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-thioethyl-purineriboside 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 microM, sufficient to activate PKA, at an extracellular concentration of 30 microM, 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.
Highlights
Cell-permeable Non-hydrolyzableCAMPDerivatives as Tools for Analysis of Signaling Pathways ControllingGene Regulationin Diety ~ ~ t e ~ ~ ~ ~ ~
A novel class of CAMPderivatives were tested for protein (G-protein)’ @-subunit( G d ), and CAMP-phOsphobinding to surfacecAMP receptors (CAR),protein ki- diesterase (PDE) (Darmon et aL, 1975; Gerisch et al, 1975; nase A (PKA), and CAMP-phosphodiesterase (PDE) Lacombe et al, 1986; Noegel et al, 1986; Klein et al, 1988; and for induction of three classes of cAMP regulated Kumagai e t aL,1989).Expression of PDE can be genes in Dictyostelium diseoideum.These derivatives induced by a constant cAMP stimulus (Yeh et al, 1978).After carry sulfur substitutions for either the axial (Sp) or aggregation, persistent stimulation with micromolar cAMP
Rationale for the Selection of CAMPDerivatives-From the Sp-5'NHcAMPS requires only 10-fold higher conrecentlysynthesizedadenosine 3'5'-monophosphorothioate centrations than CAMP, butis as slowly hydroderivatives (Jastorff and Krebs1, 972; Dostmann, 1987; Gen- lyzed as the other derivatives (Fig. 2)
Summary
Rationale for the Selection of CAMPDerivatives-From the Sp-5'NHcAMPS requires only 10-fold higher conrecentlysynthesizedadenosine 3'5'-monophosphorothioate centrations than CAMP, butis as slowly hydroderivatives (Jastorff and Krebs 972; Dostmann, 1987; Gen- lyzed as the other derivatives (Fig. 2) This emphasizes that ieser et al, 1988), we chose a subset of compounds which were the stability of phosphorothioates for degradation by PDE is aimed t o discriminate between CAR and PKA due to their low affinity for binding to theenzyme, mediated responses antdo enter thecell and activate PKA in and to theirlow intrinsic hydrolysis rate Sp and Rpisomers carry the sulfur replacementof the exo- Binding of CAMP-phosphorothiates to CAR, PKA, and cyclic oxygen at, respectively, the axial and equatorial posi- PDE-Fig. 3shows the inhibitiobny non-hydrolyzablecAMP tions. 5'-oxygen and 2'-hydroxy positions act as a hydrogen bond affinities for CAR, PKA, and PDE, respectiveiy, 300-, 170-, Gene Regulationin Dictyostelium bycAMP Phosphorothioates
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
