Mass Spectrometric Analysis of Glycine Receptor-associated Gephyrin Splice Variants

Ingo Paarmann,Bertram Schmitt,Björn Meyer,Michael Karas,Heinrich Betz

doi:10.1074/jbc.m607764200

Abstract

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRbeta 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 GlyRbeta 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 GlyRbeta loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.

Highlights

Glycine and ␥-aminobutyrate (GABA)2 are the major inhibitory neurotransmitters in the mammalian central nervous system
Since one of the cassettes, C5؅, has recently been reported to exclude glycine receptor (GlyR) from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR
Gephyrin able to interact with the GlyR was isolated from detergent extracts of spinal cord, brain, and liver by a GST pull-down approach with the gephyrin binding motif of GlyR␤ [16]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—pGEX-4T-1 and pGEX-5X-1 were obtained from Amersham Biosciences, and GlyR␤-(378 – 426)-pGEX-5X-1 [16] and gephyrin-P1-pRSET [12] have been described previously. Sample Preparation for Mass Spectrometry—To the samples, reducing SDS-PAGE loading buffer was added, followed by incubation for 5 min at 48 °C for the GlyR-associated gephyrin or at 95 °C for gephyrin purified via GST pull-down. The supernatant was collected, followed by extraction of the gel pieces for 30 min with 70% (v/v) acetonitrile, 5% (v/v) formic acid. The sample (0.4 ␮l) was mixed with 0.4 ␮l of matrix (5 mg/ml ␣-cyano-4-hydroxycinnamic acid; Bruker Daltonik, Bremen, Germany) in 50% (v/v) acetonitrile, 0.5% (v/v) trifluoroacetic acid directly on a stainless steel target (Applied Biosystems, MDS SCIEX) and dried in ambient air. Analysis of Mass Spectrometric Data—All MS spectra were smoothed, noise-filtered, and deisotoped using the Data ExplorerTM software (Applied Biosystems, MDS SCIEX). The statistical significance of differences between gephyrin protein bands for a given cassette was calculated using Fisher’s exact test [27]

RESULTS

Cassette detection and exclusion rates

SC GlyR

DISCUSSION