Elsevier

Cellular Signalling

Volume 9, Issue 7, November 1997, Pages 519-529
Cellular Signalling

Identification and Characterisation of a Human Calmodulin-Stimulated Phosphodiesterase PDE1B1

https://doi.org/10.1016/S0898-6568(97)00046-6Get rights and content

Abstract

A cDNA encoding a calmodulin-stimulated 3′,5′-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine “63 kDa” calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated ∼2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX ≅ 8-methoxymethyl-IBMX > vinpocetine ≅ zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominantly in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.

Introduction

Cyclic nucleotides regulate a variety of cellular events including ion conductance, transcription factor activity and enzyme function. cAMP and cGMP are synthesised by adenylyl and guanylyl cyclases respectively in response to a variety of cellular mediators. The cyclic nucleotide phosphodiesterases (PDEs) play a critical role in these signal transduction events by modulating the intracellular cyclic nucleotide levels through hydrolysis of cAMP and cGMP to their corresponding nucleoside 5′ phosphates. There are seven classes of phosphodiesterases, characterized by their primary structure, substrate preference, allosteric regulation and sensitivity to specific inhibitors (reviewed in reference 1).

The Type 1 PDEs (PDE1s) are allosterically regulated by calmodulin in the presence of Ca2+, leading to increased hydrolysis of both cAMP and cGMP (reviewed in reference [2]). The enzymatic activity of the PDE1s may be further modulated by phosphorylation events involving cAMP-dependent protein kinase or calmodulin-dependent protein kinase II 3, 4. The members of this class of PDEs are uniquely positioned to link events modulated by cyclic nucleotides with those involving Ca2+ and calmodulin.

Early biochemical evidence suggested the existence of multiple Ca2+/calmodulin-stimulated PDEs, differing in kinetic properties, tissue distribution and molecular weights 2, 5, 6. Current evidence suggests the existence of three genes encoding related Ca2+/calmodulin-stimulated PDEs: PDE1A, PDE1B, and PDE1C [1]. Alternative splicing of these genes is believed to lead to production of more than one protein from each locus 1, 7, 8, 9, 10. PDE1B1 corresponds to the bovine “63 kDa” calmodulin-stimulated PDE and has been cloned from bovine [7], rat 11, 12, mouse [13]and human sources 11, 14, 15.

We report here the isolation and characterization of cDNAs encoding the complete open reading frame of human PDE-1B1. We show that this recombinant protein has PDE activity that is stimulated by Ca2+/calmodulin and has an inhibitor profile consistent with a Type I PDE. In addition, we examine the distribution of human PDE1B1 mRNA in tissue by Northern analysis and in situ hybridisation. Sequence comparison between two partial genomic clones of HSPDE1B1 and rat PDE4 and Drosophila dunce genes provides evidence for conservation of intron-exon splice junctions within the catalytic domain. Lastly, we describe the mapping of the HSPDE1B1 gene to human chromosome 12.

Section snippets

Screening λ cDNA Libraries

An EcoR1 fragment of plasmid p12.3 [7]was gel purified, labelled with [32P]dCTP and [32P]dTTP (800 Ci/mmol, Dupont) by random priming (Boehringer Mannheim), and used for library screening under hydridisation conditions described previously [10]. Three positively hybridising plaques were isolated (HL2a, HL4a, HL9) from a human liver cDNA library (Clontech). The cDNA insert from HL4a was used to isolate two additional clones (FB29a, FB56a) from a foetal brain cDNA library (Stratagene). The cDNA

Cloning the Human PDE1B1 cDNA

We initiated our cloning of human PDE1B1 by using a bovine PDE1B1 cDNA [7]to screen a human liver cDNA library. Three λ clones were isolated (HL2a, HL4a, HL9a) and shown by sequence analysis to encode amino acid sequences similar to that of bovine PDE1B1. HL4a cDNA was used to screen a human foetal brain cDNA library from which two clones, FB29a and FB56a, were isolated and sequenced. Combining the sequences of cDNAs FB29a and FB56a yields a composite cDNA, 2644 nucleotides in length (Fig. 1).

Discussion

We report here the cloning, sequencing, expression, and chromosomal mapping of human PDE1B1. A full-length cDNA was constructed from overlapping cDNA clones isolated from a human brain cDNA library. The full-length cDNA encodes a protein of ∼61,300 Da which is homologous to PDE1B1 isoforms from bovine, rat and mouse 7, 11, 12, 13and has biochemical properties consistent with those of a Ca2+/calmodulin-stimulated phosphodiesterase. Recombinant HSPDE1B1 was able to hydrolyse both cGMP and cAMP.

Acknowledgements

We thank Sissy Peterman, Dina Leviten and Christi Wood for automated sequencing; Janelle Taylor and Rick Jasman for monoclonal antibody production; Kerry Fowler for synthesis of cilostamide; Guy Rosman for preparing the HSPDE1B1 riboprobe for northern analysis; Linda Watson for help with in situ hybridisation; and Kate Loughney for critically reading the manuscript.

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