Angiotensin II (Ang II) exerts important physiologic and pathophysiologic effects on cardiovascular and renal systems through its AT1 receptor subtype. Emerging evidence indicates that AT2 receptor subtype also mediates significant biological responses such as natriuresis, anti-growth, anti-proliferation, and pro-apoptosis. These inhibitory actions appear to antagonize the well-known stimulatory actions of AT1 receptor. Because a delicate balance between these opposing actions must be maintained for health and because these actions are all governed by the corresponding stimulatory and inhibitory signals, elucidation of inhibitory signaling mechanisms becomes more and more important in biomedicine and may hold keys for innovative therapeutics.Unlike the AT1 receptor for which the stimulatory signaling mechanisms are well studied, signaling and activation (R*) mechanisms of the inhibitory AT2 receptor remain unknown. Our results indicate that AT2 receptors directly activate SH2 domain containing phosphatase- 1 (SHP- 1) and b1y2 proteins, respectively, with novel mechanisms. AT2 may produce constitutively active state (R*), Ang Il-induced R*, and DTT-regulated R*. These R* states triggers activation of multiple signal effector pathways. Therefore, we hypothesize that AT2 is a novel inhibitory receptor capable of activating inhibitory signals with distinct mechanisms through multiple R* states.
The specific Aims are:1. Determine the molecular basis for physical association between SHP-1 and the AT2 receptor.2. Determine the molecular basis for physical association between gb1y2 subunits and the AT2 receptor.3. Understand activation mechanisms of the inhibitory AT2 receptor.Many techniques such as mutagenesis, co-immunoprecipitation, Western blotting, peptide binding, mammalian two-hybrid system, in vitro translation, luminescence, and BRET will be used in this project. Various cell lines (CHO-K1, L cells, and NI E- 115) and primary rabbit proximal tubular epithelial cells will be used accordingly for this project. Stable cell lines will be generated. Gq/ill chimera protein and jellyfish photoprotein apoaequorin will be used extensively as reporter system to detect AT2 receptor activation. In this proposal, tools of bioinformatics and molecular modeling will also be used to assist experimental design and interpretation. We believe this proposed study will provide important insight into mechanisms of AT2-triggered inhibitory signals and AT2 activation. This proposal has the potential to open up novel avenues for therapy of hypertrophy and fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065492-03
Application #
6802782
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Buxton, Denis B
Project Start
2002-09-01
Project End
2006-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
3
Fiscal Year
2004
Total Cost
$299,400
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Rockville
State
MD
Country
United States
Zip Code
20817
Wang, Tingting; Chen, Man; Liu, Lian et al. (2011) Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production. Toxicol Appl Pharmacol 257:328-37
Day, Regina M; Lee, Young H; Han, Li et al. (2011) Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms. Am J Physiol Lung Cell Mol Physiol 301:L772-81
Feng, Y H; Li, X; Zeng, R et al. (2006) Endogenously expressed truncated P2X7 receptor lacking the C-terminus is preferentially upregulated in epithelial cancer cells and fails to mediate ligand-induced pore formation and apoptosis. Nucleosides Nucleotides Nucleic Acids 25:1271-6
Li, Xin; Zhou, Lingying; Feng, Ying-Hong et al. (2006) The P2X7 receptor: a novel biomarker of uterine epithelial cancers. Cancer Epidemiol Biomarkers Prev 15:1906-13
Feng, Ying-Hong; Zhou, Lingyin; Sun, Yan et al. (2005) Functional diversity of AT2 receptor orthologues in closely related species. Kidney Int 67:1731-8
Feng, Ying-Hong; Zhou, Lingyin; Qiu, Rongde et al. (2005) Single mutations at Asn295 and Leu305 in the cytoplasmic half of transmembrane alpha-helix domain 7 of the AT1 receptor induce promiscuous agonist specificity for angiotensin II fragments: a pseudo-constitutive activity. Mol Pharmacol 68:347-55
Feng, Ying-Hong; Ding, Yaxian; Ren, Shuo et al. (2005) Unconventional homologous internalization of the angiotensin II type-1 receptor induced by G-protein-independent signals. Hypertension 46:419-25