This proposal studies Transforming Growth Factor Beta Receptors (TGFBRs) using novel fusion proteins for rapid, reversible, and precise inhibition of Clathrin mediated endocytosis (CME) and activation of lipid raft/Caveolin mediated endocytosis (CAVME). CME and CAVME are the predominant mechanisms for receptor internalization, and their deregulation is implicated in myriad diseases, including fibrosis of the kidney and liver. Transforming Growth Factor Beta 1 (TGF-B1) is a master regulator of liver fibrosis; therefore, signaling will be evaluated using primary mouse hepatic stellate cells, along with transformed cell lines. TGF- B1 fibrotic signaling is mediated through its two cell surface receptors, TGFBR1 and TGFBR2. Endocytosis plays a major regulatory role in TGFBR mediated signaling, as TGF-B1/TGFBR internalization via CME leads to pro-fibrotic SMAD signaling while internalization via CAVME downregulates this signaling. Despite the importance of endocytosis in fibrosis and normal physiology, distinguishing if cargo (e.g. a receptor, a drug, a pathogen) is selectively internalized via CME or CAVME remains challenging, as scientists cannot exclusively manipulate one pathway at a time. This proposal aims to overcome the limitations of currently employed endocytic inhibitors/activators by using temperature sensitive recombinant proteins composed of Clathrin light chain (CLC) or Caveolin 1 (CAV1) and a thermally responsive Elastin-like polypeptide (ELP). I hypothesize that ELP-mediated assembly of CLC will inhibit CME and down regulate TGF-B1 mediated fibrotic signaling, while CAV1-ELP assembly will activate CAVME but also down regulate this signaling. In summary, this proposal employs the TGF-B1/TGFBR pathway and primary hepatic stellate cells to further characterize CAV1-ELP and CLC-ELP fusion proteins. Completion of this project will provide the scientific community with: 1) new tools to rapidly and reversibly manipulate endocytosis; 2) a better understanding of how TGFBR trafficking contributes to a fibrotic cellular phenotype; 3) higher-fidelity knowledge about how endocytosis regulates HSC activation; and 4) new experimental assays that may identify targets for anti-fibrotic therapeutics.
This proposal employs cell lines and primary mouse hepatic stellate cells to study Transforming Growth Factor Beta Receptors (TGFBRs) and fibrotic signaling using novel temperature-sensitive proteins to control Clathrin-mediated and Caveolin-mediated endocytosis. The disease focus of this proposal is fibrotic signaling in the liver. Completion of this project will provide the scientific community with: 1) new tools to rapidly and reversibly manipulate endocytosis; 2) a better understanding of how TGFBR trafficking contributes to a fibrotic cellular phenotype; 3) higher-fidelity knowledge about how endocytosis regulates HSC activation; and 4) new experimental assays that may identify targets for anti-fibrotic therapeutics. !
