C-terminal PDZ Domain-binding Motif Research Articles

The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria

BackgroundTransmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown.ResultsIn this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria.ConclusionsThis study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.

Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period.

Copy number variants and point mutations of NEPH2 (also called KIRREL3) gene encoding an immunoglobulin (Ig) superfamily adhesion molecule have been linked to autism spectrum disorders, intellectual disability and neurocognitive delay associated with Jacobsen syndrome, but the physiological roles of Neph2 in the mammalian brain remain largely unknown. Neph2 is highly expressed in the dentate granule (DG) neurons of the hippocampus and is localized in both dendrites and axons. It was recently shown that Neph2 is required for the formation of mossy fiber filopodia, the axon terminal structure of DG neurons forming synapses with GABAergic neurons of CA3. In contrast, however, it is unknown whether Neph2 also has any roles in the postsynaptic compartments of DG neurons. We here report that, through its C-terminal PDZ domain-binding motif, Neph2 directly interacts with postsynaptic density (PSD)-95, an abundant excitatory postsynaptic scaffolding protein. Moreover, Neph2 protein is detected in the brain PSD fraction and interacts with PSD-95 in synaptosomal lysates. Functionally, loss of Neph2 in mice leads to age-specific defects in the synaptic connectivity of DG neurons. Specifically, Neph2−/− mice show significantly increased spontaneous excitatory synaptic events in DG neurons at postnatal week 2 when the endogenous Neph2 protein expression peaks, but show normal excitatory synaptic transmission at postnatal week 3. The evoked excitatory synaptic transmission and synaptic plasticity of medial perforant pathway (MPP)-DG synapses are also normal in Neph2−/− mice at postnatal week 3, further confirming the age-specific synaptic defects. Together, our results provide some evidence for the postsynaptic function of Neph2 in DG neurons during the early postnatal period, which might be implicated in neurodevelopmental and cognitive disorders caused by NEPH2 mutations.

Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na+/H+ exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.

Two novel CCDC88C mutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus

BackgroundHuman congenital non-syndromic hydrocephalus is a vastly heterogeneous condition. A subgroup of cases are not secondary to a specific cause (eg, a neural tube defect), and within this subgroup, autosomal...

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.

Synaptic removal of diacylglycerol by DGKζ and PSD-95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.

Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts via its C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the last ten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pump to the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitation assays coupled with liquid chromatography/tandem mass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing protein associated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extract using a fusion protein containing the C-terminal PDZ domain binding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized platelets demonstrated the existence of a 1,000-kDa complex containing PMCA and CLP36, and in addition, alpha-actinin and actin. Immunoflourescence microscopy confirmed the co-localization of PMCA with CLP36 in resting and activated platelets. Taken together these results suggest that PMCA is localized in non-filamentous actin complexes in resting platelets by means of PDZ domain interactions and then associates with the actin cytoskeleton during cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine and tyrosine phosphorylation events previously described in human platelets, PMCA function may be regulated by interactions with anchoring and cytoskeletal proteins.

ProSAP-interacting Protein 1 (ProSAPiP1), a Novel Protein of the Postsynaptic Density That Links the Spine-associated Rap-Gap (SPAR) to the Scaffolding Protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.

A Rich1/Amot Complex Regulates the Cdc42 GTPase and Apical-Polarity Proteins in Epithelial Cells

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Human scribble, a novel tumor suppressor identified as a target of high‐risk HPV E6 for ubiquitin‐mediated degradation, interacts with adenomatous polyposis coli

Recently, we have identified human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, as a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing leucine-rich repeats and PDZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), which is a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. Here, we investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localized with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC caused disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC.

The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation.

In the developing mammalian brain, a large fraction of excitatory synapses initially contain only N-methyl-D-aspartate receptor and thus are "silent" at the resting membrane potential. As development progresses, synapses acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs). Although this maturation of excitatory synapses has been well characterized, the molecular basis for this developmental change is not known. Here, we report that dendrite arborization and synapse maturation 1 (Dasm1), an Ig superfamily member, controls excitatory synapse maturation. Dasm1 is localized at the excitatory synapses. Suppression of Dasm1 expression by using RNA interference or expression of dominant negative deletion mutants of Dasm1 in hippocampal neurons at late developmental stage specifically impairs AMPA-R-mediated, but not N-methyl-D-aspartate receptor-mediated, synaptic transmission. The ability of Dasm1 to regulate synaptic AMPA-Rs requires its intracellular C-terminal PDZ domain-binding motif, which interacts with two synaptic PDZ domain-containing proteins involved in spine/synapse maturation, Shank and S-SCAM. Moreover, expression of dominant negative deletion mutants of Dasm1 leads to more immature silent synapses. These results suggest that Dasm1, as a transmembrane molecule, likely provides a link to bridge extracellular signals and intracellular signaling complexes in controlling excitatory synapse maturation.

NHERF2 specifically interacts with LPA2 receptor and defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation.

Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.

Nectin-dependent localization of synaptic scaffolding molecule (S-SCAM) at the puncta adherentia junctions formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the mouse hippocampus.

Two types of intercellular junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAs), are observed at the synapses between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the hippocampus. SJs are associated with active zones and postsynaptic densities (PSDs) where neurotransmission occurs, whereas PAs are not associated with either of them. We have found that the nectin-afadin unit as well as the N-cadherin-catenin unit localizes at the PAs and that both the units cooperatively organize the PAs. Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Synaptic scaffolding molecule (S-SCAM) is a neural scaffolding protein which interacts with many proteins including neuroligin, NMDA receptors, neural plakophilin-related armadillo-repeat protein/delta-catenin, a GDP/GTP exchange protein for Rap1 small G protein (PDZ-Rap-GEP), and beta-catenin. S-SCAM has been suggested to be a component of PSDs, but its precise localization at the synapses remains unknown. S-SCAM was not concentrated at the PSDs but highly concentrated and co-localized with nectins at both the sides of the PAs formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the adult mouse hippocampus. S-SCAM co-localized with nectin-1 at the primitive synapses where the SJs and the PAs were not morphologically differentiated, and they co-localized during the maturation of the SJs and the PAs. Nectin-1 had a potency to recruit S-SCAM to the nectin-1-based cell-cell adhesion sites formed in cadherin-deficient L cells as a model system. This recruitment was dependent on the C-terminal PDZ domain-binding motif of nectin-1 which is necessary for the binding of afadin, suggesting that nectins recruit S-SCAM through afadin. Consistently, S-SCAM was co-immunoprecipitated with afadin by the anti-S-SCAM antibody from the mouse brain, but S-SCAM did not directly bind afadin. These results indicate that S-SCAM localizes at the PAs in the CA3 area of the hippocampus in a nectin-dependent manner and suggest that S-SCAM serves as a scaffolding molecule at the PAs after maturation of the synapses and at the SJs during the maturation.

Link of the unique oncogenic properties of adenovirus type 9 E4-ORF1 to a select interaction with the candidate tumor suppressor protein ZO-2

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region-encoded ORF1 (E4-ORF1) protein. We report here that the PDZ domain-containing protein ZO-2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4-ORF1 but not for non-tumorigenic wild-type E4-ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C-terminal PDZ domain-binding motif of Ad9 E4- ORF1 and the first PDZ domain of ZO-2, and in cells this interaction resulted in aberrant sequestration of ZO-2 within the cytoplasm. Furthermore, transformation-defective Ad9 E4-ORF1 mutants exhibited impaired binding to and sequestration of ZO-2 in cells, and overexpression of wild-type ZO-2, but not mutant ZO-2 lacking the second and third PDZ domains, interfered with Ad9 E4-ORF1-induced focus formation. Our results suggest that the select capacity to complex with the candidate tumor suppressor protein ZO-2 is key to defining the unique transforming and tumorigenic properties of the Ad9 E4-ORF1 oncoprotein.

MAGUIN, a Novel Neuronal Membrane-associated Guanylate Kinase-interacting Protein

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile alpha motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membrane via the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.