Biochemical Characterization of the High Affinity Binding between the Glycine Receptor and Gephyrin

Abstract

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the ␤-subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR ␤-subunit (GlyR␤-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496 -654). By using isothermal titration calorimetry, a high affinity (K d ‫؍‬ 0.2-0.4 M) and low affinity (K d ‫؍‬ 11-30 M) binding site for the GlyR␤-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyR␤-loops per E domain in both cases. Binding of the GlyR␤-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.Efficient transmission of synaptic signals in the central nervous system is dependent on high local concentrations of neurotransmitter ion channels in the postsynaptic membrane of excitatory and inhibitory synapses. Glycine is an important inhibitory neurotransmitter, and its postsynaptic receptors are an essential component of inhibitory nerve terminals (1-3). Glycine receptors (GlyRs) 1 are pentameric anion channels generally consisting of two different subunits with an ␣ 3 ␤ 2 composition (4). Crucial for proper functioning of GlyRs is their clustering that generates a high packing density of neuroreceptors in the postsynaptic membrane. Gephyrin, a highly expressed neuronal protein, was found to co-localize with glycine (5, 6) and GABA A receptors (7,8). Gephyrin is crucial for the clustering of both types of inhibitory neuroreceptors (9) and anchors these receptors to the subsynaptic cytoskeleton. A direct binding of gephyrin to microtubules was shown (10), which is possibly mediated by a "tau" motif (11). So far no direct interaction with microfilaments has been demonstrated, but pharmacological studies indicate a function of actin filaments in determining the cluster size (12). Therefore, it is not surprising that binding of gephyrin to key regulators of microfilament dynamics such as profilin I, neuronal profilin IIa, and microfilament adaptors of the Mena/VASP family including neuronal Mena has been reported recently (13). The binding site of gephyrin on GlyR has been mapped to a stretch of 18 amino acids located within the large cytoplasmic loop connecting transmembrane helices three and four of the GlyR ␤-subunit (GlyR␤-loop) (14), but the binding site on ge...

Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin

“…2b, sequences no. [7][8][9]. This correlates with the Ala-scan showing that the Thr367Ala mutation increased affinity to GephE (Fig.…”

“…GephE (gephyrin P1 splice variant residues 318-736) as well as residues 378-425 of the large cytoplasmic loop of the GlyR b subunit (b49) were expressed in E. coli BL21 (DE3) (Stratagene) as an intein fusion proteins with a chitin-binding domain for affinity purification 9 . Cells were grown in lysogeny broth medium at 30°C and induced with 0.5-1 mM isopropyl-bthiogalactoside at a cell density A 600 of 0.5-1.…”

“…Gephyrin is composed of an N-terminal domain (GephG, residues 1-181) and a C-terminal domain (GephE, residues 318-736), which are connected by an unstructured linker (residues 182-317). Gephyrin was discovered 6 by co-purification with glycine receptors (GlyRs) and found to be responsible for anchoring and accumulating GlyRs at postsynaptic sites, which is accomplished by the simultaneous binding of gephyrin to the GlyR b subunit [7][8][9][10] and elements of the cytoskeleton 11,12 . A number of gene knockout studies have addressed the role of the gephyrin-GABA A R interaction in vivo.…”

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

“…However, the interaction of gephyrin with different GABA receptor subtypes is not fully understood (Kneussel et al, 2001;Lévi et al, 2004;Tretter et al, 2008). For this reason, we have focused on the diffusion of GlyRs as a read-out of the receptor-gephyrin interaction, which has been very well characterized (Meyer et al, 1995;Schrader et al, 2004). The mobility of the extrasynaptic pool of GlyRs was notably increased in the presence of the gephyrin deletion constructs, confirming the existence of extrasynaptic GlyR-gephyrin complexes that depend on the oligomerization of gephyrin.…”

Molecular architecture of glycinergic synapses

“…Through its long intracellular loop between TM3 and TM4, the subunit can bind to gephyrin (Kim et al 2006;Meyer et al 1995;Sola et al 2004), thereby enabling the formation GlyR aggregations linked to subsynaptic protein scaVolds. However, the gephyrin scaVold (see below) is believed to provide three binding sites for GlyR subunits (Schrader et al 2004). Recent evidence provides a solution to this apparent inconsistency.…”