The intracellular targeting and regulation of PDE4 cyclic AMP-specific phosphodiesterase enzymes

Cyclic adenosine 5': 3' monophosphate (cAMP) is a ubiquitous second messenger in cells which transduces the action of a wide variety of hormones and neurotransmitters. cAMP phosphodiesterase (PDE) enzymes provide the sole route for the degradation of cAMP and are thus placed in a key position for regulating cAMP-dependent processes. The cAMP-specific PDE4 enzyme family are encoded by four genes (PDE4A, -B, -C and -D) which each generate a number of isoenzymes by alternative 5' mRNA splicing. This process gives rise to PDE4 isoenzymes with unique extreme NH2-terminal splice regions, one or two upstream conserved regions (UCR1AJCR2) and a central to COOH-terminal catalytic unit. The extreme NH2-terminal splice domains confer distinct modes of regulation and intracellular targeting on the PDE4 isoenzymes. Understanding the distinct physiological roles of individual PDE4 isoenzymes may aid the development of more specific PDE4 inhibitor-based drugs for therapeutic use in a number of disease states. I have assessed the involvement of PDE4 isoenzymes in the cAMP-dependent effect of growth hormone (GH) on the differentiation of mouse 3T3 F442A preadipocyte cells (Chapter 3). In Chapter 4 of this thesis I have analysed the intracellular targeting of the rat PDE4A isoenzymes RNPDE4A1 (RDl) and RNPDE4A5 (rpde6). I have used chimeric constructs to show that the unique NH2-terminal regions of RDl (amino acids 1-100) and rpde6 (amino acids 1-256) can confer membrane association on the normally soluble protein giutathione-5'-transferase (GST). Furthermore, the region between amino acids 103 and 256 on rpde6, which is shared with the other long PDE4A splice variant rpde39 (RNPDE4A6), is sufficient for membrane binding. The NH2-terminal regions of RDl and rpde6 bind to components of the P2-membrane fraction that have different thermal stabilities suggesting that they interact with distinct lipid or protein anchoring molecules. I have further investigated the protein-protein interactions of rpde6 (Chapter 5). Evidence was obtained from yeast-2-hybrid studies that rpde6 might interact with a novel immunophilin-like protein, RB3. I confirmed this interaction in vitro, by showing that a purified GST-fusion of RB3 (but not GST) could pull down ipde6 from the cytosolic fraction of transfected COS-7 cells. Subcellular distribution analysis of transfected COS-7 cells showed that RB3 was distributed between the PI low-speed pellet fraction (-16%) and the cytosolic S fraction and was absent from the high speed P2-membrane fraction, in contrast to rpde6 which was present in all three fractions. This suggests that the in vivo interaction between RB3 and rpde6 may be limited to particular subcellular compartments. The association of GST-RB3 had a marked inhibitory effect on the catalytic activity of cytosolic rpde6 (IC50 ~0.2? M), which was also seen with a maltose binding protein (MBP)-tagged RB3, but not with GST or MBP alone. This inhibitory effect was also seen with a GST fusion of the FK506 binding protein FKBP52, the closest homologue of RB3. In contrast to cytosolic rpde6, particulate (PI and P2) fraction ipde6 was resistant to inhibition by RB3. I have shown that the COOH-terminal half of RB3 (amino acids 170-330) which contains a 34-amino acid tetratricopeptide repeat (TPR) motif in its COOH-terminal region mediates both the binding and inhibition of cytosolic rpde6.I have mapped the RB3 binding site on rpde6 to amino acids 218-259, which corresponds to the NH2-terminal half of UCR2. The NH2-terminal region of rpde6 has previously been shown to interact with the SH3 domains of Src-family tyrosyl kinases such as Lyn. I have shown that that Lyn SH3 does compete with RB3 for interaction with rpde6. The above studies suggest that the association of RB3 or a similar TPR-containing protein may represent a new mechanism for the regulation and/or intracellular targeting of rpde6 in vivo. Alternatively, this interaction may provide a means for the specific regulation of RB3 function by rpde6. (Abstract shortened by ProQuest.).