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J. Biol. Chem., Vol. 281, Issue 46, 34918-34925, November 17, 2006

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Mass Spectrometric Analysis of Glycine Receptor-associated Gephyrin Splice Variants^*

Ingo Paarmann, Bertram Schmitt, Björn Meyer, Michael Karas, and Heinrich Betz¹

From the Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt, Germany and Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, D-60438 Frankfurt am Main, Germany

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABA_A receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyR subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5–40 amino acids, are subject to alternative splicing (C1–C7, C4'–C6'). Since one of the cassettes, C5', has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyR loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6', already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5' was coisolated mainly from liver. Notably C5'-containing gephyrin bound to the GlyR loop, inconsistent with its proposed selectivity for GABA_A receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.

Received for publication, August 14, 2006 , and in revised form, September 20, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft (SFB628) and Fonds der Chemischen Industrie. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and supplemental Figs. 1–3.

¹ To whom correspondence should be addressed: Dept. of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt, Germany. Tel.: 49-69-96769-260; Fax: 49-69-96769-441; E-mail: neurochemie{at}mpih-frankfurt.mpg.de.

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