Short Term Feedback Regulation of cAMP in FRTL-5 Thyroid Cells

Abstract

Hormone or neurotransmitter signaling is mediated by transient fluctuations in intracellular cAMP within a very narrow range of concentrations (1). Maximal biological effects are elicited with only 2-3-fold changes in intracellular cAMP levels, while the cell potential for cAMP production is usually much larger. In addition, the increase in intracellular cAMP is transient despite the continuous presence of the extracellular stimulus (2, 3). This limited and short-lived nature of the activating signal is an essential feature of hormone or neurotransmitter action. This is necessary to decrease the intrinsic "noise" in the signaling mechanism, to allow iterative signaling, and to prevent excessive stimulation.The rapid and transient changes in cAMP concentrations are the result of changes in both synthesis and degradation of the second messenger cAMP, involving steps at the receptor as well as at a postreceptor level (4, 5). Following the activation of G s and adenylyl cyclase, receptor phosphorylation causes an uncoupling from G s , and therefore a decrease in cAMP synthesis. For the ␤ 2 -adrenergic receptor, ␤ 2 -adrenergic receptor kinases phosphorylate only agonist-occupied active receptors and enhance the affinity of the receptors for the inhibitor protein ␤-arrestin (6). Binding of ␤-arrestin to the phosphorylated receptors inhibits the receptor-G s interaction, thereby inducing the uncoupled or desensitized state of the receptors (7). Several kinases have also been implicated in the phosphorylation of the glycoprotein receptors including the GRK kinases, PKA 1 and PKC. While PKA efficiently phosphorylates and uncouples the ␤-adrenergic receptor (5), the involvement of this kinase in the glycoprotein receptor phosphorylation is less clear (8).In addition to receptor uncoupling from G protein and cyclase, rapid modulation of phosphodiesterases and of cAMP degradation plays an essential role in the transient accumulation of cyclic nucleotides (9). This concept was initially inferred by the use of xanthine inhibitors of PDEs and by measuring the decay of the cAMP signal in intact cells (3). The use of cAMP analogs has confirmed that cAMP-dependent PKA activation in the cell causes an activation of cAMP degradation (10 -12). The impact and physiological significance of this rapid feedback regulation is unclear.Of the many PDEs expressed in the cell, two isoforms are activated by an increase in cAMP. In platelets (13,14) and adipocytes (15, 16) a type 3 PDE is activated by a PKA-dependent phosphorylation lowering cAMP levels. Recently, a distinct PDE isoenzyme, a member of the PDE4 family, has been implicated in this feedback regulation (17). In thyroid cells, TSH causes a PKA-mediated phosphorylation and activation of a PDE4D3 variant. This conclusion is supported by studies involving PDE4-specific inhibitors and immunoprecipitation with PDE4-selective antibodies (17), and is consistent with cell-free phosphorylation and activation of the recombinant PDE4D3 enzymes (18). The site of PKA phosphorylation of PDE4D3 ...

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