**3.2 ARNO/cytohesin/Grp1: Example of ArfGEF regulated by relief of autoinhibition**

ARNO proteins are comprised of coiled coil, sec7 (catalytic), PH and polybasic (PB) domains. The ARNO group of proteins has roles in diverse cellular processes: regulation of cell adhesion and migration (Goldfinger et al., 2003;Santy and Casanova, 2001;Nagel et al., 1998; Geiger et al., 2000;Hernandez-Deviez et al., 2004); insulin signaling (Fuss et al., 2006;Hafner et al., 2006); and; vesicle transport (Hurtado-Lorenzo et al., 2006; Merkulova et al., 2010; Merkulova et al., 2011;Caumon et al., 2000). The regulation described for ARNO is complex. The effect of protein and lipid binding to the PH domain is discussed here. Protein binding to the coiled-coil domain (Esteban et al., 2006;Goldfinger et al., 2003) and PKC mediated phosphorylation (DiNitto et al., 2007) also contribute to regulating ARNO.

The molecular basis for the effect of protein and lipid binding to the PH domain has been examined in some detail (DiNitto et al., 2007; Cohen et al., 2007). ARNO is autoinhibited by the linker region between the sec7 and PH domains and a C-terminal amphipathic helix, which physically block the Arf binding site. Binding of Arl4GTP, Arf6GTP and phosphoinositides to the PH domain has two functions. One is to recruit ARNO to the membrane surface on which it is active and the second to induce a conformational change in the PH domain that relieves autoinhibition. Phosphorylation of ARNO by protein kinase C (PKC) also alleviates autoinhibition (DiNitto et al., 2007; Frank et al., 1998). The characterization was done primarily with a truncated form of Arf, lack an N-terminal extension that is unique to the Arf family of GTP binding proteins. A possible function of Nterminus – Arno interaction in regulation will be interesting to examine.

### **3.3 Brag2: PIP2 acts as allosteric modifier binding to the PH domain**

The Brag subgroup of GEF proteins has three members characterized by the presence of IQ, sec7, PH and coiled-coil domains (Casanova, 2007). Brag1 and 3 are found primarily in brain. Brag2, although enriched in brain, is ubiquitously expressed. Brag2 affects endocytosis of cell adhesion molecules, including cadherins and integrins, and has been implicated in antiangiogenic signaling in endothelial cells and invasion of breast cancer cells.

Recent work examining Brag2 supports the idea that PIP2 allosterically modifies activity by binding to the PH domain (Jian and Randazzo, manuscript in preparation). The work was an extension of work examining signaling by semaphorin. Sema3E is an antiangiogenic factor that binds to Plexin D1 resulting in recruitment of PIP kinase and increased Arf6 exchange factor activity mediated by Brag2 (Sakurai et al., 2010; Sakurai et al., 2011). Brag2 was found to bind to PIP2, which stimulated exchange factor activity in vitro. Subsequent work identified residues within the PH domain that bound to PIP2. PH domains are thought to be recruitment domains, but the two substrates for Brag2, ArfGDP and GTP, are soluble, so recruitment to a membrane by itself would not result in increased activity. PIP2 was found to increase both the Km and kcat for the exchange reaction, and, consistent with behavior as an allosteric modifier with an effect on Km, the substrate ArfGDP affected the
