PSD-95, Dlg, ZO-1 Research Articles

Voltage-induced calcium release in Caenorhabditis elegans body muscles

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Proteomic Identification and Structural Basis for the Interaction between Sorting Nexin Snx17 and Pdlim Family Proteins

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

FRMPD1 activates the Hippo pathway via interaction with WWC3 to suppress the proliferation and invasiveness of lung cancer cells.

Purpose: The expression of FERM-domain-containing protein-1 (FRMPD1)/FERM and PDZ domain-containing protein-2 (FRMD2) in malignant tumors, including lung cancer, and its underlying molecular mechanism have not been reported yet.Materials and methods: Immunohistochemistry was performed to analyze the expression of FRMPD1 in lung cancer tissues, and statistical analysis was applied to analyze the relationship between FRMPD1 expression and clinicopathological factors. The biological effects of FRMPD1 on lung cancer cell proliferation and invasion were determined by functional experiments both in vivo and in vitro. Immunoblotting, RT-qPCR, dual-luciferase assay, and immunofluorescence were performed to demonstrate whether FRMPD1 stimulates Hippo signaling. Co-immunoprecipitation assays were used to clarify the underlying role of FRMPD1 in Hippo pathway activation via interaction with WW and C2 domain containing protein-3 (WWC3).Results: We found that FRMPD1 expression in lung cancer specimens was lower than that in normal bronchial epithelium and normal submucosal glands. FRMPD1 expression had a negative correlation with age, Tumor-Node-Metastasis (TNM) stage, lymph node metastasis, as well as poor prognosis. Moreover, ectopic expression of FRMPD1 significantly inhibited the proliferation and invasion of lung cancer cells, and inhibition of FRMPD1 expression led to opposite effects. Mechanistically, we found that FRMPD1 interacted with the C-terminal PDZ binding motif of WWC3 via its PSD95/DLG/ZO1 (PDZ) domain and promoted the phosphorylation of large tumor suppressor-1 (LATS1), thus inhibiting the nuclear translocation of yes-associated protein (YAP).Conclusion: FRMPD1 could activate the Hippo pathway and ultimately inhibit the malignant behavior of lung cancer cells through its interaction with WWC3. This work will provide an important experimental basis for the discovery of novel biomarkers of lung cancer and the development of targeted drugs.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK–neurexin interaction

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

SNX27 links DGK\u03b6 to the control of transcriptional and metabolic programs in T lymphocytes

Sorting nexin 27 (SNX27) recycles PSD-95, Dlg1, ZO-1 (PDZ) domain-interacting membrane proteins and is essential to sustain adequate brain functions. Here we define a fundamental SNX27 function in T lymphocytes controlling antigen-induced transcriptional activation and metabolic reprogramming. SNX27 limits the activation of diacylglycerol (DAG)-based signals through its high affinity PDZ-interacting cargo DAG kinase ζ (DGKζ). SNX27 silencing in human T cells enhanced T cell receptor (TCR)-stimulated activator protein 1 (AP-1)- and nuclear factor κB (NF-κB)-mediated transcription. Transcription did not increase upon DGKζ silencing, suggesting that DGKζ function is dependent on SNX27. The enhanced transcriptional activation in SNX27-silenced cells contrasted with defective activation of the mammalian target of rapamycin (mTOR) pathway. The analysis of Snx27−/− mice supported a role for SNX27 in the control of T cell growth. This study broadens our understanding of SNX27 as an integrator of lipid-based signals with the control of transcription and metabolic pathways.

Sorting nexin 27 interactome in T-lymphocytes identifies zona occludens-2 dynamic redistribution at the immune synapse.

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO-1 (PDZ) domain-interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T-lymphocytes, SNX27-positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS-forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild-type and a mutant-deficient for PDZ cargo recognition identified the epithelial cell-cell junction protein zona occludens-2 (ZO-2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO-2 PDZ-dependent interaction, and demonstrated its role controlling the dynamic localization of ZO-2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.

Identification of novel transplantable GPCR recycling motif for drug discovery

β1-Adrenergic receptor (β1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β1-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312 in the third intracellular loop of the β1-AR. Progressive elongation of the β1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β1-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the β1-AR (β1-AR[+20]) produced a chimeric β1-AR that recycled rapidly and efficiently. The β1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β1-AR[+20] generated a β1-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the β1-AR to created EYFP-WT β1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β1-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps.

A molecular code for endosomal recycling of phosphorylated cargos by the SNX27-retromer complex.

Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95-Dlg-ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the β2 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor, lack these sequence determinants. Instead, we identified conserved sites of phosphorylation that substitute for acidic residues and dramatically enhance SNX27 interactions. This newly identified mechanism suggests a likely regulatory switch for PDZ interaction and protein transport by the SNX27-retromer complex. Defining this SNX27 binding code allowed us to classify more than 400 potential SNX27 ligands with broad functional implications in signal transduction, neuronal plasticity and metabolite transport.

Origins of Allostery and Evolvability in Proteins: A Case Study

Proteins display the capacity for adaptation to new functions, a property critical for evolvability. But what structural principles underlie the capacity for adaptation? Here, we show that adaptation to a physiologically distinct class of ligand specificity in a PSD95, DLG1, ZO-1 (PDZ) domain preferentially occurs through class-bridging intermediate mutations located distant from the ligand-binding site. These mutations provide a functional link between ligand classes and demonstrate the principle of "conditional neutrality" in mediating evolutionary adaptation. Structures show that class-bridging mutations work allosterically to open up conformational plasticity at the active site, permitting novel functions while retaining existing function. More generally, the class-bridging phenotype arises from mutations in an evolutionarily conserved network of coevolving amino acids in the PDZ family (the sector) that connects the active site to distant surface sites. These findings introduce the concept that allostery in proteins could have its origins not in protein function but in the capacity to adapt.

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins.

The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.

Par6 family proteins in cancer.

The regulatory networks of cell polarization and polarity effector proteins have been subjects of feverish interest in the field of cell and developmental biology but many advances in the polarity research may have flown unnoticed past the radar of mainstream cancer biologist. However, recent findings suggesting important cell cycle gatekeeping functions for polarity proteins may change that. For example, PAR6 proteins interact with classical cancer driver signaling pathways, including MAPK and PI3K and moreover, PAR6 (PARD6) genes are frequently altered in various cancers. Mammalian genomes harbor three different PARD6 genes. Recent studies in breast cancer have suggested that different PARD6 genes are not only important players but may play even opposite roles during tumorigenesis.

Deciphering the unconventional peptide binding to the PDZ domain of MAST2.

Phosphatase and tensin homologue (PTEN) and microtubule-associated serine threonine kinase 2 (MAST2) are key negative regulators of survival pathways in neuronal cells. The two proteins interact via the PDZ (PSD-95, Dlg1, Zo-1) domain of MAST2 (MAST2-PDZ). During infection by rabies virus, the viral glycoprotein competes with PTEN for interaction with MAST2-PDZ and promotes neuronal survival. The C-terminal PDZ-binding motifs (PBMs) of the two proteins bind similarly to MAST2-PDZ through an unconventional network of connectivity involving two anchor points. Combining stopped-flow fluorescence, analytical ultracentrifugation (AUC), microcalorimetry and NMR, we document the kinetics of interaction between endogenous and viral ligands to MAST2-PDZ as well as the dynamic and structural effects of these interactions. Viral and PTEN peptide interactions to MAST2-PDZ occur via a unique kinetic step which involves both canonical C-terminal PBM binding and N-terminal anchoring. Indirect effects induced by the PBM binding include modifications to the structure and dynamics of the PDZ dimerization surface which prevent MAST2-PDZ auto-association. Such an energetic communication between binding sites and distal surfaces in PDZ domains provides interesting clues for protein regulation overall.

A role for novel lipid interactions in the dynamic recruitment of SNX27 to the T-cell immune synapse.

SNX27 is a member of the sorting nexin family that plays an important role in the recycling of receptors from endosomes to the cell surface. In addition to a PX (Phox homology) domain that regulates its endosomal localization, SNX27 has a unique PDZ (Psd-95/Dlg/ZO1) domain and an atypical FERM (4.1, ezrin, radixin, moesin) domain that both function to bind short peptide sequence motifs in the cytoplasmic domains of the cargo receptors. Using the T cell immune synapse (IS) as a model for polarized protein recycling, we recently identified an additional mechanism that enhances SNX27 localization to the endosomal recycling compartment (ERC). Our study defined a phosphoinositide (PI) lipid-binding site within the SNX27 FERM domain, with a clear preference for bi- and triphosphorylated PIs, which may promote SNX27 localization to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and/or PtdIns(3,4,5)P3-enriched membrane domains. Using fluorescently tagged lipid-binding probes, we studied the kinetics of distinct PIs in living T cells during IS formation. Our results suggest that PtdIns(3,4,5)P3 accumulates at the contact site simultaneously with early SNX27 recruitment to the plasma membrane (PM), and this is partly controlled by by lipid binding through the FERM domain. These studies define 2 independent binding sites for PtdIns-derived lipids in SNX27, that contribute to the dynamic recruitment of SNX27 to distinct membranes during T cell activation.

Small‐Molecule Inhibitors of AF6 PDZ‐Mediated Protein–Protein Interactions

PDZ (PSD-95, Dlg, ZO-1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ-mediated protein-protein interactions has important implications in disease-related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1-fused gene from chromosome 6), which is an essential component of cell-cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile-Gln-Ser-Val-Glu-Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6-Bcr interaction and interfere with epidermal growth factor (EGF)-dependent signaling.

Sorting of β1-Adrenergic Receptors Is Mediated by Pathways That Are Either Dependent on or Independent of Type I PDZ, Protein Kinase A (PKA), and SAP97

The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085-5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β1-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β1-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β1-AR. β1-AR chimera containing the type I PDZ of the β2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β1-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β1-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position -3 in the PDZ regulated whether the β1-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β1-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs.

The Role of Protein Kinase A Regulation of the E6 PDZ-Binding Domain during the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 18

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

The Polarity Protein Scrib Is Essential for Directed Endothelial Cell Migration

Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

BackgroundThe anaphylatoxin C3a binds to the G protein coupled receptor (GPCR, C3aR) and activates divergent signaling pathways to induce degranulation and cytokine production in human mast cells. Adapter proteins such as the Na+/H+ exchange regulatory factor (NHERF1 and NHERF2) have been implicated in regulating functions of certain GPCRs by binding to the class I PDZ (PSD-95/Dlg/Zo1) motifs present on their cytoplasmic tails. Although C3aR possesses a class I PDZ motif, the possibility that it interacts with NHERF proteins to modulate signaling in human mast cells has not been determined.Methodology/Principal FindingsUsing reverse transcription PCR and Western blotting, we found that NHERF1 and NHERF2 are expressed in human mast cell lines (HMC-1, LAD2) and CD34+-derived primary human mast cells. Surprisingly, however, C3aR did not associate with these adapter proteins. To assess the roles of NHERFs on signaling downstream of C3aR, we used lentiviral shRNA to stably knockdown the expression of these proteins in human mast cells. Silencing the expression of NHERF1 and NHERF2 had no effect on C3aR desensitization, agonist-induced receptor internalization, ERK/Akt phosphorylation or chemotaxis. However, loss of NHERF1 and NHERF2 resulted in significant inhibition of C3a-induced mast cell degranulation, NF-κB activation and chemokine production.Conclusion/SignificanceThis study demonstrates that although C3aR possesses a class I PDZ motif, it does not associate with NHERF1 and NHERF2. Surprisingly, these proteins provide stimulatory signals for C3a-induced degranulation, NF-κB activation and chemokine generation in human mast cells. These findings reveal a new level of complexity for the functional regulation of C3aR by NHERFs in human mast cells.

Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

BackgroundThe interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level.ResultsHere, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif.ConclusionsOur refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

Deficiency of Sorting Nexin 27 (SNX27) Leads to Growth Retardation and Elevated Levels of N-Methyl-d-Aspartate Receptor 2C (NR2C)

Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.