A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange

NHERF-1, a protein adapter containing two tandem PDZ domains, was first identified as an essential cofactor required for the phosphorylation and downregulation of NHE3 activity in response to elevated intracellular cAMP. NHERF-1 contains multiple protein interaction domains, but the mechanism by which it binds NHE3 remains unknown. Yeast two-hybrid analyses demonstrated that the C-terminal sequence, STHM, of NHE3 constitutes a PDZ motif critical for its association with NHERF-1. In this assay, NHE3 bound both PDZ-I and PDZ-II when presented as isolated domains, but mutations of the individual PDZ domains in the full-length NHERF-1 suggested a significant preference of NHE3 for the PDZ-II domain. To investigate NHERF-1/NHE3 association in cells, NHERF-1 complexes were isolated from PS120 cells expressing hexahistidine-tagged NHERF-1 and NHE3 using nickel-NTA-agarose. In these experiments, mutating the C-terminal PDZ motif still allowed NHE3 binding to NHERF-1, suggesting the presence of additional mechanisms or components that stabilized a cellular NHE3/NHERF-1 complex. Transport assays in PS120 cells, however, showed that the C-terminal PDZ motif in NHE3 and a functional PDZ-II domain in NHERF-1 were required for maximal inhibition of sodium-hydrogen exchange in response to forskolin and 8-Br-cAMP. Together, the data suggested that the PDZ interaction between the NHE3 C-terminus and a NHERF-1 PDZ domain enhanced the regulation of sodium-hydrogen exchange by cAMP-elevating hormones.     

Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation.

PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na(+)/H(+) exchanger regulatory factor (NHERF-1; also called "EBP50") and its relative NHERF-2 (also called "E3KARP", "SIP-1", and "TKA-1") both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta(2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.     

Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions

The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.     

Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cbeta, and actin increases intracellular calcium in opossum kidney cells

Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C (PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+]i and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+]i that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCbeta1 and -beta3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+]i. Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+]i. Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCbeta, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium.     

Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets

NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.     

Design of a selective chemical probe for class I PDZ domains

Covalent labeling has been widely used for structural and functional analyses of proteins. To target a wide range of PDZ domains, we designed a chemical scaffold mimicking the E/D-T/S-XV peptide, which is a PDZ domain that binds ligands in higher occurrence. A chemical probe (2) that contained this moiety alkylated diverse PDZ domains, including NHERF-1 PDZ2, and differentially visualized the cellular proteome.     

Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta

The PDZ domain adaptor protein Na⁺/H⁺ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca²⁺, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca²⁺ signaling. PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation     

Receptor activity-modifying protein (RAMP) isoform-specific regulation of adrenomedullin receptor trafficking by NHERF-1

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.     

Molecular determinants for PICK1 synaptic aggregation and mGluR7a receptor coclustering: role of the PDZ, coiled-coil, and acidic domains

PSD-95/Disc-large/ZO-1 (PDZ) domain-containing proteins play a central role in synaptic organization by their involvement in neurotransmitter receptor clustering and signaling complex assembly. The protein interacting with protein kinase C (PICK1), a synaptic PDZ domain protein that also contains a coiled-coil and acidic domain, binds to several synaptic components including the metabotropic glutamate receptor mGluR7a. Coexpression of PICK1 and mGluR7a in heterologous cells induces coclustering of these two proteins. To examine the role of the different structural motifs of PICK1 in synaptic aggregation of PICK1 and mGluR7a coclustering, several PICK1 mutants were generated to analyze their distribution in transfected hippocampal cultured neurons and to test their ability to induce coclusters with mGluR7a when coexpressed in fibroblast cells. The PDZ and coiled-coil domains are both required, whereas the acidic region plays an inhibitory role in these processes. Our data suggest that synaptic aggregation and receptor coclustering depend on PICK1 binding to a target membrane receptor, e.g. mGluR7a, by a PDZ-mediated interaction and on PICK1 oligomerization through the coiled-coil domain. This study defined three structural signals within PICK1 regulating its synaptic localization and receptor coclustering activity, which could represent molecular substrates involved in synaptic development and plasticity.     

Structural Insights into Neutrophilic Migration Revealed by the Crystal Structure of the Chemokine Receptor CXCR2 in Complex with the First PDZ Domain of NHERF1

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.     

Role of the PDZ domains in Escherichia coli DegP protein

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.     

The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane.Protein kinase D (PKD)² plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). The PKD family consists of three members belonging to the Ca²⁺/calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4).PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5, 6). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DAG, and this leads to recruitment and activation of the novel PKCs and thus also PKD.PDZ (PSD-95, Discs large, ZO-1) domains are compact, globular structures of ∼90 residues, occurring in one or multiple copies within a protein, that mediate protein-protein interactions (7). These interactions occur via binding to other PDZ domains or, more commonly, by recognition of short amino acid motifs in the carboxyl termini of target proteins commonly terminating in a hydrophobic residue (8). In the case of PKD1 and PKD2, the last four amino acids are VSIL and ISVL, respectively. Here we identify Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1) as a PDZ domain-containing protein that interacts with the PDZ-binding motif of both PKD1 and PKD2.NHERF-1 was originally cloned as a critical protein component for the inhibition of Na⁺/H⁺ exchanger 3 by protein kinase A (9). NHERF-1 is 52% identical to NHERF-2, a family member with which it shares the conserved domain structure of two PDZ domains followed by an ezrin-radixin-moesin (ERM)-binding region (10). Parallel studies demonstrating its ability to strongly interact with ezrin independently identified NHERF-1 as ERM-binding phosphoprotein 50 (11). Via this ERM-binding region, NHERF-1 and NHERF-2 are predominantly localized near the actin cytoskeleton, thus poising them near the plasma membrane where they function as scaffolds. Since these original cloning reports, numerous studies have identified over 30 binding partners of these scaffold proteins including GPCRs, tyrosine kinase receptors, other adaptor proteins, signaling enzymes, and ion channels (12, 13).Here we identify PKD1 and PKD2 as NHERF-1-interacting proteins. Using a fluorescence resonance energy transfer (FRET)-based assay to assess molecular proximity, both PKD1 and PKD2 are shown to transiently associate with NHERF-1 following PKD activation. Furthermore, through use of genetically encoded reporters for PKD activity, we show a unique signature of PKD activation at the NHERF scaffold. Specifically, signaling is more tightly regulated at the scaffold than in the cytosol or bulk plasma membrane. Phosphatase activity is higher at NHERF than at the plasma membrane, resulting in a more rapidly reversible PKD response at the scaffold, and following an agonist-evoked response, PKD signaling is prolonged compared with the length of response in the cytosol. Our data identify NHERF-1 as a novel nexus of PKD signaling and raise the possibility that PKD may act as a novel regulator of proteins at the NHERF scaffold.     

Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase

Na(+)/H(+) exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pin1, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pin1 facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.     

The second PDZ domain of zonula occludens-1 is dispensable for targeting to connexin 43 gap junctions

Zonula occludens (ZO)-1 is emerging as a central player in the control of gap junction (GJ) dynamics. Previously the authors reported that ZO-1 localizes preferentially to the periphery of Cx43 GJs. How ZO-1 arrives at GJ edges is unknown, but this targeting might involve we established interaction between the Cx43 C-terminus and the PDZ2 domain of ZO-1. Here the show that despite blocking the canonical PDZ2-mediated interaction by fusion of GFP to the C-terminus of Cx43, ZO-1 continued to target to domains juxtaposed with the edges of GJs comprised solely of tagged Cx43. This edge-association was not abolished by deletion of PDZ2 from ZO-1, as mutant ZO-1 also targeted to the periphery of GJs composed of either tagged or untagged Cx43. Additionally, ZO-2 was found colocalized with ZO-1 at GJ edges. These data demonstrate that ZO-1 targets to GJ edges independently of several known PDZ2-mediated interactions, including ZO-1 homodimerization, heterodimerization with ZO-2, and direct ZO-1 binding to the C-terminal residues of Cx43.     

Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

PDZ domains of Par-3 as potential phosphoinositide signaling integrators

Multiple PDZ domain scaffold protein Par-3 and phosphoinositides (PIPs) are required for polarity in diverse cell types. We show that the second PDZ domain of Par-3 binds to phosphatidylinositol (PI) lipid membranes with high affinity. We further demonstrate that a large subset of PDZ domains in mammalian genomes are capable of binding to PI lipid membranes, indicating that lipid binding is the second most prevalent interaction mode of PDZ domains known to date. The biochemical and structural basis of Par-3 PDZ2-mediated membrane interaction is characterized in detail. The membrane binding capacity of Par-3 PDZ2 is critical for epithelial cell polarization. Interestingly, the lipid phosphatase PTEN directly binds to the third PDZ domain of Par-3. The concatenation of the PIP-binding PDZ2 and the lipid phosphatase PTEN-binding PDZ3 endows Par-3 as an ideal scaffold protein for integrating PIP signaling events during cellular polarization.     

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B,Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.     

Cooperativity between the Phosphorylation of Thr95 and Ser77 of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport

The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser⁷⁷ in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr⁹⁵, another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser⁷⁷ by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr⁹⁵. These studies indicate a remarkable cooperativity between the phosphorylation of Thr⁹⁵ and Ser⁷⁷ of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr⁹⁵ facilitates the phosphorylation of Ser⁷⁷. This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants

INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.