title: Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses.
abstract: Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA(A) receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2(SH3+) and CB2(SH3-), and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2(SH3+) and CB2(SH3-) into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2(SH3-), but not CB2(SH3+), can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Zurich, 8057 Zurich, Switzerland. tyagarajan@pharma.uzh.ch
authors.forename: Shiva K
authors.initials: SK
authors.lastname: Tyagarajan
authors.forename: Himanish
authors.initials: H
authors.lastname: Ghosh
authors.forename: Kirsten
authors.initials: K
authors.lastname: Harvey
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Fritschy
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D054351:Electrical Synapses / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D059330:GABAergic Neurons / Q000378:metabolism* / Q000648:ultrastructure; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000235:genetics; D064067:Rho Guanine Nucleotide Exchange Factors; D019076:Transgenes / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C577410:ARHGEF9 protein, human; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D020033:Protein Isoforms; D064067:Rho Guanine Nucleotide Exchange Factors; C073441:gephyrin; D020764:cdc42 GTP-Binding Protein