期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 280, 期 12, 页码 12051-12063出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M413611200
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资金
- NIDDK NIH HHS [DK58277, DK40299] Funding Source: Medline
The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K-D < 30 nM) and between GAF b modules (KD = 1-20 pM), and the sequence between the GAFs ( Thr(322)-Asp(403)) contributes to dimer stability; 2) 176 amino acids (Val(156)-Gln(331)) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K-D < 40 nM) than does the isolated R domain (KD = 110 nM) or holoenzyme (K-D = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met(1)-Glu(321)) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser(102) phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.
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