title: 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 GABAA 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 alpha1 and beta 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 beta 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 GPHNN10Y 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.
authors.affiliation: Department of Molecular Medicine, Faculty of Medical and Health Sciences, University of Auckland, Private bag 92019, New Zealand. m.rees@auckland.ac.nz
authors.forename: Mark I
authors.initials: MI
authors.lastname: Rees
authors.forename: Kirsten
authors.initials: K
authors.lastname: Harvey
authors.forename: Hamish
authors.initials: H
authors.lastname: Ward
authors.forename: Julia H
authors.initials: JH
authors.lastname: White
authors.forename: Luc
authors.initials: L
authors.lastname: Evans
authors.forename: Ian C
authors.initials: IC
authors.lastname: Duguid
authors.forename: Cynthia C-H
authors.initials: CC
authors.lastname: Hsu
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Coleman
authors.forename: Jan
authors.initials: J
authors.lastname: Miller
authors.forename: Kristin
authors.initials: K
authors.lastname: Baer
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Waldvogel
authors.forename: Francis
authors.initials: F
authors.lastname: Gibbon
authors.forename: Trevor G
authors.initials: TG
authors.lastname: Smart
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Owen
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Harvey
authors.forename: Russell G
authors.initials: RG
authors.lastname: Snell
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016472:Motor Neuron Disease / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D018009:Receptors, Glycine; C073441:gephyrin