Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes.
The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.
Fuhrmann
Jens C
JC
Max-Planck-Institute for Brain Research, Department of Neurochemistry, D-60528 Frankfurt/Main, Germany.
Kins
Stefan
S
Rostaing
Philippe
P
El Far
Oussama
O
Kirsch
Joachim
J
Sheng
Morgan
M
Triller
Antoine
A
Betz
Heinrich
H
Kneussel
Matthias
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D029721:Drosophila Proteins; D004398:Dyneins / Q000737:chemistry; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry; D024701:Myosin Type V / Q000737:chemistry; D009474:Neurons / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D029721:Drosophila Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; C513081:Myo5a protein, rat; C073441:gephyrin; C077511:MYO5A protein, human; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D004398:Dyneins