Our work over the last funding period has focused on characterization of P2Y receptor targeting in polarized cells. These studies established that seven of the eight known subtypes of P2Y receptors display polarized expression in epithelial cells, with four receptors at the basolateral membrane and three receptors at the apical membrane. We have identified and characterized the sorting signals in five of these receptors, which revealed novel mechanisms by which the receptors achieve polarized expression. These findings will be extended to define the physical and structural properties of the sorting signals in the C-terminal tails of P2Y1, P2Y4 , and P2Y14 receptors. The proteins that bind to P2Y receptor sorting signals and direct their polarized targeting are unknown. We will identify these proteins and define their roles in receptor targeting. Lipid microdomains are implicated in localization of proteins in endothelial and epithelial cells and in the regulation of signaling molecules, including P2Y receptors. We will elucidate the role(s) that lipid rafts and caveolae play in the localization and signaling responses of P2Y receptors and delimit the receptor domain(s) involved in these interactions. Successful completion of these projects will provide major new insights into the mechanistic/structural basis of cell targeting and regulation of signaling activities of a therapeutically important class of receptors in the context of a physiologically relevant cell model. The nucleotide-activated P2Y receptors are proven or potential therapeutic targets for treatment of lung diseases, hypertension, and stroke. In epithelial cells, these receptors are targeted to distinct membrane surfaces, where they regulate ion and water transport across the epithelial monolayer. Although cellular mistargeting of proteins is often associated with disease, the cellular mechanisms for proper protein localization are still not well understood. Our studies will provide novel and important information on the cellular mechanisms of P2Y receptor targeting and the regulation of P2Y receptor signaling activity in epithelial cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL071131-08
Application #
8009796
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Banks-Schlegel, Susan P
Project Start
2002-07-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
8
Fiscal Year
2011
Total Cost
$364,498
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
DuBose, D Ross; Wolff, Samuel C; Qi, Ai-Dong et al. (2013) Apical targeting of the P2Y(4) receptor is directed by hydrophobic and basic residues in the cytoplasmic tail. Am J Physiol Cell Physiol 304:C228-39
Qi, Ai-Dong; Harden, T Kendall; Nicholas, Robert A (2013) Is GPR17 a P2Y/leukotriene receptor? examination of uracil nucleotides, nucleotide sugars, and cysteinyl leukotrienes as agonists of GPR17. J Pharmacol Exp Ther 347:38-46
Wolff, Samuel C; Qi, Ai-Dong; Harden, T Kendall et al. (2010) Charged residues in the C-terminus of the P2Y1 receptor constitute a basolateral-sorting signal. J Cell Sci 123:2512-20
Qi, Ai-Dong; Wolff, Samuel C; Nicholas, Robert A (2005) The apical targeting signal of the P2Y2 receptor is located in its first extracellular loop. J Biol Chem 280:29169-75
Wolff, Samuel C; Qi, Ai-Dong; Harden, T Kendall et al. (2005) Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. Am J Physiol Cell Physiol 288:C624-32
Herold, Christopher L; Qi, Ai-Dong; Harden, T Kendall et al. (2004) Agonist versus antagonist action of ATP at the P2Y4 receptor is determined by the second extracellular loop. J Biol Chem 279:11456-64