Splice-specific Functions of Gephyrin in Molybdenum Cofactor Biosynthesis

Birthe Smolinsky,Sabrina A Eichler,Sabine Buchmeier,Jochen C Meier,Guenter Schwarz

doi:10.1074/jbc.m800985200

Abstract

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.

Highlights

Ized to the large cytoplasmic loop of the glycine receptor (GlyR) ␤-subunit [3]
Alternative splicing and tissue-specific expression of gephyrin might contribute to the functional diversity including receptor binding [31], clustering [36], phosphorylation [37], binding to other proteins involved in gephyrin-mediated synapse formation, and/or controlling the gephyrin metabolic function in molybdenum cofactor (Moco) synthesis
The latter was investigated in this study using eight splice variants that have been identified in rat spinal cord with modifications in both domains (G and E) essential for Moco synthesis as well as the C domain, which is believed to be crucial for brain-specific functions because it contains binding sites for a number of interacting proteins [8]

Summary

Introduction

Ized to the large cytoplasmic loop of the GlyR ␤-subunit [3]. Recently, GABAA receptor binding to gephyrin has been reported, which involved the ␣2-subunit [4]. Encoding GephG with the central domain (Geph-GC) and subdomains 3 and 4 of the GephE (Geph-GCE3/4), GephE (Geph-E) and a chimeric construct containing a second E domain fused to the C terminus of gephyrin (Geph-EE) [5] were transfected into L929 cells, and Moco and MPT content were determined by the nit-1 reconstitution assay [24].

Results

Conclusion