PNUTS forms a trimeric protein complex with GABAC receptors and protein phosphatase 1

Institut für Biochemie (Emil-Fischer-Zentrum), Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Molecular and Cellular Neuroscience (Impact Factor: 3.84). 05/2008; 37(4):808-19. DOI: 10.1016/j.mcn.2008.01.004
Source: PubMed


Phosphorylation and dephosphorylation of neurotransmitter receptors represent an important mechanism to regulate synaptic signal transduction. Here, we identified PNUTS, a targeting subunit of protein phosphatase 1 (PP1) as a new binding partner of GABA(C) receptors. In the mammalian retina, PNUTS is co-expressed with GABA(C) receptors and PP1 in bipolar cells. PNUTS and PP1 were detected in membrane protein preparations of the retina and precipitate with GABA(C) receptor specific antibodies. Furthermore, PNUTS shuttles from the nucleus to the membrane in cells co-expressing GABA(C) receptors. We show simultaneous binding of PP1 and GABA(C) receptors to different domains of PNUTS, demonstrating that PNUTS cross-links PP1 and GABA(C) receptors. Finally, modeling studies showed that the PP1 docking motif of PNUTS fits into the binding pocket on the enzyme surface, despite a C-terminal adjacent proline. We suggest that PNUTS targets PP1 to synaptic sites, acting as a temporary bridge between the phosphatase and GABA(C) receptors.

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    • "Immunocytochemistry HEK-293 cells were grown on poly-(L-lysine)-coated glass coverslips to approximately 50% confluency, transfected for 18–24 h with 1 lg DNA using lipofection, washed, fixed and blocked for antibody stainings as described (Rose et al. 2008). Spartin was detected by the fused GFP-tag, ZIP1/3 by fused T7-tags using T7- specific antibodies (1 : 1000; Novagen) and PKC-f by PKC-f specific antibodies (1 : 5000; Sigma-Aldrich). "
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    ABSTRACT: J. Neurochem. (2011) 118, 737–748. Protein kinase C-ζ interacting proteins (ZIP1-3) recruit the enzymatic activity of the atypical protein kinase C isoforms PKC-λ/ι or PKC-ζ to target proteins. In this study, we searched for binding partners of ZIP3 in the CNS and identified spartin, a multifunctional protein that is mutated in spastic paraplegia type 20. In transfected cells, spartin was present on the surface of lipid droplets (LD), whereas ZIP proteins appeared in intracellular speckles. In the presence of spartin, ZIP1 and ZIP3 were translocated to spartin-positive LD. This translocation was mediated by amino acids 196–393 of spartin that interacted with an N-terminal region of ZIP proteins. Furthermore, ZIP proteins interacted simultaneously with spartin and PKC-ζ, resulting in an enrichment of PKC-ζ on spartin/ZIP-labelled LD. Without spartin, neither ZIP proteins nor PKC-ζ were detected on LD. Interestingly, the presence of the spartin/ZIP/PKC-ζ complex increased LD size. This effect was most pronounced upon incorporation of the ZIP3 isoform into the trimer. Finally, we co-localized spartin, ZIP proteins and PKC-ζ in axon terminals of neurons in the mammalian retina. In summary, we describe spartin as new binding partner of the ZIP/PKC-ζ dimer that recruits PKC-ζ to LD and show that the expressed ZIP isoform regulates LD size.
    Journal of Neurochemistry 06/2011; 118(5):737-48. DOI:10.1111/j.1471-4159.2011.07367.x · 4.28 Impact Factor
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    • "Recently, it has also been shown that PNUTS co-localizes with PP1γ1 and GABA C receptors in the axon termini of rod bipolar cells where it forms a trimeric protein complex with these proteins, suggesting that PNUTS regulates PP1 at retinal synapses (Rose et al., 2008). However, despite these insights, to date the precise physiological role of PNUTS remains elusive. "
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    ABSTRACT: Protein phosphatase-1 (PP1) nuclear targeting subunit (PNUTS), also called PP1R10, p99, or CAT 53 was originally isolated as a mammalian nuclear PP1-binding protein. In this study, we performed yeast two-hybrid screens to identify PNUTS-interacting proteins. Here, we report that LCP1 (epidermal Langerhans cell protein 1), a novel member of the HMG-box protein family, binds tightly to PNUTS. Co-immunoprecipitation of deletion constructs revealed that the C-terminus of LCP1 is sufficient for the interaction with an N-terminal region of PNUTS that is distinct from its PP1-binding domain. Furthermore, immunofluorescence studies showed that a subpopulation of LCP1 co-localizes with PNUTS in nuclear speckles. Importantly, we found that the N-terminus of LCP1 has a strong trans-activation activity in a GAL4-based heterologous transcription assay. The transcriptional activity of LCP1 is markedly suppressed by its interaction with PNUTS, in a PP1-independent manner. These findings suggest that the coordinated spatial and temporal regulation of LCP1 and PNUTS may be a novel mechanism to control the expression of genes that are critical for certain physiological and pathological processes.
    Experimental and Molecular Medicine 04/2009; 41(3). DOI:10.3858/emm.2009.41.3.022 · 3.45 Impact Factor
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    • "Cell surface proteins were biotinylated in COS-7 cells that were grown in 10 cm dishes and transfected with a mixture of 5 lg mGluR8a/b and 5 lg 4.1B expression plasmids as above. For individual expression of mGluR8a/b, the plasmid amount of 4.1B was substituted by an unrelated expression plasmid encoding for PNUTS (Rose et al. 2008) in order to achieve comparable conditions in the transfection/expression procedure. Biotinylation, cell lysis, and purification of biotinylated proteins were performed "
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    ABSTRACT: The function of the CNS depends on the correct regulation of neurotransmitter receptors by interacting proteins. Here, we screened a retinal cDNA library for proteins interacting with the intracellular C-terminus of the metabotropic glutamate receptor isoform 8a (mGluR8a). The band 4.1B protein binds to the C-termini of mGluR8a and mGluR8b, co-localizes with these glutamate receptors in transfected mammalian cells, facilitates their cell surface expression and inhibits the mGluR8 mediated reduction of intracellular cAMP concentrations. In contrast, no interaction with 4.1B was observed for other mGluRs tested. Amino acids encoded by exons 19 and 20 of 4.1B and a stretch of four basic amino acids present in the mGluR8 C-termini mediate the protein interaction. Besides binding to 4.1B, mGluR8 isoforms interact with 4.1G, 4.1N, and 4.1R. Because band 4.1 transcripts undergo extensive alternative splicing, we analyzed the splicing pattern of interacting regions and detected a 4.1B isoform expressed specifically in the retina. Within this tissue, mGluR8 and 4.1B, 4.1G, 4.1N, and 4.1R show a comparable distribution, being expressed in both synaptic layers and in somata of the ganglion cell layer. In summary, our studies identified band 4.1 proteins as new players for the mGluR8 mediated signal transduction.
    Journal of Neurochemistry 04/2008; 105(6):2375-87. DOI:10.1111/j.1471-4159.2008.05331.x · 4.28 Impact Factor
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