c-Src is a PDZ interaction partner and substrate of the E3 ubiquitin ligase Ligand-of-Numb protein X1

The very C-terminus of c-Src is a ligand for PDZ domains. In a screen for PDZ domains that interact with c-Src, we identified one of the PDZ domains of the Ligand-of-Numb protein X1 (LNX1), a multiple PDZ domain scaffold and RING type E3 ubiquitin ligase. We demonstrate that the interaction of c-Src with LNX1 depends on the C-terminal PDZ ligand of c-Src. Furthermore, we show that c-Src phosphorylates LNX1. Moreover, c-Src itself is ubiquitinated by LNX1, suggesting an interdependent regulation of c-Src and LNX1.     

Evaluation of energetic and dynamic coupling networks in a PDZ domain protein

A number of computational and experimental studies have identified intramolecular communication "pathways" or "networks" important for transmitting allostery. Here, we have used mutagenesis and NMR relaxation methods to investigate the scope and nature of the communication networks found in the second post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain of the human protein tyrosine phosphatase 1E protein (hPTP1E) (PDZ2). It was found that most mutations do not have a significant energetic contribution to peptide ligand binding. Three mutants that showed significant changes in binding also displayed context-dependent dynamic effects. Both a mutation at a partially exposed site (H71Y) and a buried core position (I35V) had a limited response in side-chain (2)H-based dynamics when compared to wild-type PDZ2. In contrast, a change at a second core position (I20F) that had previously been shown to be part of an energetic and dynamic network, resulted in extensive changes in side-chain dynamics. This response is reminiscent to that seen previously upon peptide ligand binding. These results shed light on the nature of the PDZ2 dynamic network and suggest that position 20 in PDZ2 acts as a "hub" that is energetically and dynamically critical for transmitting changes in dynamics throughout the PDZ domain.     

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.     

Interaction of the human somatostatin receptor 3 with the multiple PDZ domain protein MUPP1 enables somatostatin to control permeability of epithelial tight junctions

The presence of heterotrimeric G-proteins at epithelial tight junctions suggests that these cellular junctions are regulated by so far unknown G-protein coupled receptors. We identify here an interaction between the human somatostatin receptor 3 (hSSTR3) and the multiple PDZ protein MUPP1. MUPP1 is a tight junction scaffold protein in epithelial cells, and as a result of the interaction with MUPP1 the hSSTR3 is targeted to tight junctions. Interaction with MUPP1 enables the receptor to regulate transepithelial permeability in a pertussis toxin sensitive manner, suggesting that hSSTR3 can activate G-proteins locally at tight junctions.

Structured summary:

MINT-6800756, MINT-6800770: MUPP1 (uniprotkb:O75970) and hSSTR3 (uniprotkb:P32745) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800587:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800562:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by two hybrid (MI:0018)MINT-6800622:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with PIST (uniprotkb: Q9HD26), Hsp70 (uniprotkb:P08107), Maguk p55 (uniprotkb: Q8N3R9), MAGI3 (uniprotkb:Q5TCQ9), ZO-2 (uniprotkb:Q9UDY2), ZO-1 (uniprotkb:Q07157) and MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800607, MINT-6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)     

The PDZ/coiled-coil domain containing protein PIST modulates insulin secretion in MIN6 insulinoma cells by interacting with somatostatin receptor subtype 5

The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic beta-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.     

Semaphorin 4B interacts with the post-synaptic density protein PSD-95/SAP90 and is recruited to synapses through a C-terminal PDZ-binding motif

The semaphorins are a large family of proteins that act as guidance signals for axons and dendrites. The class 4 semaphorins are integral membrane proteins that are widely expressed throughout the nervous system. Here, we show that a subclass of these semaphorins is characterized by a PDZ-binding motif at their carboxy-terminus. This sequence mediates the interaction with the post-synaptic density protein PSD-95/SAP90. Co-expression of Sema4B with PSD-95 in COS 7 cells results in the clustering of Sema4B. Sema4B co-localizes with PSD-95 at synaptic contacts between cultured hippocampal neurons. This synaptic localization depends on the presence of the PDZ-binding motif.     

Characterisation of the interaction between syndecan-2, neurofibromin and CASK: Dependence of interaction on syndecan dimerization

Neurofibromin and calcium/calmodulin-dependent serine protein kinase (CASK) are membrane-associated signalling and scaffolding proteins which are mutated in human genetic neurological disorders. Syndecan-2 is a highly glycosylated transmembrane protein whose intracellular C-terminus has previously been shown to interact with the post-synaptic density 95/discs large/zonula occludens-1 (PDZ) domain of CASK and with two separate regions of neurofibromin. These three proteins collaborate to orchestrate the induction of filopodia and dendritic spines. We have used systematic mutagenesis of the intracellular region of syndecan-2 and a quantitative yeast two-hybrid (Y2H) assay to study the determinants of their interactions. We show that syndecan’s interactions with both CASK and neurofibromin are dependent on syndecan homodimerization and that neurofibromin largely interacts with the membrane-proximal part of the dimeric syndecan intracellular domain, leaving the membrane-distal C-terminus free to interact with CASK. We conducted a phylogenetic study of syndecan sequences, finding correspondence between conserved residues and mutations affecting both dimerization and interactions; we also find that fish have a very different syndecan repertoire from tetrapods. Further Y2H screens reveal that syndecan-2 interacts with a third distinct region of neurofibromin, and that the multiple neurofibromin regions bind competitively, rather than co-operatively, to syndecan. We combine these results to propose a model for the ternary syndecan-neurofibromin-CASK complex.     

Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos

TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development.     

Cell polarization: From epithelial cells to odontoblasts

Cell polarity identifies the asymmetry of a cell. Various types of cells, including odontoblasts and epithelial cells, polarize to fulfil their destined functions. Odontoblast polarization is a prerequisite and fundamental step for tooth development and tubular dentin formation. Current knowledge of odontoblast polarization, however, is very limited, which greatly impedes the development of novel approaches for regenerative endodontics. Compared to odontoblasts, epithelial cell polarization has been extensively studied over the last several decades. The knowledge obtained from epithelia polarization has been found applicable to other cell types, which is particularly useful considering the remarkable similarities of the morphological and compositional features between polarized odontoblasts and epithelia. In this review, we first discuss the characteristics, the key regulatory factors, and the process of epithelial polarity. Next, we compare the known facts of odontoblast polarization with epithelial cells. Lastly, we clarify knowledge gaps in odontoblast polarization and propose the directions for future research to fill the gaps, leading to the advancement of regenerative endodontics.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Mitigation of radiation injury by selective stimulation of the LPA2 receptor

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA₂ receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA₂ receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24 h after radiation exposure. Our findings suggest that by specifically activating LPA₂ receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.