Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase.
The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.
Liu
Li
L
Cell Pathways, Inc., 702 Electronic Drive, Horsham, PA 19044, USA. lliu@cellpathways.com
Underwood
Tashandra
T
Li
Han
H
Pamukcu
Rifat
R
Thompson
W Joseph
WJ
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D004791:Enzyme Inhibitors / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D009712:Nucleotides, Cyclic; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP