Epithelial cells form barriers that can selectively regulate transport between different compartments. An extensive network of junctions joins the cells into sheets and limits their mobility under normal circumstances. However these cells do become migratory under both normal and pathological conditions. Epithelial cells must migrate during normal development and during the repair of damage. In addition cancerous epithelial cells aberrantly activate pro-migratory pathways during metastasis. Epithelial migration involves a remodeling of the cell’s structure and behavior that starts by redirecting polarity in the direction of migration. At the leading edge, actin rich protrusions and new cell-matrix adhesions anchor the cell to help propel the cell forward and the trailing edge retracts . Epithelial cells can adopt several different types of migration depending on the biological circumstances at hand . During tissue morphogenesis, development and wound healing, epithelial cells move in sheets. In this case, they maintain their cell-cell junctions . Epithelial cells can also detach from each other and migrate individually during development or cancer metastasis .
Epithelial cell motility is initiated by various growth factors, such as HGF, EGF, PDGF, VEGF, CSF-1, FGF and TGF-β [5–10]. HGF, also known as Scatter Factor (SF), is a potent motogen for numerous epithelial cells expressing the c-Met receptor . It induces scattering of multiple epithelial cell lines in 2D culture [12–14]. When epithelial cells are grown in 3D cultures, addition of HGF to the growth media initiates tubulogenesis [14, 15]. HGF production by mesenchymal cells  is increased in the event of injury to epithelia . In addition, HGF is involved in the invasive behaviors of some cancers .
A number small GTPases, including members of the Ras, Rho and Arf families regulate the cell shape changes that underlie motility. There are six Arf proteins, and Arf6 in particular has been implicated in the regulation of cell shape and motility. Initially, Arf6 was shown to regulate intracellular trafficking processes like endocytosis and recycling of membrane proteins [19, 20]. But it has subsequently been shown that Arf6 is also involved in regulating the actin cytoskeleton during migration and phagocytosis [21–26]. Arf6 is required for HGF stimulated epithelial cell motility . HGF will induce MDCK cells in culture to scatter from islands and increased Arf6 activation is observed as soon as 1 hour post HGF treatment [23, 26–28]. More recently, we found that CNK3/IPCEF, a scaffold that binds the Arf-activating cytohesin proteins, is necessary for the activation of Arf6 downstream of HGF and for HGF-stimulated migration .
While there are 6 Arf proteins in mammalian cells, a much larger number of proteins have been identified as Arf activating guanosine exchange factors (GEFs). There are 15 identified sec7 Arf GEFs divided into 5 subfamilies. It is thought that the various Arf-GEFs activate Arfs at different subcellular locations and in response to different signals. One class of Arf-GEFs, the cytohesins, has been extensively implicated in the regulation of cell shape and migration. There are 4 cytohesins. Cytohesin1 and 4 are mostly hematopoetic whereas cytohesin 2/ARNO and cytohesin 3/Grp-1 are ubiquitously expressed .
Overexpression of cytohesin 2/ARNO enhances cell motility in MDCK cells , and the phenotype is strikingly reminiscent of the response of these cells to HGF. Cytohesin 2-induced scattering of MDCK cells requires the activation of Rac1 by the Rac-GEF, Dock180 . Cytohesin 2-dependent Rac activation also depends on the coiled-coil domain in cytohesin 2 . We previously found that cytohesin 2 and Dock180 associate within a larger complex and can be co-immunoprecipitated. IPCEF/CNK3 and GRASP, two scaffold proteins that both bind the coiled-coil domain of cytohesin 2, are necessary for the assembly of this complex, and for cytohesin dependent Rac activation . These data led us to propose a model where one scaffold recruits cytohesin 2 to the membrane in response to upstream signals, while the other acts as a bridge linking cytohesin 2 and Dock180. Our demonstration that CNK3/IPCEF is required for activation of Arf6 by HGF suggests that it is the scaffold that recruits cytohesin 2 in response to upstream signals.
Here, we test the hypothesis that GRASP binds to both Dock180 and cytohesin 2 and bridges the two GEFs. We find that GRASP interacts with Dock180 independently of its ability to bind cytohesin 2. Dock180 and GRASP interact via the SH3 domain of Dock180 and the proline rich domain of GRASP. Furthermore, in addition to physically bridging cytohesin 2 and Dock180, GRASP affects cell migration directly. Knockdown of GRASP inhibits HGF-induced migration in MDCK cells.