SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post‐synaptic specializations of inhibitory synapses

Abstract

Chemical synapses are specialized sites of the communication between neurons where information is processed and integrated. Electron microscopy has allowed morphological Received November 24, 2010; revised manuscript received December 8, 2010; accepted December 9, 2010. Address correspondence and reprint requests to Hiroyuki Sakagami, MD, PhD, Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan. E-mail: sakagami@med.kitasato-u.ac.jp Abbreviations used: Arf, ADP ribosylation factor; BRAG, brefeldin A-resistant Arf-GEF; DGC, dystrophin-associated glycoprotein complex; GABA A R, GABA A receptor; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GGA1, Golgi-localizing, c-adaptin ear homology domain, Arf-binding protein 1; GST, glutathione S-transferase; HA, hemagglutinin; IRSP, insulin receptor tyrosine kinase substrate of 53 kDa; MAGI, membrane-associated guanylate kinase with inverted orientation; PDZ, PSD-95/Discs large/Zona occludens 1; PSD, post-synaptic density; SDS, sodium dodecyl sulfate; SDS-PAGE, SDSpolyacrylamide gel electrophoresis; S-SCAM, synaptic scaffolding molecule; synArfGEF(Po), potential synaptic Arf-GEF; VGAT, vesicular c-aminobutyric acid transporter. AbstractSynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/ MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both b-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.

The BRAG/IQSec family of Arf GEFs

“…30 Interestingly, both BRAG1 and BRAG2 are abundant in the PSDs of excitatory synapses, while BRAG3 localizes exclusively to inhibitory GABAergic synapses. 31,32 Consistent with their localization, both BRAG1 and BRAG2 bind the excitatory synapse scaffolding protein PSD95 via their C-terminal PDZ motifs 33,34 while BRAG3 interacts with the inhibitory synapse scaffold gephyrin. 35 Mutations in BRAG1 have been discovered in families with non-syndromic X-linked intellectual disability (XLID).…”

Molecular and functional heterogeneity of GABAergic synapses

“…Recently S-SCAM was shown to bind also SynArfGEF, which selectively activates Arf6, a GTP-binding protein member of the ADP ribosylation factor family. S-SCAM is able to interact also with several MAGUKs (including PSD-95, SAP97 and Homer) [103]. Further, these authors showed that SynArfGEF also interacts with dystrophin and utrophin via its PDZ domain and that it colocalizes with dystrophin and gephyrin at GABAergic synapses.…”

“…Interaction between α-dystroglycan and α-/β-neurexins by means of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains has been reported several years ago [101], and was proposed to compete with α-latrotoxin binding on neurexins. In addition, binding of synaptic scaffolding molecule (S-SCAM) to β-dystroglycan and NL2 has been characterized in studies showing the selective presence of S-SCAM at inhibitory synaptic sites [102,103]. S-SCAM has been identified for its synaptogenic role and interactions with NMDA-receptor subunits and NLs.…”

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

“…Of these, BRAG1, BRAG2, and BRAG3 (brefeldin-resistant Arf GEFs) comprise a subfamily that is characterized by the presence of an IQ motif N-terminal to the catalytic domain. Although BRAG1 (6) and BRAG3 (7) are predominantly expressed in the brain, BRAG2 appears to be ubiquitously expressed (8).…”