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J. Biol. Chem., Vol. 283, Issue 25, 17370-17379, June 20, 2008

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Splice-specific Functions of Gephyrin in Molybdenum Cofactor Biosynthesis^*

Birthe Smolinsky, Sabrina A. Eichler, Sabine Buchmeier^¶, Jochen C. Meier¹, and Guenter Schwarz¹²

From the Institute of Biochemistry and Center for Molecular Medicine, University of Cologne, 50674 Cologne, Germany, the Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin-Buch, Germany, and the ^¶Institute of Zoology, Technical University Braunschweig, 38106 Braunschweig, Germany

Gephyrin is a multifunctional protein involved in the clustering of inhibitory neuroreceptors. In addition, gephyrin catalyzes the last step in molybdenum cofactor (Moco) biosynthesis essential for the activities of Mo-dependent enzymes such as sulfite oxidase and xanthine oxidoreductase. Functional complexity and diversity of gephyrin is believed to be regulated by alternative splicing in a tissue-specific manner. Here, we investigated eight gephyrin variants with combinations of seven alternatively spliced exons located in the N-terminal G domain, the central domain, and the C-terminal E domain. Their activity in Moco synthesis was analyzed in vivo by reconstitution of gephyrin-deficient L929 cells, which were found to be defective in the G domain of gephyrin. Individual domain functions were assayed in addition and confirmed that variants containing either an additional C5 cassette or missing the C6 cassette are inactive in Moco synthesis. In contrast, different alterations within the central domain retained the Moco synthetic activity of gephyrin. The recombinant gephyrin G domain containing the C5 cassette forms dimers in solution, binds molybdopterin, but is unable to catalyze molybdopterin (MPT) adenylylation. Determination of Moco and MPT content in different tissues showed that besides liver and kidney, brain was capable of synthesizing Moco most efficiently. Subsequent analysis of cultured neurons and glia cells demonstrated glial Moco synthesis due to the expression of gephyrins containing the cassettes C2 and C6 with and without C3.1

Received for publication, February 6, 2008 , and in revised form, April 1, 2008.

^* This work was supported in part by the DFG (Schw759/2-2 and FOR471 (to G. S.) and ME2075/3-1 (to J. C. M.)), Helmholtz Association (VH-NG-246, to J. C. M.), and the 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 Figs. S1 and S2 and Table S1.

¹ Both authors contributed equally to this report.

² To whom correspondence should be addressed: Institut für Biochemie, Universität zu Köln, Otto-Fischer-Str. 12–14, 50674 Köln, Germany. Tel.: 49-221-470-6432; Fax: 49-221-470-6731; E-mail: gschwarz{at}uni-koeln.de.

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