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Originally published In Press as doi:10.1074/jbc.M202446200 on December 2, 2002

J. Biol. Chem., Vol. 278, Issue 6, 3606-3614, February 7, 2003
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Cyclic AMP-independent Involvement of Rap1/B-Raf in the Angiotensin II AT2 Receptor Signaling Pathway in NG108-15 Cells*

Louis GendronDagger §||, Jean-François OlignyDagger ||, Marcel Daniel Payet§, and Nicole Gallo-PayetDagger §**¶¶

From the Dagger  Service of Endocrinology and the § Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

The angiotensin II (Ang II) type 2 (AT2) receptor is an atypical seven-transmembrane domain receptor. Controversy surrounding this receptor concerns both the nature of the second messengers produced as well as its associated signaling mechanisms. Using the neuronal cell line NG108-15, we have reported previously that activation of the AT2 receptor induced morphological differentiation in a p21ras-independent, but p42/p44mapk-dependent mechanism. The activation of p42/p44mapk was delayed, sustained, and had been shown to be essential for neurite elongation. In the present report, we demonstrate that activation of the AT2 receptor rapidly, but transiently, activated the Rap1/B-Raf complex of signaling proteins. In RapN17- and Rap1GAP-transfected cells, the effects induced by Ang II were abolished, demonstrating that activation of these proteins was responsible for the observed p42/p44mapk phosphorylation and for morphological differentiation. To assess whether cAMP was involved in the activation of Rap1/B-Raf and neuronal differentiation induced by Ang II, NG108-15 cells were treated with stimulators or inhibitors of the cAMP pathway. We found that dibutyryl cAMP and forskolin did not stimulate Rap1 or p42/p44mapk activity. Furthermore, adding H-89, an inhibitor of protein kinase A, or Rp-8-Br-cAMP-S, an inactive cAMP analog, failed to impair p42/p44mapk activity and neurite outgrowth induced by Ang II. The present observations clearly indicate that cAMP, a well known stimulus of neuronal differentiation, did not participate in the AT2 receptor signaling pathways in the NG108-15 cells. Therefore, the AT2 receptor of Ang II activates the signaling modules of Rap1/B-Raf and p42/p44mapk via a cAMP-independent pathway to induce morphological differentiation of NG108-15 cells.

* This work was supported in part by grants from the Canadian Institute for Heath Research (to N. G.-P. and M. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a studentship from the Fonds de la recherche en santé du Québec.

|| These authors contributed equally to this work.

** To whom correspondence should be addressed: Service of Endocrinology, Faculty of Medicine, University of Sherbrooke, 3001 12th Ave., Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5243; Fax: 819-564-5292; E-mail: Nicole.Gallo_Payet@USherbrooke.ca.

¶¶ Holder of the Canadian Research Chair in endocrinology of the adrenal gland.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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