PNUTS forms a trimeric protein complex with GABA(C) receptors and protein phosphatase 1.
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.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.Frontiers in Molecular Neuroscience 01/2012; 5:52.
- [Show abstract] [Hide abstract]
ABSTRACT: 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. · 3.97 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Male germ cells with aberrant DNA damage are the weighted factor contributing to male infertility. Mounting evidence shows that DNA damage in male germ cells impairs spermatogenesis and lowers fecundity. MicroRNAs (miRNAs) regulating expression of multiple genes play a significant role in spermatogenesis. Our previous results have shown that microRNA-383 (miR-383) is one of the notable down-regulated microRNAs in the testes of sterile males with maturation arrest (MA) and is located predominantly in spermatogonia and primary spermatocytes. However, the role that miR-383 plays in DNA damage during spermatogenesis remains unknown. In this study, we found that miR-383 inhibited the focal formation and abundance of γH2AX, which is the major marker of sites of DNA damage, with or without ultraviolet irradiation and cisplatin in testicular embryonal carcinoma (NT-2) cells. In addition, NT-2 cells were remarkably sensitized to DNA damage reagent (cisplatin) by forcing expression of miR-383 and silencing expression of protein phosphatase 1, regulatory subunit 10 (PNUTS). By constructing Renilla luciferase reporters and co-transfecting miR-383 and reporters in NT-2 cells, we identified that PNUTS was a valid target of miR-383. Further results demonstrated that the repression of the phosphorylated form of H2AX by miR-383 was due to independent depletion of PNUTS and cell cycle arrest. In conclusion, we found a novel function of miR-383 in the DNA damage pathway. miR-383 impairs the phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest independently, as well as sensitizing NT-2 cells to cisplatin.Cellular signalling 01/2014; · 4.09 Impact Factor