Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disability. BRAG1 is a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif, however the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors (-Rs), depresses AMPA-R-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic X-linked intellectual disability.

Summary Background Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including AP-1, AP-3 and GGAs. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. Results Here we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade which results in the sequential recruitment of the Arf-like protein Arl1, the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2, and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1/2 activity with Brefeldin A results in the loss of AP-1, AP-3 and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains and blocks recruitment of BIG1/2, Arfs and adaptor complexes to the endosome. Conclusion Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery.

Background:We previously showed that BRAG2, a known activator of Arf6, regulates cell surface levels of ␤1 integrin. Results: BRAG2 can also activate Arf4 and Arf5, but it is Arf5 that regulates integrin endocytosis. Conclusion: BRAG2 interacts with clathrin and activates Arf5 to enhance integrin internalization. Significance: This is the first evidence of a role for Arf5 at the plasma membrane in the regulation of endocytosis.

Adhesion signaling between epithelial cells and the extracellular matrix plays a critical role in maintaining tissue homeostasis and the response to tissue damage. Focal adhesion kinase (FAK) and its close relative Pyk2 are non-receptor tyrosine kinases that mediate adhesion signaling to promote cell proliferation, motility and survival. FAK has also been shown to act as a mechanosensor by modulating cell proliferation in response to changes in tissue compliance. We previously showed that mice lacking FAK in the intestinal epithelium are phenotypically normal under homeostatic conditions but hypersensitive to experimental colitis induced by dextran sulfate sodium (DSS). Here we report that Pyk2-deficient mice are also phenotypically normal under homeostatic conditions and are similarly hypersensitive to DSS-induced colitis. These data indicate that normal intestinal development and homeostatic maintenance can occur in the presence of either FAK or Pyk2, but that both kinases are necessary for epithelial repair following injury. In contrast, mice lacking both FAK and Pyk2 develop spontaneous colitis with 100% penetrance by 4 weeks of age. Normal colonic phenotype and function are restored upon treatment of the double knockout mice with antibiotics, implicating commensal bacteria or bacterial products in the etiology of the spontaneous colitis exhibited by these mice.