| 2002 |
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. |
Yeast two-hybrid screen, co-immunoprecipitation, biochemical binding assays |
The Journal of biological chemistry |
High |
11950846
|
| 2003 |
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. |
GST pull-down assay with phosphorylation-mimicking mutants and phosphorylated peptides |
Biochemical and biophysical research communications |
Medium |
12615054
|
| 2006 |
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. |
Yeast two-hybrid screen, GST pull-down (in vitro translated NHERF4 and Xenopus oocyte lysates), co-immunoprecipitation (HEK293 cells), immunolocalization |
Pflugers Archiv : European journal of physiology |
High |
16565876
|
| 2006 |
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. |
Bacterial and mammalian two-hybrid, coexpression in opossum kidney (OK) cells, confocal immunofluorescence, phosphate transport assays, PKC inhibition/activation pharmacology |
American journal of physiology. Renal physiology |
High |
16926447
|
| 2006 |
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. |
Uptake assays in HEK293 cells, cell surface expression assay, deletion mutagenesis, co-immunolocalization in mouse kidney |
Drug metabolism and disposition |
High |
16896066
|
| 2007 |
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. |
Yeast two-hybrid screen, GST pull-down, deletion mutagenesis, patch-clamp electrophysiology, siRNA knockdown in HEK293 cells |
Biochemical and biophysical research communications |
High |
17645868
|
| 2008 |
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. |
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 |
Cellular physiology and biochemistry |
High |
19088451
|
| 2010 |
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. |
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 |
American journal of physiology. Gastrointestinal and liver physiology |
Medium |
20634435
|
| 2012 |
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. |
Co-immunoprecipitation, GST pull-down, cell surface expression assay, internalization assay, anion exchange activity assay, phosphorylation site mutagenesis |
Cellular signalling |
High |
22627094
|
| 2012 |
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. |
Co-immunoprecipitation, co-localization, siRNA knockdown, ligand binding assay, cAMP assay, endothelial migration and angiogenesis assays |
Biochimica et biophysica acta |
High |
22884631
|
| 2016 |
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. |
In vitro interaction assay, immunoblot of membrane fractions, Usp2-KO mouse phenotype, co-immunoprecipitation with Clathrin Heavy Chain |
PloS one |
Medium |
26756164
|
| 2021 |
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. |
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 |
Cancer letters |
High |
34216689
|
| 2025 |
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. |
In vitro GCC activity assay, peptide competition assay, 3D structural modeling, FRET, mouse and human enteroid fluid secretion assay, ST stimulation |
The Journal of biological chemistry |
High |
40759370
|