title: Identification and analysis of phosphodiesterase isoenzymes
Creators: name: Rena, Neil Graham
Publisher: name: University of Glasgow
Year: 1998

description: The second messenger cAMP is involved in the mediation of a wide range of signals in response to a wide range of external stimuli, including processes as varied as neurotransmission, metabolism and embryonic development. Each of the components of the cAMP signalling system are encoded by families of genes. In the case of cAMP phosphodiesterases, the enzymes that hydrolyse cAMP, this variety produces proteins with different targeting, expression and kinetic characteristics. A full understanding of the differing functions of these isoenzymes may in the longer term allow drugs to be designed which allow specific interventions in human pathologies. To realise such an approach, it is important that each isoenzyme be identified and characterised. This thesis concentrates on the characterisation of four of these isoenzymes, namely PDE1B, PDE3B, PDE4A1 and PDE4A10. In chapter three of this thesis, I use immunoblotting of cell fractions to examine the particulate association of transiently expressed full-length PDE3B. I show solubilisation of full-length PDE3B for the first time. In addition to this I determine the solubility of two deletion mutants which delete a putative membrane association domain. I demonstrate that a mutant with a deleted putative membrane association domain is still particulate, which strongly suggests that PDE3B possesses additional membrane association determinants. I also present data which suggests that PDE3B can be stimulated by protein kinase B. In chapter four I examine a PKC-mediated induction of PDE1 in Chinese hamster ovary cells. There was no sequence information for Chinese hamster PDEs. This compelled me to design molecular probes to phylogenetically stable pieces of PDE1. Sequencing of amplification products of these probes reveal that PDE1B is the induced PDE1 form in Chinese hamster ovary cells. Selective isoforms of PKC appear to mediate this stimulation. Chapters five and six of this thesis are devoted to furthering knowledge of the number and primary structure of PDEs. In chapter five I identify mammalian homologues for the rat PDE RD1. Thus, RD1 becomes only the second PDE4A which has been identified in human and rat. The putative membrane association sequences of RD1 are perfectly conserved across a range of mammalian species, supporting the notion that the sequences may have a conserved functional role. Serendipitously, this work exposes a rodent-lineage specific deletion of apparently ancient PDE4A sequence. This finding, which may be associated with functional alterations, may be a first step in the production of single state PDE inhibitors, since it suggests a possible target for such inhibitors. The deletion may also be of considerable interest to molecular taxonomists as a phylogenetic marker with the potential to solve previously intractable controversies over rodent phylogeny. In chapter six of this thesis I identify, clone and express a novel human PDE4A splice variant, PDE4A10. I then make a survey of endogenous expression of this PDE in native brain and cell lines by RT-PCR. Furthermore, I identify almost the full open reading frame of the rat homologue of this splice variant. This PDE is therefore the third PDE4A which has been identified in both rat and human. The work in chapter six also makes a wider contribution, by describing a novel method for producing precise overlaps in clones of DNA made by a modification of shotgun cloning. Shotgun cloning was already the easiest way to fragment a large piece of DNA into smaller ones. The innovation to shotgun cloning that I describe makes it easier and extends its utility. The overlaps of DNA fragments that result will allow the shotgun fragments to be ordered as they appeared in the original DNA sequence and if the ordered fragments are sized, this will produce a restriction map for the contig. This approach is considerably faster than existing methods for producing DNA contigs and restriction maps. If it is taken up, the ease and speed of this new technique should significantly hasten sequencing projects such as the Human Genome Project.