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J. Biol. Chem., Vol. 283, Issue 33, 22430-22442, August 15, 2008

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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, and Marco Conti¹

From the Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143 and the Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305

One of the defining properties of β₂-adrenergic receptor (β₂AR) 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 β-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 β₂AR 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 β₂AR desensitization as well as the interaction of the receptor with G_i. These findings demonstrate that PDE4D plays a major role in shaping the β₂AR signal.

Received for publication, September 10, 2007 , and in revised form, April 30, 2008.

Note Added in Proof—During the revision of this manuscript, a study consistent with a critical role of PDE in β₂-adrenergic signaling was published (Violin, J. D., DiPilato, L. M., Yildirim, N., Elston, T. C., Zhang, J., and Lefkowitz, R. J. (2008) J. Biol. Chem. 283, 2949–2961).

^* This work was supported, in whole or in part, by National Institutes of Health Grant HD20788 (to M. C.). This work was also supported by a grant from the Leducq Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: Dept. of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, 513 Parnassus Ave., HSW 1656, Box 0556, San Francisco, CA 94143-0556. Tel.: 415-476-9214; Fax: 415-502-7866; E-mail: contim{at}obgyn.ucsf.edu.

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