Title

Identification and characterization of the BRAG family of guanine nucleotide exchange factors at excitatory synapses

Date of Completion

January 2008

Keywords

Biology, Neuroscience

Degree

Ph.D.

Abstract

The postsynaptic density (PSD) at excitatory synapses is a highly specialized, dynamic protein complex located adjacent to the plasma membrane at the tips of dendritic spines. The PSD contains receptors with associated signaling and scaffolding proteins that govern signal transduction in the postsynaptic cell. Activation of signaling pathways in the PSD is necessary for synaptic transmission and has been shown to promote synaptic modifications, or synaptic plasticity, which are hypothesized to underlie learning and memory formation. Thus, it is critical to gain a better understanding of how signaling pathways are coordinated within the PSD to produce these changes in synaptic structure. In large-scale proteomic analyses to identify components of the PSD and elucidate its functions, the Brefeldin A-Resistant Arf GEFs (BRAGs), BRAG1, BRAG2, and BRAG3, were identified as constituents of the PSD. The BRAGs are a novel family of guanine nucleotide exchange factors (GEFs) that contain a coiled-coil (CC) motif, an isoleucine-glutamine (IQ) motif, a Sec7 domain, and a pleckstrin homology (PH) domain. BRAG1 and BRAG3 also contain a C-terminal sequence predicted to bind to PDZ domains. The Sec7 domain classifies the BRAGs as GEFs for ADP-ribosylation factors (Arfs), a family of small GTPases. The Arfs are divided into three classes. Class I and II Arfs are mainly localized to the Golgi apparatus where they regulate vesicle trafficking. Arf6, the sole class III Arf, is primarily involved in actin cytoskeletal reorganization. From immunoblot analyses, we demonstrate that BRAG1 and BRAG2 are brain specific and are highly enriched in the PSD. Their expression begins during synaptogenesis and remains high in the adult. Furthermore, immunocytochemical studies reveal that BRAG1 and BRAG2 colocalize with postsynaptic markers in cultured hippocampal neurons. We have also verified that BRAG1 can activate Arf1 in vitro and Arf1 and Arf6 in vivo. Through this activation of Arf, we demonstrate that BRAG1 regulates transport of the PSD proteins PSD-95 and NR2B and dendritic spine structure. Thus, the BRAGs are prominent components of the PSD that may modulate synaptic structure by regulating the trafficking of synaptic proteins and remodeling of the actin cytoskeleton in spines. ^