptors and thereby tune their surface expression and activity [#0, #2, #6]. Through distinct PDZ domains it engages a broad set of partners: PDZ1 binds and directly inhibits the catalytic activity and oligomerization of guanylyl cyclase C (GCC), positioning NHERF4 as a negative-feedback regulator of heat-stable enterotoxin-induced fluid secretion in enteroids [#0, #12]; the fourth PDZ domain (with a contribution from PDZ1) binds the epithelial Ca2+ channels TRPV5/TRPV6 and is an essential modulator of channel current [#2, #5]; and PDZ3 (via serine 329) binds SLC26A3/DRA to drive its internalization and reduce anion exchange [#8]. NHERF4 produces partner-specific functional outcomes — it mediates Ca2+-dependent stimulation of NHE3 surface expression and activity [#6], increases cell-surface expression of the carnitine transporter OCTN2 [#4], and enhances functional expression and cAMP signaling of the prostacyclin receptor hIP to support endothelial migration and angiogenesis [#9]. Several of these interactions are governed by phosphorylation of either the partner's PDZ ligand or of NHERF4 itself, including PKC-dependent regulation of MRP2 binding [#1] and PKA/PKC phosphorylation upon hIP activation [#9]. Beyond ion transport, NHERF4 binds the Mas receptor and suppresses its PLC/Ca2+ and AKT signaling, inhibiting renal carcinoma cell migration, invasion, and metastasis [#11]. NHERF4 protein abundance is itself controlled post-translationally by the deubiquitinase USP2-45 in a circadian manner, linking NHERF4 levels to intestinal Ca2+ absorption [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established the first molecular role of NHERF4 by showing it binds guanylyl cyclase C and dampens enterotoxin-stimulated cyclase activity without affecting apical targeting, defining it as a regulatory scaffold rather than a trafficking determinant for GCC.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, and GCC activity assays\",\n      \"pmids\": [\"11950846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain mediating GCC binding not yet mapped\", \"Mechanism of catalytic inhibition unresolved at this stage\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed that NHERF4's interaction with a partner (MRP2) is phosphorylation-dependent, introducing the principle that PKC-driven modification of a PDZ ligand controls NHERF4 binding.\",\n      \"evidence\": \"GST pull-down with phospho-mimetic mutants and phosphorylated peptides\",\n      \"pmids\": [\"12615054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence for MRP2 activity or trafficking demonstrated\", \"Single-lab in vitro binding without cellular validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified NHERF4 as a binder and modulator of epithelial Ca2+ channels TRPV5/TRPV6 and of the renal phosphate transport machinery, mapping the fourth PDZ domain as the principal interaction module and revealing partner-specific routing via MAP17 to the TGN.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, co-IP, immunolocalization, OK-cell coexpression with phosphate transport assays\",\n      \"pmids\": [\"16565876\", \"16926447\", \"16896066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NHERF4 directly gates channel activity vs. trafficking unresolved at this stage\", \"In vivo relevance of MAP17-NHERF4 TGN routing not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated that NHERF4 is a physiological modulator of TRPV6 channel current, moving from binding to a functional electrophysiological readout.\",\n      \"evidence\": \"GST pull-down, deletion mutagenesis, patch-clamp, and siRNA knockdown in HEK293 cells\",\n      \"pmids\": [\"17645868\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which PDZ binding alters channel gating unknown\", \"In vivo TRPV6 regulation not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined NHERF4 as a NHERF-isoform-specific mediator of Ca2+-dependent stimulation of NHE3, showing that a Ca2+-induced decrease in NHERF4-NHE3 association increases NHE3 surface expression and activity.\",\n      \"evidence\": \"Overlay assay, FRET, sucrose gradient, NHE3 activity and surface biotinylation in PS120 cells\",\n      \"pmids\": [\"19088451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling link between Ca2+ rise and complex remodeling not defined\", \"Physiological context in intact epithelium not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended NHERF4's regulatory repertoire to anion exchange and GPCR signaling, mapping PDZ3 (Ser329) to SLC26A3/DRA internalization and PDZ1/2 to prostacyclin receptor potentiation with defined endothelial phenotypes.\",\n      \"evidence\": \"Co-IP, GST pull-down, surface/internalization and transport assays, phospho-site mutagenesis; siRNA, ligand binding, cAMP, migration and angiogenesis assays\",\n      \"pmids\": [\"22627094\", \"22884631\", \"20634435\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single scaffold drives opposite outcomes (internalization vs. surface stabilization) for different partners not mechanistically reconciled\", \"Lipid-raft dependence of DRA regulation correlative\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed that NHERF4 abundance is itself regulated post-translationally by the circadian deubiquitinase USP2-45, linking scaffold levels to rhythmic intestinal Ca2+ absorption via TRPV6.\",\n      \"evidence\": \"In vitro interaction, membrane-fraction immunoblot, Usp2-KO mouse phenotype, co-IP with Clathrin Heavy Chain\",\n      \"pmids\": [\"26756164\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination/deubiquitination of NHERF4 not demonstrated\", \"TRPV6 link to the calcium phenotype inferred rather than proven\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified the Mas receptor as a NHERF4 partner and established NHERF4 as a negative regulator of PLC/AKT signaling that suppresses renal carcinoma metastasis, broadening NHERF4's role into oncogenic signaling.\",\n      \"evidence\": \"SPR-MS, GST pull-down, co-IP, overexpression/knockdown, migration/invasion and in vivo metastasis assays, AKT and Ca2+ readouts\",\n      \"pmids\": [\"34216689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PDZ domain responsible for Mas binding not mapped\", \"Whether suppression occurs via receptor sequestration or trafficking unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the molecular basis of GCC inhibition first seen in 2002, showing PDZ1 peptides interfere with GCC oligomerization at the dimer interface and reduce GCC-mediated fluid secretion in enteroids, defining NHERF4 as a negative-feedback brake on enterotoxin-driven secretion.\",\n      \"evidence\": \"In vitro GCC activity assay, peptide competition, structural modeling, FRET, mouse/human enteroid fluid secretion with ST stimulation\",\n      \"pmids\": [\"40759370\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the NHERF4-PDZ1/GCC complex lacking\", \"Therapeutic translation of inhibitory peptides untested in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NHERF4's distinct PDZ domains are selected and coordinated to produce partner-specific, sometimes opposite, regulatory outcomes — and how its phosphorylation state integrates these decisions — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length NHERF4 engaging multiple partners\", \"Logic of activation vs. internalization choice undefined\", \"In vivo loss-of-function phenotypes largely uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 6, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 6, 8, 9, 11, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 6, 7]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [4, 6, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 11, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GUCY2C\", \"TRPV5\", \"TRPV6\", \"SLC9A3\", \"SLC26A3\", \"SLC22A5\", \"PTGIR\", \"MAS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}