**4. Conclusions**

206 Crosstalk and Integration of Membrane Trafficking Pathways

which provides an additional test of the model for allostery. We described this below for

The enzymology of two ArfGEFs has been examined using some of these principles.

mediated phosphorylation (DiNitto et al., 2007) also contribute to regulating ARNO.

terminus – Arno interaction in regulation will be interesting to examine.

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

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 N-

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

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

**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

Brag2.

cells.

The knowledge of kinetic parameters is limited to a few GAPs and GEFs. The information available has provided a number of insights into the biological function of the proteins and potential regulation. For instance, the effect of cotaomer and cargo on ArfGAP1 led to the idea that it may act prior to transport vesicle formation rather than after vesicle formation as has been generally accepted. Activation of ARNO by Arf6 and Arl4 has led to the idea of sequential signaling functions of Arf proteins.

Other aspects of the known enzymology of GAPs and GEFs, such as the discrepant turnover numbers among the GAPs, are intriguing. The slow turnover number could result from a lack of understanding of optimal conditions for a particular ArfGAP, including potential allosteric modifiers that may stimulate activity. Also possible, the different turnover numbers may be related to the biological process being controlled. In addition to examination of additional GAPs and GEFs and further characterization of individual proteins to find optimal conditions for enzymatic activity, identification of GEF/GAP pairs will be important for understanding the function of the Arf proteins in biological processes.

## **5. Acknowledgment**

The work was supported by the intramural program of the National Cancer Institute, National Institutes of Health, USA.
