Cell-permeable Non-hydrolyzable cAMP Derivatives as Tools for Analysis of Signaling Pathways Controlling Gene Regulation in Dictyostelium

Abstract

A novel class of cAMP derivatives were tested for binding to surface cAMP receptors (CAR), protein kinase A (PKA), and cAMP-phosphodiesterase (PDE) and for induction of three classes of cAMP regulated genes in Dictyostelium discoideum. These derivatives carry sulfur substitutions for either the axial (Sp) or equatorial (Rp) exocyclic oxygen atoms, while further modifications were introduced to provide specificity for binding to either CAR or PKA, and/or to increase lipophilicity and render the derivatives membrane-permeable. All derivatives bind weakly to PDE and are almost not degraded during incubation with Dictyostelium cells. One cAMP derivative, 6-thioethylpurineriboside 3',5‘-monophosphorothioate, Sp-isomer (Sp-6SEtcPuMPS), fulfills the criteria for selective activation of PKA in vivo. The compound enters Dictyostelium cells and reaches an intracellular concentration of 1 µM, sufficient to activate PKA, at an extracellular concentration of 30 µM, which is insufficient to activate CAR. Expression of cAMP-regulated prespore and prestalk genes and the aggregative PDE gene are effectively induced by CAR agonists and very poorly by PKA agonists. Even Sp-6SEtcPuMPS is ineffective to induce gene expression. These data not only indicate that surface cAMP receptors are the first targets for cAMP-induced gene expression, but argue against direct induction of expression of these genes by cAMP-induced PKA activation.