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ABSTRACT: The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and SUMOylation (SUMO is small ubiquitin-related modifier). In the present study, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the SUMOylation and NEDDylation machinery. The SUMO E3 ligases Pias1 [Pias is protein inhibitor of activated STAT (signal transducer and activator of transcription)] and Pias3L interacted strongly with mGluR8b, and were co-localized with the E2-conjugating Ubc9, SUMO1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. SUMO1 conjugation of Lys882, present in a bona fide consensus sequence for SUMOylation (VKSE) in the mGluR8b C-terminus, was enhanced by addition of Pias1, consistent with an interaction between both proteins. Mutation of Lys882 to arginine reduced, but did not abolish, mGluR8b SUMOylation. Co-mutating a second lysine residue (Lys903) located in the mGluR8b isoform-specific C-terminus largely prevented SUMO1 conjugation by Ubc9. Modelling studies suggested that Lys903 contacts Ubc9 and thus is part of the non-canonical SUMOylation site VKSG. In summary, the results of the present study show in vivo SUMOylation of the complete mGluR8b and co-localize proteins of the SUMOylation machinery in the retina.
Biochemical Journal 02/2011; 435(2):365-71. · 4.90 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Neuroscience 05/2008; 37(4):808-19. · 3.66 Impact Factor
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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. · 4.06 Impact Factor
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ABSTRACT: 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 GABAC receptors. In the mammalian retina, PNUTS is co-expressed with GABAC receptors and PP1 in bipolar cells. PNUTS and PP1 were detected in membrane protein preparations of the retina and precipitate with GABAC receptor specific antibodies. Furthermore, PNUTS shuttles from the nucleus to the membrane in cells co-expressing GABAC receptors. We show simultaneous binding of PP1 and GABAC receptors to different domains of PNUTS, demonstrating that PNUTS cross-links PP1 and GABAC 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 GABAC receptors.
Molecular and Cellular Neuroscience.
