Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. Receptor levels on the plasma membrane are dependent upon the rates of internalization and recycling back to the cell surface. We and others have recently identified a novel Eps15 homology (EH)-domain containing protein, EHD1, as a critical component of the endocytic recycling pathway. The physiologic importance of EHD-mediated function has been clearly demonstrated, as loss of EHD1 function leads to impaired recycling of many critical receptors. The long- term goals of this project are to understand the fundamental mechanisms controlling intracellular trafficking and transport of proteins to the plasma membrane, with emphasis on understanding the mode by which EHD structure impacts its function. The central hypothesis of this proposal is that the electrostatic surface charge of the EH-domain of EHD1 (EH-1) and its C-terminal EHD paralogs is responsible for the selectivity of interactions with NPF-containing protein partners and phosphoinositides, and that these interactions are important for the regulation of endocytic transport. We provide compelling evidence for our hypothesis based on our strong preliminary data that EH-1 selectively interacts with proteins containing NPF motifs followed by a cluster of negatively charged residues. In addition, our new preliminary data also reflect the functional importance of the EHD/phosphoinositide interactions by demonstrating that EH-1 is capable of interacting directly with phosphatidylinositol moieties, and that this interaction is required to allow localization of EHD1 to tubular and vesicular membrane structures.
Our Specific Aims for the proposal are: 1) To identify the molecular and structural basis for the selectivity of C-terminal EH-domains for specific NPF-containing proteins, and 2) To elucidate the specific phosphoinositide(s) that are enriched/comprise EHD tubular membranes and determine the significance of EHD/phosphoinositide binding in the regulation of endocytic trafficking. The knowledge to be obtained from this study will provide critical new information on the mode by which the C-terminal EHD proteins associate with endosomal membranes and coordinate control of recycling. This will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease.

Public Health Relevance

Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. The knowledge to be gained from undertaking this proposal will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087455-03
Application #
8274823
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2010-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$285,856
Indirect Cost
$92,806
Name
University of Nebraska Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
168559177
City
Omaha
State
NE
Country
United States
Zip Code
68198
Giridharan, Sai Srinivas Panapakkam; Caplan, Steve (2014) MICAL-family proteins: Complex regulators of the actin cytoskeleton. Antioxid Redox Signal 20:2059-73
Cai, Bishuang; Xie, Shuwei; Liu, Fengming et al. (2014) Rapid degradation of the complement regulator, CD59, by a novel inhibitor. J Biol Chem 289:12109-25
Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava et al. (2014) Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1. J Cell Sci 127:1684-98
Xie, Shuwei; Naslavsky, Naava; Caplan, Steve (2014) Diacylglycerol kinase ? regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling. J Biol Chem 289:31914-26
Cai, Bishuang; Giridharan, Sai Srinivas Panapakkam; Zhang, Jing et al. (2013) Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes. J Biol Chem 288:30172-80
Giridharan, Sai Srinivas Panapakkam; Cai, Bishuang; Vitale, Nicolas et al. (2013) Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis. Mol Biol Cell 24:1776-90, S1-15
Zhang, Jing; Reiling, Calliste; Reinecke, James B et al. (2012) Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function. Traffic 13:745-57
Giridharan, Sai Srinivas Panapakkam; Rohn, Jennifer L; Naslavsky, Naava et al. (2012) Differential regulation of actin microfilaments by human MICAL proteins. J Cell Sci 125:614-24
Zhang, Jing; Naslavsky, Naava; Caplan, Steve (2012) Rabs and EHDs: alternate modes for traffic control. Biosci Rep 32:17-23
Cai, Bishuang; Caplan, Steve; Naslavsky, Naava (2012) cPLA2ýý and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes. Mol Biol Cell 23:1874-88

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