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    Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene
    (Portland Press Ltd, 1997) Bolger, Graeme B.; Erdogan, Suat; Jones, Randy E.; Loughney, Kate; Scotland, Grant; Hoffmann, Ralf; Wilkinson, Ian
    We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IV PDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 ?M), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 ?M respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.
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    UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions
    (2000) Beard, Matthew B.; Olsen, Aileen E.; Jones, Randy E.; Erdogan, Suat; Houslay, Miles D.; Bolger, Graeme B.
    The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique 'signature' regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR1 and UCR2 are located between the extreme amino- terminal region and the catalytic region of the PDE4 enzymes. The UCR1 of the PDE4D3 isoform was used as a 'bait' in a two-hybrid screen, which identified a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and 'pull down' analysis of constructs incorporating various regions of the PDE4D3 cDNA demonstrated that the carboxyl-terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2. The interaction was blocked by mutations of two positively charged amino acids (Arg-98 and Arg-101 to alanine) located within an otherwise largely hydrophobic region of UCR1. Mutation of three negatively charged amino acids in UCR2 (Glu-146, Glu-147, and Asp-149, all to alanine) also blocked the interaction. The phosphorylation of UCR1 by cAMP-dependent protein kinase (PKA) in vitro attenuated the ability of UCR1 to interact with UCR2. Mutation of the PKA substrate site in UCR1 (Ser-54) to aspartic acid, which mimics the activation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 and UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic interactions and which may be disrupted by PKA phosphorylation. We suggest that the UCR1 and UCR2 domains may form a module that interacts with and regulates the PDE4 catalytic region.