Proteomic Analysis of Glyrβ Interacting Proteins: Evidence for Syndapin I Regulating Synaptic Glycine Receptors

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brain stem. 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 pull-down experiments with rat brain extracts using a glutathione Stransferase 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 co-immunoprecipitates 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 SH3 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 knockeddown 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. Postsynaptic inhibition by glycine is http://www.jbc.org/cgi/doi/10.1074/jbc.M113.504860 The latest version is at JBC Papers in Press. Published on February 7, 2014 as Manuscript M113.504860 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 3, 2017 hp://w w w .jb.org/ D ow nladed from