This is a revised proposal to investigate the involvement of a protein called the guanylate cyclase regulatory protein (GCRP) in the regulation of ANF-stimulated guanylate cyclase activity. This protein which was recently cloned and expressed in the applicant's laboratory has been shown to amplify ANF-dependent cyclase activity in cotransfection experiments. The applicant hypothesizes that; (1) guanylate cyclase exists in an active and inactive form and that the presence of ANF and ATP promotes the development of the former; (2) the inactive form is suppressed by an autoinhibitory site present in the kinase-like domain of the cyclase and (3) in the presence of ANF and ATP, GCRP, and/or its splice variant GCRP-2, associates with the cyclase and induces a conformational change which releases the inhibitory constraint imposed on the active site. To evaluate this hypothesis the applicant proposes to: (1) locate the autoinhibitory site on the guanylate cyclase molecule using site-directed mutagenesis and to determine whether ANF and ATP activate the cyclase by releasing the inhibitory constraint, (2) investigate whether ANF induces the association of GCRP and GCRP-2 with guanylate cyclase and (3) examine whether limited proteolysis or binding at the autoinhibitory site induces a conformational change at the active site of the cyclase.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
3R01HL056791-04S1
Application #
6436131
Study Section
Special Emphasis Panel (ZRG4 (03))
Program Officer
Lin, Michael
Project Start
1997-07-01
Project End
2002-06-30
Budget Start
2000-07-01
Budget End
2002-06-30
Support Year
4
Fiscal Year
2001
Total Cost
$44,876
Indirect Cost
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Ding, Yaxian; Chen, Zi-Jiang; Liu, Shiguo et al. (2005) Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone. J Pharm Pharmacol 57:111-6
Chen, Zi-Jiang; Vetter, Michael; Chang, Geen-Dong et al. (2004) Cyclophilin A functions as an endogenous inhibitor for membrane-bound guanylate cyclase-A. Hypertension 44:963-8
Chen, Zi-Jiang; Vetter, Michael; Chang, Geen-Dong et al. (2003) Quercetin, a phytoestrogen and dietary flavonoid, activates different membrane-bound guanylate cyclase isoforms in LLC-PK1 and PC12 cells. J Pharm Pharmacol 55:353-8
Chen, Zi-Jiang; Vetter, Michael; Chang, Geen-Dong et al. (2003) Non-genomic effects of tamoxifen on the activation of membrane-bound guanylate cyclase GC-A. J Pharm Pharmacol 55:1539-45
Vetter, Michael; Chen, Zi-Jiang; Chang, Geen-Dong et al. (2003) Cyclosporin A disrupts bradykinin signaling through superoxide. Hypertension 41:1136-42
Chen, Zi Jiang; Vetter, Michael; Che, Danian et al. (2002) The bradykinin/soluble guanylate cyclase signaling pathway is impaired in androgen-independent prostate cancer cells. Cancer Lett 177:181-7
Chen, Z J; Song, D L; Miao, Z et al. (2001) Proteolytic activation of membrane-bound guanylate cyclase. Biochem Pharmacol 61:915-20
Chen, Z J; Che, D; Chang, C H (2001) Antioxidants, vitamin C and dithiothreitol, activate membrane-bound guanylate cyclase in PC12 cells. J Pharm Pharmacol 53:243-7
Chen, Z J; Che, D; Vetter, M et al. (2001) 17beta-estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells. J Steroid Biochem Mol Biol 78:451-8
Chen, Z J; Miao, Z H; Vetter, M et al. (2000) Molecular cloning of a regulatory protein for membrane-bound guanylate cyclase GC-A. Biochem Biophys Res Commun 278:106-11

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