Isoform Heterogeneity of the Human Gephyrin Gene (GPHN), Binding Domains to the Glycine Receptor, and Mutation Analysis in Hyperekplexia

Abstract

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABA A receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR ␣ 1 and ␤ subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR ␤ subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHN N10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.Neuronal postsynaptic membranes contain a wide variety of ion channels and receptor proteins that contribute to the balance of excitatory and inhibitory neurotransmission in the human central nervous system. Mature postnatal neuroinhibition is mediated by postsynaptic glycinergic and GABA A 1 receptor systems that are structurally related to the nicotinic receptors with a typical pentameric structure constructed from heterogeneous subunit constituents containing four transmembrane (TM) domains and a large intracellular loop between TM3 and TM4 (1, 2). These subunits combine to form fast response, ligand-gated chloride channels that modify excitatory neurotransmission in the human brainstem and spinal cord (3-5). Inhibitory glycine receptors (GlyRs) are composed of three ligand-binding ␣ 1 subunits (GlyR ␣ 1 ) and two structural ␤ subunits (GlyR ␤) that are anchored and clustered on the postsynaptic membrane of inhibitory neurons by the interaction of a motif within the TM3-TM4 loop of the GlyR␤ subunit with anchoring protein, gephyrin (6 -8). In contrast to GlyRs, there is a lack of evidence for a direct physical interaction between gephyrin and GABA A receptor subunit(s) (7, 9), although immunohistochemical studies have detected co-localization of gephyrin with major subtypes of GABA A receptors at GABAergic postsynaptic...

Splice-specific Glycine Receptor Binding, Folding, and Phosphorylation of the Scaffolding Protein Gephyrin

“…This very different behavior of Geph-C3, which is highly expressed in Glia (28), liver, and kidney (27,31), could link Geph-C3 to the non-neuronal metabolic function of gephyrin, the biosynthesis of the molybdenum cofactor, as seen already in previous studies (28). Finally, the observed membrane enrichment of Geph and Geph-C4, with Geph-C3 more homogeneously distributed in the cytosol, suggests that the high oligomeric forms prefer spatial proximity to the plasma membrane.…”

“…Beside phosphorylation, alternative splicing of the gephyrin gene is also believed to contribute to the functional complexity of gephyrin. To date more than 10 different variants have been reported (13,(25)(26)(27) affecting each of the three domains. The majority of modifications reside in the C domain, pointing toward its crucial contribution in controlling gephyrin folding and clustering.…”

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

“…Currently, the major known genetic cause of hyperekplexia is missense, nonsense, frameshift, or splice site mutations in the glycine receptor (GlyR) 4 ␣1 gene (GLRA1) (8 -10), although large GLRA1 deletions are also common in patients of Kurdish descent (11,12). Rare mutations in the genes encoding the GlyR ␤ subunit (GLRB) (13,14) and the synaptic clustering proteins gephyrin (GPHN) (15) and collybistin (ARHGEF9) (16) have also been linked to hyperekplexia.…”

Glycine Receptor: Molecular Organization and Pathology

“…Нині доведено, що гіпер-плексія виникає внаслідок мутацій не лише GLRA1 (хоча ураження саме цього гена найчастіше стає причиною хвороби), але і GLRВ [66,67] та SLC6A5, що кодує пресинап-тичний транспортер гліцину другого типу [68,69]. Окрім того, було виявлено, що мутації геферину та колібістину, білків, що забезпе-чують кластеризацію гліцинових рецепторів у постсинапсі, також можуть бути причиною гіперплексії [70,71].…”