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J. Biol. Chem., Vol. 281, Issue 12, 7919-7926, March 24, 2006

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Regulation of Neuroendocrine Exocytosis by the ARF6 GTPase-activating Protein GIT1^*

Maria Zeniou Meyer¹, Nadine Déliot¹, Sylvette Chasserot-Golaz, Richard T. Premont, Marie-France Bader, and Nicolas Vitale²

From the Department of Neurotransmission and Neuroendocrine Secretion, Institut des Neurosciences Cellulaires et Intégratives UMR-7168 CNRS/Université Louis Pasteur, 5 rue Blaise Pascal, 67084 Strasbourg, France and the Department of Medicine (Gastroenterology), Duke University Medical Center, Durham, North Carolina 27710

Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release.

Received for publication, January 4, 2006 , and in revised form, January 26, 2006.

^* This work was supported by Association de la Recherche sur le Cancer Grant 3208 (to M.-F. B.), Ministère de la Recherche Grant ACI BCMS 015 (to M.-F. B.), National Institutes of Health Grant R01 GM59989 (to R. T. P.), and Agence Nationale pour la Recherche Grant ANR-05-BLAN-0326-01 (to N. V.). 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.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 33-388-45-67-12; Fax: 33-388-60-16-64; E-mail: vitalen{at}neurochem.u-strasbg.fr.

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