cGMP has been implicated in modulation of numerous mammalian systems pertinent to pathophysiology, including smooth muscle tone in blood vessels and other tissues, vision, and platelet aggregation. The objective of this proposal is to examine the regulation of a cGMP-binding phosphodiesterase (B-PDE) which is a probable target of agents that affect the cGMP cascade. This will be done by purifying the bovine lung enzyme and determining its structure and regulatory features. The functions of the B-PDE may be regulated both by an allosteric mechanism and by phosphorylation. The evidence suggests that the enzyme contains at least two functional domains--a highly specific cGMP binding site and a separate hydrolytic site for cGMP. While there is evidence for communication between these putative domains, there is no dir""""""""ct evidence for a function of the binding site. The quaternary structure of B-PDE, number of cGMP binding domains and cyclic nucleotide binding properties of these domains will be ascertained. Effects of binding site- or hydrolytic site-specific cGMP analogs on the enzyme will be tested. Purified protein kinases will be used to study phosphorylation of the enzyme and to determine the mechanism whereby phosphorylation is regulated in a substrate-directed manner by cGMP binding to B-PDE. Preliminary results indicate that the B-PDE is a relatively specific and potent substrate of cGMP-dependent protein kinase, an enzyme for which a physiological substrate has not yet been established. The functional domains of B-PDE will also be studied by generating proteolytic fragments which contain binding activity, hydrolytic activity, or the phosphorylation site. Fragments will be labeled by prior phosphorylation or (32P)cGMP photoaffinity- labeling of B-PDE, and characterized in order to orient the functional domains within the B-PDE. Partial amino acid sequencing of the fragments may reveal features required for function or phosphorylation. Peptides modeled after the sequences of phosphorylation site(s) will be synthesized and tested as substrates for protein kinases in order to identify determinants important for phosphorylation. Oligonucleotide probes based on sequences of peptide fragments, and polyclonal antibodies generated against B-PDE, will be used to screen a bovine lung cDNA library for purposes of cloning. This will permit deduction of the complete primary sequence, assessment of the domain structure, and may indicate evolutionary homologies with other proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041269-02
Application #
3299350
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Francis, S H; Turko, I V; Corbin, J D (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acid Res Mol Biol 65:1-52
Francis, S H; Turko, I V; Grimes, K A et al. (2000) Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5. Biochemistry 39:9591-6
Fink, T L; Francis, S H; Beasley, A et al. (1999) Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5). J Biol Chem 274:34613-20
Corbin, J D; Francis, S H (1999) Cyclic GMP phosphodiesterase-5: target of sildenafil. J Biol Chem 274:13729-32
Turko, I V; Ballard, S A; Francis, S H et al. (1999) Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodiesterase (Type 5) by sildenafil and related compounds. Mol Pharmacol 56:124-30
Turko, I V; Francis, S H; Corbin, J D (1998) Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation. Biochem J 329 ( Pt 3):505-10
Loughney, K; Hill, T R; Florio, V A et al. (1998) Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase. Gene 216:139-47
Granovsky, A E; Natochin, M; McEntaffer, R L et al. (1998) Probing domain functions of chimeric PDE6alpha'/PDE5 cGMP-phosphodiesterase. J Biol Chem 273:24485-90
Wyatt, T A; Naftilan, A J; Francis, S H et al. (1998) ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells. Am J Physiol 274:H448-55
Turko, I V; Francis, S H; Corbin, J D (1998) Hydropathic analysis and mutagenesis of the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5). cGMP versus cAMP substrate selectivity. Biochemistry 37:4200-5

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