Critical Role of PDE4D in β2-Adrenoceptor-dependent cAMP Signaling in Mouse Embryonic Fibroblasts

Matthew D Bruss,Wito Richter,Kathleen Horner,S.-L Catherine Jin,Marco Conti

doi:10.1074/jbc.m803306200

Abstract

One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.

Highlights

By transducing the action of catecholamines into changes in intracellular cAMP levels, ␤-adrenergic receptors (␤ARs)2 play a critical role in cellular homeostasis [1, 2]
At higher concentrations of ligand, GRK-mediated phosphorylation of the occupied receptor increases the affinity of the receptor for the scaffold protein ␤-arrestin, which precludes the interaction with G proteins and promotes receptor internalization via binding to ADP-ribosylation factors (ARFs), dynamin, and clathrin-coated pits [5]
The ␤2AR is the predominant ␤-adrenergic receptor subtype expressed in mouse embryonic fibroblasts (MEFs), as cAMP elevation in response to ISO is blocked by a ␤2AR antagonist (ICI118551) but not by a ␤1AR antagonist (CGP20712A)

Summary

Introduction

By transducing the action of catecholamines into changes in intracellular cAMP levels, ␤-adrenergic receptors (␤ARs) play a critical role in cellular homeostasis [1, 2]. Occupancy of the ␤2AR causes rapid phosphorylation of the receptor by different kinases, with PKA- and GRK-mediated phosphorylation having been the most widely studied in terms of their effects. These phosphorylations directly modulate receptor coupling to G proteins and constitute a signal for the recruitment of additional proteins involved in receptor signaling and trafficking of the receptor in and out of the plasma membrane [3]. Regulation of ␤2-Adrenergic Signaling by PDE4D shaped by the presence of a large array of cyclic nucleotide phosphodiesterases (PDEs) (8 –10) These enzymes compose a superfamily of proteins that mediate cyclic nucleotide degradation. Most PDE genes are expressed as multiple variants through the use of different promoters or alternative splicing, generating up to 100 individual PDE proteins, which implies a complex array of functions for these enzymes

Methods

Results

Conclusion