Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.
The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.
Grosskreutz
Y
Y
Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt/Main, Germany.
Hermann
A
A
Kins
S
S
Fuhrmann
J C
JC
Betz
H
H
Kneussel
M
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011963:Receptors, GABA-A / Q000378:metabolism; D018009:Receptors, Glycine / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors
C577410:ARHGEF9 protein, human; D002352:Carrier Proteins; C114561:GLRB protein, human; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; D064067:Rho Guanine Nucleotide Exchange Factors; C073441:gephyrin