PDZ Domain Scaffolding Protein Research Articles

Mechanisms of PDZ domain scaffold assembly illuminated by use of supported cell membrane sheets.

PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners. The kinetics of the binding were remarkably slow and binding strength two-three orders of magnitude higher than the intrinsic affinity for the isolated PDZ interaction. Interestingly, discrete changes in the intrinsic PICK1 PDZ affinity did not affect overall binding strength but instead revealed dual scaffold modes for PICK1. Our data supported by simulations suggest that intrinsic PDZ domain affinities are finely tuned and encode specific cellular responses, enabling multiplexed cellular functions of PDZ scaffolds.

Scaffolding protein GOPC regulates tight junction structure.

GOPC (FIG/PIST/CAL) is a PDZ-domain scaffolding protein that regulates the trafficking of a wide array of proteins, including small GTPases, receptors and cell surface molecules such as cadherin 23 and cystic fibrosis transmembrane regulator. In Madin-Darby canine kidney (MDCK) cells, we find that GOPC localizes to the trans-Golgi network (TGN) but not to the cis- or trans-Golgi cisternae. Colocalization occurs with the early endosome Rab GTPase Rab5 and a TGN/endosome marker Rab14 but not with Rab11, a marker of recycling endosomes. No localization of GOPC was detected to the lateral membranes or tight junctions. Knockdown of GOPC in MDCK cells results in decreased transepithelial resistance and increased paracellular flux. This might be attributable to the compromised trafficking of tight junction components from the TGN, as GOPC-knockdown cells have decreased lateral labeling of the tight junction protein claudin-1 and decreased protein levels of claudin-2. GOPC might mediate the trafficking of newly synthesized tight junction proteins from the TGN to the cell surface or the recycling of these proteins from specialized endosomal compartments.

Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.

Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

Photosensitive TRPs.

The Drosophila "transient receptor potential" channel is the prototypical TRP channel, belonging to and defining the TRPC subfamily. Together with a second TRPC channel, trp-like (TRPL), TRP mediates the transducer current in the fly's photoreceptors. TRP and TRPL are also implicated in olfaction and Malpighian tubule function. In photoreceptors, TRP and TRPL are localised in the ~30,000 packed microvilli that form the photosensitive "rhabdomere"-a light-guiding rod, housing rhodopsin and the rest of the phototransduction machinery. TRP (but not TRPL) is assembled into multimolecular signalling complexes by a PDZ-domain scaffolding protein (INAD). TRPL (but not TRP) undergoes light-regulated translocation between cell body and rhabdomere. TRP and TRPL are also found in photoreceptor synapses where they may play a role in synaptic transmission. Like other TRPC channels, TRP and TRPL are activated by a G protein-coupled phospholipase C (PLCβ4) cascade. Although still debated, recent evidence indicates the channels can be activated by a combination of PIP2 depletion and protons released by the PLC reaction. PIP2 depletion may act mechanically as membrane area is reduced by cleavage of PIP2's bulky inositol headgroup. TRP, which dominates the light-sensitive current, is Ca(2+) selective (P Ca:P Cs >50:1), whilst TRPL has a modest Ca(2+) permeability (P Ca:P Cs ~5:1). Ca(2+) influx via the channels has profound positive and negative feedback roles, required for the rapid response kinetics, with Ca(2+) rapidly facilitating TRP (but not TRPL) and also inhibiting both channels. In trp mutants, stimulation by light results in rapid depletion of microvillar PIP2 due to lack of Ca(2+) influx required to inhibit PLC. This accounts for the "transient receptor potential" phenotype that gives the family its name and, over a period of days, leads to light-dependent retinal degeneration. Gain-of-function trp mutants with uncontrolled Ca(2+) influx also undergo retinal degeneration due to Ca(2+) cytotoxicity. In vertebrate retina, mice knockout studies suggest that TRPC6 and TRPC7 mediate a PLCβ4-activated transducer current in intrinsically photosensitive retinal ganglion cells, expressing melanopsin. TRPA1 has been implicated as a "photo-sensing" TRP channel in human melanocytes and light-sensitive neurons in the body wall of Drosophila.

PDZ-domain scaffold proteins differentially modulate neuronal membrane expression of C-terminal splice variants of the human L-type calcium channel CaV1.3

PDZ-domain scaffold proteins differentially modulate neuronal membrane expression of C-terminal splice variants of the human L-type calcium channel CaV1.3

Abstract 24: Thrombus Formation is Impaired in Glutamate Receptor Interacting Protein 1 Deficient Mice

Platelets contribute to the pathogenesis of myocardial infarction and stroke. Understanding mechanisms of platelet activation and thrombus formation may provide new targets for anti-platelet therapies. GRIP1 (glutamate receptor interacting protein 1) is a 7 PDZ domain scaffolding protein which localizes receptors at the membrane in a regulated manner. Previous work has shown that glutamate receptors increase platelet activation through glutamate receptor signaling and we wanted to investigate the regulation of these receptors. In the neuron, GRIP1 regulates surface expression of GluR2, however, GRIP1 KO mice are embryonic lethal due to hemorrhage and hypovolemia indicating severe vascular defects. Endothelial cells do not appear to express GRIP1, therefore, we hypothesize that GRIP1 mediates platelet activation by PDZ domain coordination of protein-protein interactions. We have discovered that GRIP1 is present in mouse and human platelets. To explore the role of GRIP1 in platelets we have created a platelet-specific GRIP1 -/- mouse. GRIP1 -/- mice have normal platelet counts but significantly decreased thrombus formation (p<0.05) during ferric chloride injury and their bleeding time is significantly longer (p<0.05) in tail bleeding assays. They have no observed spontaneous bleeding or premature death. CD62P and JON/A expression after stimulation with thrombin, 2-meADP, U46619 , or convulxin was unchanged compared to WT mice. To determine which protein-protein interactions could be mediating the GRIP1 effects in platelets, we performed IP of GRIP1 and mass spectrometry. GRIP1 showed an association with the GP1b complex. Aggregation after stimulation with thrombin and 2-meADP is also impaired. Ongoing studies will determine whether specific members of the GP1b complex associate with GRIP1 in IP assays, flow chamber adhesion, and botrocetin aggregation to complement our in vivo data. Based on our data, GRIP1 has a central role in platelet thrombus formation through non-glutamate receptor dependent interactions.

Abstract 94: Scaffolding Protein EBP50 Regulates Vascular Smooth Muscle Nox1 Activity by Binding p47 phox

The NADPH oxidase (Nox) enzyme family is implicated in the pathogenesis of cardiovascular diseases (CVDs) including hypertension. In large vessel smooth muscle cells (SMCs), a unique hybrid Nox1 oxidase exists which propagates SMC dysfunction and hypertrophy. We recently demonstrated that these actions involve activation of Nox1 by hydrogen peroxide (H 2 O 2 ) leading to feed-forward production of reactive oxygen species and activation of apoptosis signal regulating kinase 1 (Ask1). Here we postulate that Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), a PDZ-domain scaffolding protein, is required for H 2 O 2 -mediated activation of SMC Nox1. In SMCs, knockdown of EBP50 by shRNA abolished superoxide (O 2 •- ) production in 50 μM H 2 O 2 -treated cells (37.7 ± 4.5, 79.9 ± 2.3 and 29.0 ± 3.5 x10 4 O 2 •- -mediated RLU for GFP plasmid vehicle-treated, GFP plasmid H 2 O 2 -treated and EBP50 shRNA plasmid H 2 O 2 -treated groups, respectively). Furthermore, H 2 O 2 -induced O 2 •- production was absent in SMCs isolated from EBP50 knockout (KO) mice (29.0 ± 2.7 vs 79.1 ± 2.4 x10 4 RLU for EBP50 KO vs wildtype after H 2 O 2 treatment). Intriguingly, p47 phox , an essential cytosolic subunit of the hybrid SMC Nox1 system, upon phosphorylation co-immunoprecipitated with EBP50 in COS-7 cells transfected with both proteins (Fig 1). Taken together, our data suggest that EBP50 promotes Nox activity by binding active p47 phox . These studies identify a novel partner in Nox1 activation and potentially provide new insights into the broader regulation of this enzyme family. Moreover, this added level of complexity could yield new therapeutic strategies to disrupt Nox in the treatment of hypertension and other CVDs.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Phototransduction mechanisms in Drosophila microvillar photoreceptors

AbstractPhototransduction in Drosophila is mediated by a G‐protein coupled phospholipase C (PLC) cascade leading to graded membrane depolarization. It is the preferred model for phototransduction in microvillar photoreceptors and an important model for the ubiquitous phosphoinositide signaling cascade. The photoreceptors respond sensitively to single photons 10–100× more rapidly than vertebrate rods, yet still light adapt to signal under full sunlight. Incident light is absorbed and transduced in the rhabdomere, a 1–2 µm diameter, 100 µm long waveguide composed of ∼30,000 tightly packed microvilli, which contain the visual pigment rhodopsin and all the major components of the cascade. PLC hydrolyzes phosphatidyl‐inositol (4,5) bisphosphate (PIP2) to generate diacylglycerol (DAG), inositol (1,4,5) trisphosphate (InsP3), and also a proton. This results in activation of two classes of Ca2+ permeable cation channels, TRP and TRPL—the prototypical members of the transient receptor potential (TRP) superfamily of cation channels. Recent evidence suggests that the channels may be activated in a combinatorial manner by two neglected consequences of PLC activity, namely simultaneous depletion of PIP2 and acidification. Several components of the cascade, including TRP, PLC, and protein kinase C are assembled into multimolecular signaling complexes by the PDZ‐domain scaffolding protein INAD. Ca2+ influx via TRP channels is essential for rapid kinetics, amplification, and light adaptation and mediates both positive and negative feedback via multiple downstream targets, including the channels, rhodopsin, and PLC. This Ca2+‐dependent feedback along with the ultracompartmentalization provided by the microvillar design and molecular scaffolding is critical for the combination of sensitivity, rapid kinetics, and broad dynamic range. WIREs Membr Transp Signal 2012, 1:162–187. doi: 10.1002/wmts.20For further resources related to this article, please visit the WIREs website.

Scribblemutants promote aPKC and JNK-dependent epithelial neoplasia independently of Crumbs

BackgroundMetastatic neoplasias are characterized by excessive cell proliferation and disruptions to apico-basal cell polarity and tissue architecture. Understanding how alterations in cell polarity can impact upon tumour development is, therefore, a central issue in cancer biology. The Drosophila gene scribble (scrib) encodes a PDZ-domain scaffolding protein that regulates cell polarity and acts as a tumour suppressor in flies. Increasing evidence also implicates the loss of human Scrib in cancer. In this report, we investigate how loss of Scrib promotes epithelial tumourigenesis in Drosophila, both alone and in cooperation with oncogenic mutations.ResultsWe find that genetically distinct atypical protein kinase C (aPKC)-dependent and Jun N-terminal kinase (JNK)-dependent alterations in scrib mutants drive epithelial tumourigenesis. First, we show that over-expression of the apical cell polarity determinants Crumbs (Crb) or aPKC induces similar cell morphology defects and over-proliferation phenotypes as scrib loss-of-function. However, the morphological and proliferative defects in scrib mutants are independent of Crb function, and instead can be rescued by a dominant negative (kinase dead) aPKC transgene. Secondly, we demonstrate that loss of Scrib promotes oncogene-mediated transformation through both aPKC and JNK-dependent pathways. JNK normally promotes apoptosis of scrib mutant cells. However, in cooperation with oncogenic activated Ras or Notch signalling, JNK becomes an essential driver of tumour overgrowth and invasion. aPKC-dependent signalling in scrib mutants cooperates with JNK to significantly enhance oncogene-mediated tumour overgrowth.ConclusionThese results demonstrate distinct aPKC and JNK-dependent pathways through which loss of Scrib promotes tumourigenesis in Drosophila. This is likely to have a direct relevance to the way in which human Scrib can similarly restrain an oncogene-mediated transformation and, more generally, on how the outcome of oncogenic signalling can be profoundly perturbed by defects in apico-basal epithelial cell polarity.

Melatonin: Signaling mechanisms of a pleiotropic agent

Melatonin acts both as a hormone of the pineal gland and as a local regulator molecule in various tissues. Quantities of total tissue melatonin exceed those released from the pineal. With regard to this dual role, to the orchestrating, systemic action on various target tissues, melatonin is highly pleiotropic. Numerous secondary effects result from the control of the circadian pacemaker and, in seasonal breeders, of the hypothalamic/pituitary hormonal axes. In mammals, various binding sites for melatonin have been identified, the membrane receptors MT(1) and MT(2), which are of utmost chronobiological importance, ROR and RZR isoforms as nuclear receptors from the retinoic acid receptor superfamily, quinone reductase 2, calmodulin, calreticulin, and mitochondrial binding sites. The G protein-coupled receptors (GPCRs) MT(1) and MT(2) are capable of parallel or alternate signaling via different Galpha subforms, in particular, Galpha(i) (2/) (3) and Galpha(q), and via Gbetagamma, as well. Multiple signaling can lead to the activation of different cascades and/or ion channels. Melatonin frequently decreases cAMP, but also activates phospholipase C and protein kinase C, acts via the MAP kinase and PI3 kinase/Akt pathways, modulates large conductance Ca(2+)-activated K(+) and voltage-gated Ca(2+) channels. MT(1) and MT(2) can form homo and heterodimers, and MT(1) interacts with other proteins in the plasma membrane, such as an orphan GPCR, GPR50, and the PDZ domain scaffolding protein MUPP1, effects which negatively or positively influence signaling capacity. Cross-talks between different signaling pathways, including influences of the membrane receptors on nuclear binding sites, are discussed. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites

PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

The Cystic Fibrosis Transmembrane Regulator Forms Macromolecular Complexes with PDZ Domain Scaffold Proteins

Cystic fibrosis transmembrane regulator (CFTR), an epithelial Cl- channel defective in cystic fibrosis, is localized at the apical membrane of epithelial cells. CFTR interacts with a number of ion channels (outwardly rectifying chloride channels, epithelial sodium channels, and inwardly rectifying potassium channels), protein kinases A and C, and putative protein phosphatases and ATP transporters. These data suggest that CFTR may exist in macromolecular complexes with these and other scaffolding proteins at the apical membrane. PDZ domain proteins have been shown to localize and cluster CFTR in the Golgi and at the plasma membrane. This review highlights the role of PDZ domain proteins in regulating the trafficking and processing of CFTR.

Postsynaptic signaling of new players at the neuromuscular junction.

The neuromuscular junction (NMJ) represents the most well studied synapse and is widely regarded as structurally and functionally less complicated than neuronal synapses in the brain. Recent studies, however, have identified the localization and function of new signaling molecules at the NMJ. Surprisingly, many synaptic proteins previously identified in the brain are indeed also concentrated on the postsynaptic muscle side of the NMJ. These include the serine/threonine kinase Cdk5, the neurotrophin receptor TrkB, Eph receptors and ephrins, NMDA receptors and nitric oxide synthase, various PDZ-domain scaffold proteins, and beta-amyloid precursor protein. These observations indicate that the molecular composition of NMJ is much more intricate than we originally thought. The potential significance of these new signaling molecules at the NMJ will be discussed.

Organization of signaling complexes by PDZ-domain scaffold proteins.

Transduction of biological signals from receptors at the plasma membrane to their targets in cytoplasm and nucleus relies on specific protein-protein interactions. A common strategy used by cells is to organize proteins in the same signaling cascade into large molecular weight, multiprotein complexes. PDZ domain proteins have been shown to play important roles in assembling various signaling complexes. Here, we first present biophysical basis of the advantages of organizing proteins in a signaling cascade into a clustered multiprotein complex. We then discuss the structure, ligand binding, and function of PDZ domains in organizing synaptic signaling complexes.

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development.

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.