Complex Formation between the Postsynaptic Scaffolding Protein Gephyrin, Profilin, and Mena: A Possible Link to the Microfilament System

Abstract

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.

Gephyrin: a central GABAergic synapse organizer

“…75,[82][83][84] Profilin and Mena A study seeking the linkage between gephyrin and microfilament identified profilin, Mena and G-actin as gephyrin-binding proteins. 85 Endogenous profilin and Mena were found to colocalize with gephyrin in the inhibitory synapses of spinal cord neurons, and were enriched in gephyrin-rich domains of the neuronal plasma membrane. 85 Gephyrin and profilin 1 were shown to form a complex, 86 but via an unexpected binding mode that required the E domain of gephyrin and the actin/ PIP 2 -binding site of profilins (Giesemann et al, 85 but also see Bausen et al 87 ).…”

“…85 Endogenous profilin and Mena were found to colocalize with gephyrin in the inhibitory synapses of spinal cord neurons, and were enriched in gephyrin-rich domains of the neuronal plasma membrane. 85 Gephyrin and profilin 1 were shown to form a complex, 86 but via an unexpected binding mode that required the E domain of gephyrin and the actin/ PIP 2 -binding site of profilins (Giesemann et al, 85 but also see Bausen et al 87 ). A study examining the potential impact of these multipartite interactions on the function of gephyrin showed that cytochalasin D (an actin-depolymerizing agent) had differential effects on gephyrin clusters that were lost following cytochalasin D treatment of immature cultures but not following the same treatment of more mature cultures.…”

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

“…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.…”

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

“…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 gephyrin is not known.…”