期刊
MOLECULAR PHARMACOLOGY
卷 70, 期 5, 页码 1822-1831出版社
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.106.028688
关键词
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资金
- NIDDK NIH HHS [DK40299, DK58277] Funding Source: Medline
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, Anabaena adenylyl cyclases, Escherichia coli FhIAs ( 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: PDE5 Delta 1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5 Delta 1-419 contained GAF-B and C domain; PDE5 Delta 1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5 Delta 1-534 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. K m 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.
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