Abstract
Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaenaadenylyl cyclases, Escherichia coliFhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Δ1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Δ1-419 contained GAF-B and C domain; PDE5Δ1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Δ1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. Km values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.
Footnotes
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This work was supported by National Institutes of Health grants DK40299 and DK58277 and by American Heart Association Postdoctoral Fellowship 032525B.
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ABBREVIATIONS: PDE, cyclic nucleotide phosphodiesterase; GAF, mammalian cGMP-binding phosphodiesterase, Anabaenaadenylyl cyclases, Escherichia coliFhlA; IBMX, 3-isobutyl-1-methylxanthine; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid; KP, 10 mM potassium phosphate, pH 6.8; KPM, 10 mM potassium phosphate, pH 6.8, containing 15 mM β-mercaptoethanol.
- Received July 7, 2006.
- Accepted August 22, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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