Date of Award


Degree Type


Degree Name

Doctor of Philosophy


A potential role for cAMP in regulating the differentiation of myoblasts has lead to the examination of the components of the cAMP signalling system, in this case a cyclic nucleotide phosphodiesterase (PDE). A Type IV PDE isozyme was identified in L6 myoblasts using consensus sequence PCR primers, and its regulation by cAMP studied. Sequencing of RT-PCR products showed the presence of a Type IV PDE mRNA known as ratPDE 3.1. High levels of cAMP induced dramatic increases in the PDE mRNA levels as demonstrated by northern blot analysis. Run-off transcription experiments showed increased rates of transcription. L6 myoblasts which had been transfected with the cDNA for a mutant protein kinase A (PKA) regulatory subunit were used to look at the role of PKA. The reduction of PKA activity caused by the mutant prevented both the cAMP-induced increase in PDE mRNA levels and PDE activity. Hence ratPDE 3.1 transcription is regulated by cAMP via PKA.;To study the PDE itself, a full length cDNA and several truncated forms were subcloned into the bacterial expression vector pGEX-KG. The PDE was expressed and purified with yields of 0.8-2 mg/l. The purified PDE was active, inhibited by both Type IV specific inhibitors (RO 20-1724 and rolipram) and the general PDE inhibitor MIX, and had normal kinetic characteristics for a PDE of the Type IV subfamily. The enzyme had different cation requirements than those previously reported for Type V PDE; Zn{dollar}\sp{lcub}2+{rcub}{dollar} had no effect on activity. Thus the type IV PDE is not a zinc hydrolase as has been suggested for the Type V PDE.;Structure-function relationships of conserved domains within the enzyme were studied using expressed truncations of the C- and N-terminal regions. Two areas of the enzyme which are distinct from the catalytic domain and and responsible for (a) the regulation of PDE activity, and, (b) the quaternary structure have been identified. Possible regulation through phosphorylation was also examined using the purified PDE. Phosphorylation of ratPDE 3 by protein kinase C, p34{dollar}\sp{lcub}\rm cdc2{rcub}{dollar} and casein kinase II was demonstrated, but had no affect on PDE activity.;These approaches allow the construction of a model for the regulation of the PDE enzyme and provide the basis for detailed molecular analysis.



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