Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3′,5′-cyclic nucleotide phosphodiesterase¹

Abstract

Human cyclic GMP-stimulated 3′,5′-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105 715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.

Introduction

The cyclic nucleotide phosphodiesterases (PDEs) are essential regulatory components of the cellular response to hormones, neurotransmitters and autacoids. There are seven PDE families (PDE1-PDE7) identified by their distinctive catalytic and regulatory properties, amino acid sequences, and sensitivities to PDE inhibitors (Beavo, 1995). PDEs contain an approximately 250 amino acid catalytic region in the carboxy-terminal portion of the protein (Charbonneau, 1990).

The cGMP-stimulated PDEs (PDE2) hydrolyze both cAMP and cGMP, although they have a higher affinity for cGMP than for cAMP (Manganiello et al., 1990). The enzyme's catalytic activity is regulated allosterically by the binding of cGMP at site(s) remote from the catalytic site thereby increasing the enzyme's affinity for cAMP.

The cGMP-stimulated PDE has been purified from a number of sources (reviewed in Manganiello et al., 1990). It is a dimer of 102–105 kDa subunits and can be found in either the soluble or particulate fractions of tissue homogenates. The amino acid sequence of the bovine heart PDE2A has been determined and a cGMP-binding domain identified (Le Trong et al., 1990; Charbonneau et al., 1986). Bovine adrenal and rat brain cDNAs encoding PDE2A have been isolated (Sonnenburg et al., 1991; Tanaka et al., 1991, Repaske et al., 1992; Yang et al., 1994). Thus far the PDE2 amino acid and cDNA sequences that have been determined are derived from a single gene (PDE2A) in each species. Over most of their lengths the bovine (PDE2A1) and rat (PDE2A2) amino acid sequences were similar. However, PDE2A1 and PDE2A2 contained completely different amino-terminal regions (25 and 37 amino acids, respectively). A third amino-terminal sequence, bovine PDE2A3, has recently been reported (Genbank accession No. L49503: Juilfs, D.M, Sonnenburg, W.K., Seraji, S. and Beavo, J.A., unpublished). These amino-terminal differences are believed to result from alternative exon splicing and it has been suggested that the different amino termini may be responsible for particulate or soluble localization of the enzyme (Yang et al., 1994; Sonnenburg et al., 1991; Murashima et al., 1990).

In this paper we report the isolation of human PDE2A cDNAs (PDE2A3) and a preliminary characterization of the biochemical properties of the recombinant protein.