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First published online 20 March 2007
doi: 10.1242/jcs.003905

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Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering

Barbara Lardi-Studler¹, Birthe Smolinsky², Caroline M. Petitjean¹, Franziska Koenig³, Corinne Sidler¹, Jochen C. Meier⁴, Jean-Marc Fritschy^1,*, and Guenter Schwarz^2,*

¹ Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland
² Institute of Biochemistry, University of Cologne, D-50674 Köln, Germany
³ Institute of Plant Biology, Technical University Braunschweig, D-38023 Braunschweig, Germany
⁴ Max-Delbrück-Center for Molecular Medicine, D-13125 Berlin-Buch, Germany

Author for correspondence (e-mail: fritschy{at}pharma.unizh.ch)

Accepted 21 February 2007

Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABA_A receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA_A-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA_A receptors at the expense of PSD-95 clusters – a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.

Key words: Inhibitory neurotransmission, GABAergic synapse, GABA_A receptors, Glycine receptors, Postsynaptic density, Gene transfection

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