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J. Biol. Chem., Vol. 279, Issue 18, 18733-18741, April 30, 2004

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Biochemical Characterization of the High Affinity Binding between the Glycine Receptor and Gephyrin^*

Nils Schrader¶, Eun Young Kim, Jan Winking, Jens Paulukat||, Hermann Schindelin**, and Günter Schwarz

From the Department of Plant Biology, Technical University Braunschweig, D-38023 Braunschweig, Germany and the Department of Biochemistry, Center for Structural Biology, State University of New York, Stony Brook, New York 11794-5115

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the -subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR -sub-unit (GlyR-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496–654). By using isothermal titration calorimetry, a high affinity (K_d = 0.2–0.4 µM) and low affinity (K_d = 11–30 µM) binding site for the GlyR-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyR-loops per E domain in both cases. Binding of the GlyR-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.

Received for publication, October 13, 2003 , and in revised form, February 11, 2004.

^* This work was supported in part by Deutsche Forschungsgesellschaft Grant Schw759/2-1 (to G. S.) and National Institutes of Health Grants DK54835 and NS48605 (to H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by Deutsche Akademische Austauschdienst Fellowship D/00/05497.

^|| Present address: Pharmacenter Frankfurt, Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.

^** To whom correspondence may be addressed. Tel.: 1 631 632 1022; Fax: 1 631 632 1555; E-mail: hermann.schindelin{at}sunysb.edu. Supported by Deutsche Akademische Austauschdienst Fellowship D/00/22272. To whom correspondence may be addressed. Tel.: 49 531 391 5891; Fax: 49 531 391 8208; E-mail: g.schwarz{at}tu-bs.de.

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