The maintenance of polarized plasma membrane domains with distinct protein and lipid compositions is critical for efficient renal cell function. The distribution of receptors and ion transporters in these cells is regulated in part by the rate at which these proteins are internalized from the apical and basolateral cell surfaces. The mechanisms that underlie cargo recruitment to clathrin coated pits where this process is initiated are poorly understood. Localized synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) at endocytic sites plays multiple roles in endocytosis, and modulation of surface PIP2 levels in nonpolarized cells can differentially affect the internalization of distinct proteins. The central hypothesis of this proposal is that apical and basolateral PIP2 metabolism in renal epithelial cells is independently regulated to selectively modulate clathrin-dependent endocytosis from these domains. Consistent with this idea, that the three isoforms of PI5-kinase (the enzymes that generate cell surface PIP2) are differentially localized in polarized renal epithelial cells.
The specific aims of our proposal are to determine whether apical and basolateral pools of PIP2 are independently modulated in renal epithelial cells;to examine how changes in PIP2 levels upon agonist stimulation of G protein coupled receptors affect endocytosis from polarized membrane domains;and to determine how the apically localized PI5-kinase mPI5KI? regulates apical endocytosis. The results of our studies have important implications for our understanding of how polarized cells compartmentalize PI synthesis and catabolism and for how this process is affected during normal signaling and in renal disease.

Public Health Relevance

The surface of kidney cells is segregated into distinct domains with unique protein and lipid compositions that face the urine and the bloodstream. This asymmetric distribution of surface components is essential for kidney cells to properly reabsorb water, ions, and metabolites from the forming urine. These processes are controlled by receptors and ion transporters whose delivery to and removal from the cell surface is regulated by numerous cellular factors. Our goal is to understand how kidney cells differentially regulate the removal of these proteins from the surface, and how these mechanisms are selectively adjusted during the response to specific physiological signals. The results of our research will provide new information about how the trafficking of kidney proteins is regulated under normal conditions and under conditions of physiological stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK064613-08
Application #
8288901
Study Section
Special Emphasis Panel (ZRG1-RUS-C (03))
Program Officer
Mullins, Christopher V
Project Start
2003-07-01
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
8
Fiscal Year
2012
Total Cost
$303,810
Indirect Cost
$90,639
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Raghavan, Venkatesan; Rbaibi, Youssef; Pastor-Soler, Núria M et al. (2014) Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia. Proc Natl Acad Sci U S A 111:8506-11
Szalinski, Christina M; Guerriero, Christopher J; Ruiz, Wily G et al. (2013) PIP5KI? selectively modulates apical endocytosis in polarized renal epithelial cells. PLoS One 8:e53790
Welling, Paul A; Weisz, Ora A (2010) Sorting it out in endosomes: an emerging concept in renal epithelial cell transport regulation. Physiology (Bethesda) 25:280-92
Cui, Shanshan; Guerriero, Christopher J; Szalinski, Christina M et al. (2010) OCRL1 function in renal epithelial membrane traffic. Am J Physiol Renal Physiol 298:F335-45
Weisz, Ora A; Rodriguez-Boulan, Enrique (2009) Apical trafficking in epithelial cells: signals, clusters and motors. J Cell Sci 122:4253-66
Folsch, Heike; Mattila, Polly E; Weisz, Ora A (2009) Taking the scenic route: biosynthetic traffic to the plasma membrane in polarized epithelial cells. Traffic 10:972-81
Butterworth, Michael B; Weisz, Ora A; Johnson, John P (2008) Some assembly required: putting the epithelial sodium channel together. J Biol Chem 283:35305-9
Weixel, Kelly M; Edinger, Robert S; Kester, Lauren et al. (2007) Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells. J Biol Chem 282:36534-42
Guerriero, Christopher J; Weisz, Ora A (2007) N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels. Am J Physiol Cell Physiol 292:C1562-6
Chen, Chaoping; Jin, Jing; Rubin, Marc et al. (2007) Association of gag multimers with filamentous actin during equine infectious anemia virus assembly. Curr HIV Res 5:315-23

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