Regulation of a Drosophila cGMP-PDE by prenylation and interaction with a prenyl-binding protein
Résumé
Post-translational modification by isoprenylation is a pivotal process for the correct functioning of many signalling proteins. The Drosophila melanogaster cGMP-specific phosphodiesterase, DmPDE5/6, possesses a CaaX-box prenylation signal-motif, as do several novel cGMP-PDEs from insect and echinoid species. DmPDE5/6 is prenylated in vivo at Cys1128 and is localised to the plasma membrane when expressed in Drosophila S2 cells. Site-directed mutagenesis of the prenylated cysteine residue (C1128S-DmPDE5/6), pharmacological inhibition of prenylation or co-expression of the Drosophila prenyl-binding protein, DmPrBP/δ, each alters the sub-cellular localisation of DmPDE5/6. Thus prenylation constitutes a critical post-translational modification of DmPDE5/6 for membrane-targeting. Co-immunoprecipitation and sub-cellular fractionation experiments show that DmPDE5/6 interacts with DmPrBP/δ in Drosophila S2 cells. Transgenic lines allow targeted expression of tagged, prenylation deficient C1128S-DmPDE5/6 to Type I (principal) cells in Drosophila Malpighian tubules, an in vivo model for DmPDE5/6 function. In contrast to wild-type DmPDE5/6, which was exclusively associated with the apical membrane, the C1128S-DmPDE5/6 mutant form was located primarily in the cytosol, although some residual association occurred at the apical membrane. Despite the profound change in intracellular localisation of C1128S-DmPDE5/6, active transport of cGMP is affected as by DmPDE5/6. This suggests that in addition to prenylation and interaction with DmPrBP/δ, further functional membrane-targeting signals exist within DmPDE5/6.
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