The overall objective of this proposal is to define the molecular machinery that supports receptor-mediated vesicle trafficking processes in hepatocytes. These processes are essential for normal iron homeostasis, growth factor receptor signaling, and cell survival. Further, their disruption can lead to a variety of prevalent liver diseases. Here we focus on the vesicle-based machinery utilized by the hepatocyte at the plasma membrane (PM) and trans-Golgi network (TGN) to sort and traffic distinct receptors and cargo proteins. These include the transferrin receptors (TfRs), the epidermal growth factor receptor (EGFR), and lysosomal hydrolases used in their degradation. We have substantial preliminary data implicating the action of a conserved receptor sorting and vesicle formation machinery in hepatocytes that is centered around the membrane-severing GTPase dynamin 2 (Dyn2). We have found that this contractile polymer associates with the actin cytoskeleton and specific adaptor proteins at both the PM to regulate receptor endocytosis and the TGN to mediate trafficking of nascent hydrolases to the lysosome. Most recently, we have found that this complex can sort cargo through interactions with ubiquitin-associated proteins and is regulated by non- receptor tyrosine kinases. Thus, the CENTRAL HYPOTHESIS of this proposal is that a polymeric Dyn2 contractile scaffold associates with the actin cytoskeleton and specific adaptor proteins to mediate the dual processes of cargo sequestration and vesicle formation from the PM and TGN in hepatocytes. We will use state of the art imaging, biochemical, and molecular methods to explore three related but independent aims.
In Aim One we will test how the regulated assembly of a Dyn2-actin endocytic complex mediates differential internalization and trafficking of the two different TfRs from the hepatocyte PM.
In Aim Two we will define how the Dyn2 complex interacts with the ubiquitin-based sorting machinery in hepatocytes to mediate internalization and post-endocytic trafficking of the EGFR, targeting this receptor to lysosomes for degradation. Finally, in Aim Three we will define how the Dyn2 complex interacts with a novel ubiquitin-based sorting machinery at the hepatocyte TGN to maintain Golgi structure and mediate transport of newly synthesized hydrolases to the lysosome for EGFR degradation. This proposal is conceptually and technically innovative in that it provides major advances toward understanding the molecular mechanism of how essential hepatocyte receptors and cargo proteins are differentially sorted into vesicle carriers for subsequent trafficking to distinct organelles. The information gathered by these studies will help identify strategies for the treatment and prevention of diseases that affect basic liver function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK044650-22
Application #
8444419
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Serrano, Jose
Project Start
1992-02-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
22
Fiscal Year
2013
Total Cost
$324,840
Indirect Cost
$120,538
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Schulze, Ryan J; Weller, Shaun G; Schroeder, Barbara et al. (2013) Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. J Cell Biol 203:315-26
Cao, Hong; Krueger, Eugene W; McNiven, Mark A (2011) Hepatocytes internalize trophic receptors at large endocytic "Hot Spots". Hepatology 54:1819-29
Cao, Hong; Chen, Jing; Krueger, Eugene W et al. (2010) SRC-mediated phosphorylation of dynamin and cortactin regulates the "constitutive" endocytosis of transferrin. Mol Cell Biol 30:781-92
Weller, Shaun G; Capitani, Mirco; Cao, Hong et al. (2010) Src kinase regulates the integrity and function of the Golgi apparatus via activation of dynamin 2. Proc Natl Acad Sci U S A 107:5863-8
Chi, Susan; Cao, Hong; Chen, Jing et al. (2008) Eps15 mediates vesicle trafficking from the trans-Golgi network via an interaction with the clathrin adaptor AP-1. Mol Biol Cell 19:3564-75