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
Cao, Hong; Schroeder, Barbara; Chen, Jing et al. (2016) The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase. J Biol Chem 291:16424-37
Schroeder, Barbara; Schulze, Ryan J; Weller, Shaun G et al. (2015) The small GTPase Rab7 as a central regulator of hepatocellular lipophagy. Hepatology 61:1896-907
Inoue, Jun; Krueger, Eugene W; Chen, Jing et al. (2015) HBV secretion is regulated through the activation of endocytic and autophagic compartments mediated by Rab7 stimulation. J Cell Sci 128:1696-706
Schulze, Ryan J; McNiven, Mark A (2014) A well-oiled machine: DNM2/dynamin 2 helps keep hepatocyte lipophagy running smoothly. Autophagy 10:388-9
Rasineni, Karuna; McVicker, Benita L; Tuma, Dean J et al. (2014) Rab GTPases associate with isolated lipid droplets (LDs) and show altered content after ethanol administration: potential role in alcohol-impaired LD metabolism. Alcohol Clin Exp Res 38:327-35
Schroeder, Barbara; McNiven, Mark A (2014) Importance of endocytic pathways in liver function and disease. Compr Physiol 4:1403-17
McNiven, Mark A (2013) Breaking away: matrix remodeling from the leading edge. Trends Cell Biol 23:16-21
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
Schroeder, Barbara; Srivatsan, Subhashini; Shaw, Andrey et al. (2012) CIN85 phosphorylation is essential for EGFR ubiquitination and sorting into multivesicular bodies. Mol Biol Cell 23:3602-11
Cao, Hong; Krueger, Eugene W; McNiven, Mark A (2011) Hepatocytes internalize trophic receptors at large endocytic "Hot Spots". Hepatology 54:1819-29

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