application): Angiotensin-converting enzyme (ACE) is a key component of the renin-angiotensin system that regulates blood pressure. Studies with ACE knockout mice have revealed additional roles of ACE in renal physiology and male fertility. Although ACE exists primarily as a cell-surface protein, a soluble form is present under normal conditions in serum and other body fluids. Because information in the literature suggests that the specific physiological function of cell-bound ACE may differ from those of ACE in circulation, production of soluble ACE from cell-bound ACE could be significant in biological regulation. Analysis of ACE cleavage-secretion processes using natural ACE-producing cells and cells transfected with expression vectors of ACE or its mutants, revealed that the ectodomain of ACE is cleaved at a specific site near the plasma membrane, but the cleavage specificity is maintained not by sequence at or around the cleavage site but by the presence of the distal ectodomain of the ACE protein. ACE-secretase is a membrane-anchored metalloprotease and yeasts contain an ACE-cleaving activity with the properties of the mammalian ACE-secretase. The activity of the mammalian secretase can be upregulated by treatment of cells with phorbol esters, calmodulin inhibitors or protein tyrosine phosphatase inhibitors.
The aims of this application are: 1) to define the sequence in the ectodomain of ACE that activates the secretase, 2) to clone and characterize the ACE-secretase, and 3) to determine whether the phosphorylation states of the cytoplasmic domains of ACE and the secretase regulates the rate of cleavage-secretion of ACE. Secretion of deletion and substitution mutants of ACE-CD4 chimeras will be monitored for identifying the domain in ACE that triggers its cleavage-secretion. For cloning human ACE secretase, genetic complementation of yeast and human cells will be carried out. A yeast mutant that lacks the ACE-secretase activity will be generated by systematically mutating its known metalloproteases. The mutant will then be used for expressing a human cDNA library and scored for the restoration of ACE-secretion. Similarly, mammalian cells devoid of ACE-secretase will be generated by chemical mutagenesis followed by FACS sorting and these mutant cells will be complemented with the clone for the human ACE-secretase. The mechanism of regulation of ACE secretion by PMA, calmodulin inhibitors and tyrosine phosphatase inhibition will be explored by determining whether the relevant protein kinases bind to and phosphorylate the cytoplasmic domains of ACE and the secretase or its associated proteins. Involvement of specific tyrosine and serine/threonine protein kinase pathways in this regulation will be investigated by using specific chemical inhibitors, dominant-negative mutant kinases and cell lines devoid of specific kinases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054297-08
Application #
6604258
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Barouch, Winifred
Project Start
1996-08-01
Project End
2005-06-30
Budget Start
2003-07-01
Budget End
2005-06-30
Support Year
8
Fiscal Year
2003
Total Cost
$259,000
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Chattopadhyay, Saurabh; Karan, Goutam; Sen, Indira et al. (2008) A small region in the angiotensin-converting enzyme distal ectodomain is required for cleavage-secretion of the protein at the plasma membrane. Biochemistry 47:8335-41
Chattopadhyay, Saurabh; Santhamma, Kizhakkekara R; Sengupta, Saubhik et al. (2005) Calmodulin binds to the cytoplasmic domain of angiotensin-converting enzyme and regulates its phosphorylation and cleavage secretion. J Biol Chem 280:33847-55
Santhamma, Kizhakkekara R; Sadhukhan, Ramkrishna; Kinter, Michael et al. (2004) Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme. J Biol Chem 279:40227-36
Santhamma, K R; Sen, I (2000) Specific cellular proteins associate with angiotensin-converting enzyme and regulate its intracellular transport and cleavage-secretion. J Biol Chem 275:23253-8
Sadhukhan, R; Santhamma, K R; Reddy, P et al. (1999) Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice. J Biol Chem 274:10511-6
Sadhukhan, R; Sen, G C; Ramchandran, R et al. (1998) The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface. Proc Natl Acad Sci U S A 95:138-43