Disruption of the PDZ-domain binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis and reward motivation

The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine adaptations and changes in dopamine-related behaviors different from those seen in other genetic DAT mouse models. Dopamine levels in striatum are reduced to ~45% of wild-type (WT), accompanied by elevated dopamine turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked dopamine release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced dopamine D2 receptor levels while immunohistochemistry and autoradiography show unchanged dopamine D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alters DA clearance dynamics.

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MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

10.1101/2020.07.08.194100 ◽

2020 ◽

Author(s):

Ryan Ard ◽

Jean-Christian Maillet ◽

Elias Daher ◽

Michael Phan ◽

Radoslav Zinoviev ◽

...

Keyword(s):

Catalytic Activity ◽

Molecular Mechanisms ◽

Rho Gtpase ◽

Pdz Domain ◽

Diacylglycerol Kinase ◽

Binding Motif ◽

Membrane Blebbing ◽

Rhoa Activity ◽

C Terminus ◽

Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

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Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

The Journal of Cell Biology ◽

10.1083/jcb.200407072 ◽

2005 ◽

Vol 168 (2) ◽

pp. 303-313 ◽

Cited By ~ 121

Author(s):

Doris Meder ◽

Anna Shevchenko ◽

Kai Simons ◽

Joachim Füllekrug

Keyword(s):

Free Surface ◽

Apical Membrane ◽

Mdck Cells ◽

Single Cells ◽

Basolateral Membrane ◽

Pdz Domain ◽

Binding Motif ◽

Membrane Domains ◽

Regulatory Factor ◽

Pdz Protein

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

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Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins

Journal of Virology ◽

10.1128/jvi.74.20.9680-9693.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9680-9693 ◽

Cited By ~ 179

Author(s):

Siu Sylvia Lee ◽

Britt Glaunsinger ◽

Fiamma Mantovani ◽

Lawrence Banks ◽

Ronald T. Javier

Keyword(s):

High Risk ◽

Cellular Proliferation ◽

Pdz Domain ◽

Viral Proteins ◽

Cellular Protein ◽

Human Papillomaviruses ◽

Binding Motif ◽

Cellular Factor ◽

Domain Protein ◽

Hpv 18

ABSTRACT A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

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Slitrk2 controls excitatory synapse development via PDZ-mediated protein interactions

Scientific Reports ◽

10.1038/s41598-019-53519-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Kyung Ah Han ◽

Jinhu Kim ◽

Hyeonho Kim ◽

Dongwook Kim ◽

Dongseok Lim ◽

...

Keyword(s):

Protein Interactions ◽

Intracellular Signaling ◽

Hippocampal Neurons ◽

Pdz Domain ◽

Protein Tyrosine Phosphatases ◽

Excitatory Synapse ◽

Receptor Protein ◽

Binding Motif ◽

Synapse Development

AbstractMembers of the Slitrk (Slit- and Trk-like protein) family of synaptic cell-adhesion molecules control excitatory and inhibitory synapse development through isoform-dependent extracellular interactions with leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs). However, how Slitrks participate in activation of intracellular signaling pathways in postsynaptic neurons remains largely unknown. Here we report that, among the six members of the Slitrk family, only Slitrk2 directly interacts with the PDZ domain-containing excitatory scaffolds, PSD-95 and Shank3. The interaction of Slitrk2 with PDZ proteins is mediated by the cytoplasmic COOH-terminal PDZ domain-binding motif (Ile-Ser-Glu-Leu), which is not found in other Slitrks. Mapping analyses further revealed that a single PDZ domain of Shank3 is responsible for binding to Slitrk2. Slitrk2 forms in vivo complexes with membrane-associated guanylate kinase (MAGUK) family proteins in addition to PSD-95 and Shank3. Intriguingly, in addition to its role in synaptic targeting in cultured hippocampal neurons, the PDZ domain-binding motif of Slitrk2 is required for Slitrk2 promotion of excitatory synapse formation, transmission, and spine development in the CA1 hippocampal region. Collectively, our data suggest a new molecular mechanism for conferring isoform-specific regulatory actions of the Slitrk family in orchestrating intracellular signal transduction pathways in postsynaptic neurons.

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Regulation of the endosomal SNX27-retromer by OTULIN

Nature Communications ◽

10.1038/s41467-019-12309-z ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Aurelia Stangl ◽

Paul R. Elliott ◽

Adan Pinto-Fernandez ◽

Sarah Bonham ◽

Luke Harrison ◽

...

Keyword(s):

Cell Surface ◽

Membrane Trafficking ◽

Pdz Domain ◽

Binding Motif ◽

Ubiquitin Chain ◽

Sorting Nexin ◽

Catalytic Function ◽

Retromer Complex ◽

Mass Spectrometric Identification ◽

Affinity Interaction

Abstract OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

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Human Papillomavirus Type 16 E6 Activates NF-κB, Induces cIAP-2 Expression, and Protects against Apoptosis in a PDZ Binding Motif-Dependent Manner

Journal of Virology ◽

10.1128/jvi.01942-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5301-5307 ◽

Cited By ~ 96

Author(s):

Michael A. James ◽

John H. Lee ◽

Aloysius J. Klingelhutz

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Transcriptional Activation ◽

Pdz Domain ◽

Airway Epithelial Cells ◽

Binding Motif ◽

Dependent Manner ◽

Airway Epithelial ◽

Transcript Accumulation ◽

Human Telomerase Reverse Transcriptase

ABSTRACT Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-κB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-κB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-κB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-κB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-κB activation and protection from apoptosis in airway epithelial cells.

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Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca2+/calmodulin-dependent protein kinase II

European Journal of Neuroscience ◽

10.1111/j.1460-9568.2006.04846.x ◽

2006 ◽

Vol 23 (11) ◽

pp. 2864-2876 ◽

Cited By ~ 35

Author(s):

Qing-Bao Tian ◽

Tatsuo Suzuki ◽

Takashi Yamauchi ◽

Hiroyuki Sakagami ◽

Yoshiyuki Yoshimura ◽

...

Keyword(s):

Protein Kinase ◽

Pdz Domain ◽

Ldl Receptor ◽

Scaffold Proteins ◽

Binding Motif ◽

Related Protein ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein

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An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking

Biochemical Journal ◽

10.1042/bj20081422 ◽

2009 ◽

Vol 418 (2) ◽

pp. 345-367 ◽

Cited By ~ 29

Author(s):

Heather L. Wieman ◽

Sarah R. Horn ◽

Sarah R. Jacobs ◽

Brian J. Altman ◽

Sally Kornbluth ◽

...

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Glucose Uptake ◽

Pdz Domain ◽

Binding Motif ◽

Lysosomal Degradation ◽

Interacting Protein ◽

Surface Localization ◽

Cell Surface Localization ◽

Growth Factor Regulation

Cell surface localization of the Glut (glucose transporter), Glut1, is a cytokine-controlled process essential to support the metabolism and survival of haemopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. In the present study, we show that a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ-domain protein, GIPC (Gα-interacting protein-interacting protein, C-terminus), bound to Glut1 in part via the Glut1 C-terminal PDZ-binding motif, and we found that GIPC deficiency decreased Glut1 surface levels and glucose uptake. Unlike the Glut1 degradation observed on mutation of the Glut1 PDZ-binding domain, however, GIPC deficiency resulted in accumulation of intracellular Glut1 in a pool distinct from the recycling pathway of the TfR (transferrin receptor). Blockade of Glut1 lysosomal targeting after growth factor withdrawal also led to intracellular accumulation of Glut1, a portion of which could be rapidly restored to the cell surface after growth factor stimulation. These results indicate that the C-terminal PDZ-binding motif of Glut1 plays a key role in growth factor regulation of glucose uptake by both allowing GIPC to promote Glut1 trafficking to the cell surface and protecting intracellular Glut1 from lysosomal degradation after growth factor withdrawal, thus allowing the potential for a rapid return of intracellular Glut1 to the cell surface on restimulation.

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Kalirin, a Key Player in Synapse Formation, Is Implicated in Human Diseases

Neural Plasticity ◽

10.1155/2012/728161 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 35

Author(s):

Prashant Mandela ◽

Xin-Ming Ma

Keyword(s):

Learning And Memory ◽

Cortical Neurons ◽

Molecular Mechanisms ◽

Synapse Formation ◽

Pdz Domain ◽

Long Term Potentiation ◽

Human Diseases ◽

Binding Motif ◽

Spine Density ◽

Morphological Alterations

Synapse formation is considered to be crucial for learning and memory. Understanding the underlying molecular mechanisms of synapse formation is a key to understanding learning and memory. Kalirin-7, a major isoform of Kalirin in adult rodent brain, is an essential component of mature excitatory synapses. Kalirin-7 interacts with multiple PDZ-domain-containing proteins including PSD95, spinophilin, and GluR1 through its PDZ-binding motif. In cultured hippocampal/cortical neurons, overexpression of Kalirin-7 increases spine density and spine size whereas reduction of endogenous Kalirin-7 expression decreases synapse number, and spine density. In Kalirin-7 knockout mice, spine length, synapse number, and postsynaptic density (PSD) size are decreased in hippocampal CA1 pyramidal neurons; these morphological alterations are accompanied by a deficiency in long-term potentiation (LTP) and a decreased spontaneous excitatory postsynaptic current (sEPSC) frequency. Human Kalirin-7, also known as Duo or Huntingtin-associated protein-interacting protein (HAPIP), is equivalent to rat Kalirin-7. Recent studies show that Kalirin is relevant to many human diseases such as Huntington’s Disease, Alzheimer’s Disease, ischemic stroke, schizophrenia, depression, and cocaine addiction. This paper summarizes our recent understanding of Kalirin function.

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