title: Heat shock cognate protein 70 regulates gephyrin clustering.
abstract: Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.
authors.affiliation: Institut de Biologie de l'Ecole Normale Supérieure, Paris, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Machado
authors.forename: Philippe
authors.initials: P
authors.lastname: Rostaing
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Guigonis
authors.forename: Marianne
authors.initials: M
authors.lastname: Renner
authors.forename: Andréa
authors.initials: A
authors.lastname: Dumoulin
authors.forename: Michel
authors.initials: M
authors.lastname: Samson
authors.forename: Christian
authors.initials: C
authors.lastname: Vannier
authors.forename: Antoine
authors.initials: A
authors.lastname: Triller
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000235:genetics; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D047468:Immunoprecipitation / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D058105:Polymerization / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D040901:Proteomics / Q000379:methods; D051381:Rats; D018009:Receptors, Glycine / Q000235:genetics / Q000648:ultrastructure; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D000244:Adenosine Diphosphate; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D050883:HSC70 Heat-Shock Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018009:Receptors, Glycine; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein; C073441:gephyrin