PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering

Eur J Neurosci. 2010 Apr;31(7):1173-84. doi: 10.1111/j.1460-9568.2010.07149.x. Epub 2010 Mar 19.

Abstract

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor (GEF) that is essential for the synaptic clustering of gephyrin and GABAA receptors in selected regions of the mammalian central nervous system. It has been previously proposed that Cb regulates gephyrin clustering by activating Cdc42, and thus acts as a signal transducer in a membrane activation process which labels postsynaptic membrane domains for inhibitory synapse formation. Here, we dissected the functional roles of the Dbl-homology (DH) and pleckstrin homology (PH) domains of the constitutively active splice variant Cb II by substituting conserved amino acid residues that are required for GEF activity towards Cdc42 and phosphoinositide binding, respectively. A Cb II mutant lacking any detectable GEF activity towards Cdc42 was still fully active in inducing gephyrin scaffold formation, both in transfected NIH-3T3 cells and in cultured hippocampal neurons. Furthermore, mice with a forebrain-specific inactivation of the Cdc42 gene displayed normal densities of gephyrin and GABA(A) receptor clusters in the hippocampus. In contrast, substitution of Cb II PH-domain residues essential for phosphoinositide binding abolished gephyrin recruitment to synaptic sites. Our results provide evidence that the formation of gephyrin scaffolds at inhibitory synapses requires an intact Cb II PH-domain but is Cdc42-independent.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / metabolism*
  • Cells, Cultured
  • Embryo, Mammalian
  • Green Fluorescent Proteins / genetics
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Hippocampus / cytology
  • Homeodomain Proteins / genetics
  • Humans
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Models, Molecular
  • Mutation / genetics
  • Neurons / cytology
  • Protein Structure, Tertiary / physiology
  • Pseudopodia / physiology
  • Receptors, GABA-A / genetics
  • Receptors, GABA-A / metabolism
  • Rho Guanine Nucleotide Exchange Factors
  • Synapses / metabolism*
  • Transcription Factors / genetics
  • Transfection / methods
  • Vesicular Inhibitory Amino Acid Transport Proteins / metabolism
  • cdc42 GTP-Binding Protein / deficiency
  • cdc42 GTP-Binding Protein / metabolism*

Substances

  • Arhgef9 protein, mouse
  • Carrier Proteins
  • Guanine Nucleotide Exchange Factors
  • Homeodomain Proteins
  • Membrane Proteins
  • Receptors, GABA-A
  • Rho Guanine Nucleotide Exchange Factors
  • Transcription Factors
  • Vesicular Inhibitory Amino Acid Transport Proteins
  • Viaat protein, mouse
  • empty spiracles homeobox proteins
  • gephyrin
  • Green Fluorescent Proteins
  • cdc42 GTP-Binding Protein