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 reporte d here, we have identified two new regions within the primary structur e of these two related isozymes that are important for regulation by C a2+/CaM, PDE1A1 is identical to the PDE1A2 isozyme except for the amin o terminal 18 residues, In agreement with earlier studies, the CaM con centration required for half maximal activation (K-CaM) of recombinant PDE1A1 (0.3 nM) was approximate to 10-fold less than the K-CaM for re combinant PDE1A2 (4 nM), A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminotermi nal residues were constructed and expressed using the baculovirus syst em, Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide tha t was still activated 3-fold by CaM (K-CaM approximate to 3 nM), Howev er, complete CaM-independent activation occurred when residues 4-98 we re deleted, To determine the location of the additional CaM-binding do main(s), the inhibitory potency of seven overlapping, synthetic peptid es spanning amino acids 76-149 of PDE1A2 was tested using the CaM-acti vated enzyme, One peptide spanning amino acids 114-137 of PDE1A2 appea red to be the most potent inhibitor of CaM-stimulated activity, These results reveal the existence of a CaM-binding domain located approxima tely 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 ac tive state at low Ca2+ concentrations is located between the two CaM-b inding domains.