Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.
Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.
Department of Pharmacology, University of Washington, Seattle 98195, USA.
W K
WK
Sonnenburg
D
D
Seger
K S
KS
Kwak
J
J
Huang
H
H
Charbonneau
J A
JA
Beavo
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D018076:DNA, Complementary; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D010766:Phosphorylation; D016415:Sequence Alignment; D013379:Substrate Specificity
D002147:Calmodulin; D018076:DNA, Complementary; D007527:Isoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1