Acute kidney injury is a common hospital complication that leads death or long-term kidney damage if recovery is not complete. Hepatocyte growth factor (HGF) promotes the recovery from kidney damage at multiple steps. While many of the proximal signals activated by HGF and the downstream targets that remodel cell shape in response to HGF are known, the details of the circuits that link them together are unclear. This limits the possibility of selectively modulating HGF signaling without impacting the actions of other growth factors. Work in the applicant's lab has recently demonstrated that protein- protein interactions are critical for building a pro-migratory circuit that links activatin of Arf6 by cytohesin-2 to the downstream activation of Rac by Dock180. Preliminary data suggest that this signaling circuit is required for HGF-stimulated Rac activation and migration. The overall objective of this application is to determine the molecular details of this cytohesin-dependent Arf-to-Rac signaling module and to test the degree to which it mediates HGF-induced motility and the recovery from acute kidney injury. This is the next step toward the long-term goal of understanding the initiation of motility in epithelial cells by small GTPase cascades. This application will test a hypothesized model for the cytohesin-dependent Arf-to-Rac signaling module. This module will be tested by pursuing 4 specific aims: 1) Determine how cytohesin-2 activity is controlled;2) Determine the mechanism of interaction of cytohesin-2 and Dock180 and test the degree to which this interaction mediates HGF dependent Rac activation;3) Determine the role of active Arf6 in cytohesin-dependent Rac activation;4) Determine the extent to which cytohesin-dependent Rac activation regulates HGF-stimulated wound healing and recovery of the kidney from ischemia reperfusion injury.
Aim 1 will test the hypothesis that cytohesin-2 intramolecular interactions prevent its interaction with membranes until these interactions are disrupted by phosphorylation.
Aim 2 will use mutagenesis to identify the regions necessary for the interaction of the cytohesin-binding scaffold GRASP with Dock180. The importance of this interaction for HGF- induced Rac activation will be tested using knockdown of GRASP and expression of GRASP mutants.
Aim 3 will test the hypothesis that Arf6-dependent lipid modifications promote activation of Rac by Dock180.
Aim 4 will use siRNA, mutant and pharmacologic modulation of cytohesin-dependent Arf-to- Rac signaling to test its involvement in HGF-stimulated wound healing and recovery of the kidney from ischemia reperfusion injury. A proven model of this pro-migratory circuit will allow the design of interventions to modulate this circuit without impacting other Arf6 or Rac-dependent functions. Such treatments could promote or inhibit HGF-dependent migration without disrupting the actions of a broad range of growth factors.
HGF-dependent migration of epithelial cells is important for wound healing, normal development and has been implicated in metastatic progression. The proposed research will test a model for a signaling circuit involved in this migration. Understanding the molecular details of this circuit will allow the development of tools to alter it activity, which could be used to promote kidney repair after injury and fight metastatic progression.
|Koubek, Emily J; Santy, Lorraine C (2016) ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation. Small GTPases :1-18|
|Salem, Joseph C; Reviriego-Mendoza, Marta M; Santy, Lorraine C (2015) ARF-GEF cytohesin-2/ARNO regulates R-Ras and Î±5-integrin recycling through an EHD1-positive compartment. Mol Biol Cell 26:4265-79|
|Reviriego-Mendoza, Marta M; Santy, Lorraine C (2015) The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice. Physiol Rep 3:|
|Attar, Myriam A; Santy, Lorraine C (2013) The scaffolding protein GRASP/Tamalin directly binds to Dock180 as well as to cytohesins facilitating GTPase crosstalk in epithelial cell migration. BMC Cell Biol 14:9|
|Hiester, Kevin G; Santy, Lorraine C (2013) The cytohesin coiled-coil domain interacts with threonine 276 to control membrane association. PLoS One 8:e82084|