Variants of the receptor/channel clustering molecule gephyrin in brain: Distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain

Abstract

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). Both antibodies recognized an antigen with the expected molecular weight of 93-95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GN(N) was markedly distinct from that labeled by anti-GN(C), a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48-50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis.

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

“…2) within the constitutional 736-amino acid reading frame. Degeneracy is observed at two sites, I240L and A242R, amino acid changes that lie immediately 5Ј adjacent to the C3 linker insertion site (30). The amino acid conservation between chicken and human GPHN is reduced to 98.7%, with all but one of the nine degenerate changes occurring at the MoeA domain, and a reversal of the rodent I240L and A242R changes is observed (Fig.…”

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses

“…Cell nuclei were counterstained with DAPI. The illustrated expression patterns in ( (Kawasaki et al 1997;Paarmann et al 2006). In a reciprocal coimmunoprecipitation experiment, the rabbit anti-gephyrin antibody, but not the corresponding PIS, coprecipitated the 75 kDa GRIP1c4-7 (Fig.…”

“…The mouse mAb to gephyrin (clone mAb 7a) used in immunocytochemistry was from Cedarlane (Accurate Chemical & Scientific Corporation, Westbury, NY, USA). The rabbit antigephyrin C-terminus antibody used in immunoblots was prepared as described elsewhere (Kawasaki et al 1997). The mouse mAb to the hemagglutinin (HA) epitope was from Covance (Princeton, NJ, USA).…”

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

“…The specificity of the guinea pig anti-rat ␥2 antibody has been determined previously (21)(22)(23)(24)(25)(26)(27)(28). The Rb anti-gephyrin antiserum used in immunoblotting was a gift from Dr. Ben Bahr (University of Connecticut) (29), and the mouse monoclonal antibody to gephyrin (clone 7a) used in rat brain immunocytochemistry was from Synaptic Systems (Gottingen, Germany). The sheep anti-GAD antiserum was a gift from Dr. Kopin (National Institutes of Health).…”