title: Proteomic analysis of glycine receptor β subunit (GlyRβ)-interacting proteins: evidence for syndapin I regulating synaptic glycine receptors.
abstract: Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.
authors: Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60438 Frankfurt/Main.
authors: Isabel
authors: I
authors: Del Pino
authors: Institute for Biochemistry I, Jena University Hospital, Friedrich Schiller University Jena, D-07743 Jena.
authors: Dennis
authors: D
authors: Koch
authors: Department for Theoretical and Computational Biophysics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, and.
authors: Rudolf
authors: R
authors: Schemm
authors: Institute for Biochemistry I, Jena University Hospital, Friedrich Schiller University Jena, D-07743 Jena.
authors: Britta
authors: B
authors: Qualmann
authors: Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60438 Frankfurt/Main,; Max-Planck Institute for Medical Research, 69120 Heidelberg, Germany. Electronic address: heinrich.betz@mpimf-heidelberg.mpg.de.
authors: Heinrich
authors: H
authors: Betz
authors: Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60438 Frankfurt/Main,. Electronic address: Ingo.Paarmann@gmx.net.
authors: Ingo
authors: I
authors: Paarmann
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008817:Mice, Mutant Strains; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D040901:Proteomics; D051381:Rats; D017208:Rats, Wistar; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*; D013569:Synapses / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C491948:Glrb protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; C114726:Pacsin1 protein, mouse; C117738:Pacsin1 protein, rat; D010750:Phosphoproteins; D018009:Receptors, Glycine
keywords: Cys Loop Receptors; Glycine Receptors; Inhibitory Synapse; Knockdown; Mass Spectrometry (MS); Membrane Protein Trafficking; Molecular Modeling; Protein-Protein Interactions; Spinal Cord; Syndapin