Characterisation and analysis of PDE4A phosphodiesterase isoforms

Wilson, Moira Ann (1995) Characterisation and analysis of PDE4A phosphodiesterase isoforms. PhD thesis, University of Glasgow.

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Abstract

Studies were performed on a human cyclic AMP specific phosphodiesterase (PDE) of the PDE4A family. The cDNA for this isoform had been isolated from a human T-lymphocyte cDNA library and engineered for expression in Saccharomyces cerivisiae (Dr M.Sullivan, Sandoz). Characterisation of the PDE activity of h6.1 demonstrated that both pellet and soluble forms were kinetically identical, hydrolysing cyclic AMP specifically. Substrate hydrolysis was unaffected by low concentrations (2muM) of cyclic GMP or calcium / calmodulin but was selectively inhibited by the PDE4 class selective inhibitors rolipram and RO-20-1724. The PDE3 class inhibitor cilostimide and the non-selective inhibitor IBMX served as weak inhibitors. Both rolipram and IBMX were shown to inhibit pellet and soluble h6.1 activities in a simple competitive manner. EDTA-treated h6.1 could be dose- dependently activated by the divalent cations Mg2+ and Mn2+. This suggested that there was a site specific interaction between the divalent cations and h6.1. Pelleted h6-1 was not affected by NaCl or detergent treatment. Thus PDE activity was neither peripherally bound or an integral membrane protein. Indeed, the only difference found between pellet and soluble h6.1 was that the pellet activity was more thermostable. In each case, activity decayed as a single exponential, which suggested that only a single population of enzyme was expressed in each compartment. The function of the distinct N and C terminal domains of h6.1 were investigated. The N-terminal region of h6.1 was shown to be important in such functions as substrate hydrolysis, inhibitor interaction and thermostability. This analysis of a human PDE4A isoform (10AORF) which differed from h6.1 in having a larger N-terminal domain, showed that it exhibited a significantly lower Km for cyclic AMP (1.9+/-0.2muM, p=0.025) compared to h6.1 (4.1+1.7muM). In addition, 10AORF exhibited an IC50 (0.19+/-0.02muM, p=0.004) for rolipram which was considerably lower than that for h6.1(0.5+/-0.15muM). Importantly, it has been suggested that a conformational change, induced by the larger N-terminal region of 10AORF occurred, which resulted in a change in the way in which rolipram inhibited enzyme activity. Whilst rolipram was a simple competitive inhibitor of h6.1, partial competitive inhibition was seen with 10AORF. The enzyme 10AORF also showed increased thermostability with a t0.5 (>30 mins) significantly greater than that of h6.1 at 45°C (10mins). The role of the N-terminal region in modulating PDE properties was emphasised further upon the analysis of HYB1. The chimeric PDE, HYB1 was a construct formed by the substitution of the N-terminus of h6.1 with that of the inactive splice variant 2EL. Whilst this had no significant effect on the Kmcyclic AMP, it significantly increased the IC50 for rolipram (1.93+/-0.23muM) and reduced the thermostability of HYB1 (3 mins), compared to h6.1. Thus, the distinct N-terminal domain of 10AORF contributed to changing the potency of rolipram action, the Km for cyclic AMP hydrolysis and thermostability. The C-terminal domain was shown to be involved in the interaction with rolipram also, since the truncation of this region (YMS12) resulted in a significantly greater IC50 (1.7+/-0.14muM, p=0.0001) for rolipram inhibition compared to h6.1. The cellular expression of an analogous PDE4A rat isoform, RD1, was investigated in rat brain. The in vivo expression of the rat PDE4A splice variant, RD1 was studied in rat cerebellum synaptosomal fractions. Subfractionation of those synaptosomal fractions which contained the greatest amount of RD1 immunoreactivity, determined that RD1 was enriched in the plasma membrane fraction. The location of RD1 was identified with the plasma membrane markers 5'-nucleotidase and adenylyl cyclase. This has been previously shown in COS-1 cells transfected with RD1 cDNA (Shakur et al 1993). Location at the plasma membrane, along with adenylyl cyclase, places RD1 at a site alongside other components of the cyclic AMP signalling pathway, and provides a route for the termination of the signal generated by cyclic AMP.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Miles Houslay
Keywords: Biochemistry
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71682
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 17 May 2019 09:31
URI: http://theses.gla.ac.uk/id/eprint/71682

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