Regulated Exocytosis in Chromaffin Cells

doi:10.1074/jbc.272.5.2788

Regulated Exocytosis in Chromaffin Cells

A POTENTIAL ROLE FOR A SECRETORY GRANULE-ASSOCIATED ARF6 PROTEIN*

From the Institut National de la Santé et de la Recherche Médicale, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France, and
^§ Biochemie I, Universitaet Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

^‡ To who correspondence should be addressed. Tel.: 33 3 88 45 67 13; Fax: 33 3 88 60 08 06; E-mail: bader{at}neurochem.u-strasbg.fr

Abstract

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with βγ subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF₄⁻ which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

Footnotes

↵^¶ Supported by the Université Louis Pasteur.
↵* This study was supported by INSERM (“Equipements lourds”), by the Fondation pour la Recherche Médicale, and by the Association de la Recherche contre le Cancer. 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.
↵¹ The abbreviations used are:

ARF

ADP-ribosylation factor

SLO

streptolysin-O

ELISA

enzyme-linked immunosorbent assay

PAGE

polyacrylamide gel electrophoresis

GTPγS

guanosine 5′-3-O-(thio)triphosphate

myr

myristoylated.
- Received July 31, 1996.
- Revision received November 11, 1996.