Received for publication July 10, 2006.
Revised August 21, 2006.
Accepted for publication August 22, 2006.

A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in PDE5 dimerization

Abstract

Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two GAFs (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPK). 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: PDE51-321 contained the sequence between GAF-A and -B, GAF-B, and C domain; PDE51-419 contained GAF-B and C domain; PDE51-465 contained the C-terminal portion of GAF-B and C domain; PDE51-534 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_ms 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 to 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.

Key words: cGMP, Phosphodiesterases, Receptor binding studies, Protein-binding, Structure/function/mechanism

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