(provided by candidate): This application has evolved from some preliminary observation that HGF (that acts as a mitogen, morphogen and a motogen, in addition to its anti-apoptotic properties) does not induce branching morphogenesis in inner medullary collecting duct cells that specifically lack Gpc3-/ and present low levels of Gpc4. The long term goal is to better understand the role of (Gpcs) in HGF mediated epithelial cell morphogenesis (and signaling downstream of the receptor c-met), and eventually in the developing kidney. The application has been divided into two major specific aims. 1) Identify whether total expression of any Gpc or the presence of a specifc Gpc is necessary for HGF mediated branching morphogenesis? 2) Examine which signaling pathways known to be important for HGF induced morphogenesis are dependent on this interaction of HGF with Gpc? The initial experiments will be designed to address specific aim 1. HA tagged WT Gpc3 and Gpc4 cDNAs will be subcloned into retroviral vector and then transduced into Gpc3-/ cells. The expression of the protein on the cell surface will be confirmed by immunostaining for HA, Flow staining of HA and also by Weternblotting the lysates from these cells for anti-HA tag. Following this, cells will be subject to HGF mediated morphogenesis in collagen matrix to determine whether both Gpc3 or Gpc4 expression can rescue the lack of effect observed in the preliminary data. Depending on the results of this specific aim 1 we will then attempt to identify the specific regions of Gpc (core protein and the GAG side chains) that are necessary for their interaction with c-met. After determining the specific aim1 , we will then proceed to examine the signaling pathways known to be involved in HGF mediated morphogenesis. Once again, based on the preliminary data, we will initially focus on MAPK pathway and determine if there is any change in the kinetic of its activation by HGF in presence or absence of Gpc expression. We will then proceed to examine the kinetics of Gpc and c-met interactions in the same setting. Depending upon the results from these two broad experiments we might proceed to examine other pathways such as PI3-K, PLC-gamma etc.
These specific aims are designed to hopefully lead to a better understanding of the role of Gpc expression during kidney development vis-a-vis HGF.
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