Trimeric G Proteins Control Exocytosis in Chromaffin Cells: Go REGULATES THE PERIPHERAL ACTIN NETWORK AND CATECHOLAMINE SECRETION BY A MECHANISM INVOLVING THE SMALL GTP-BINDING PROTEIN Rho

Stéphane Gasman,Sylvette Chasserot-Golaz,Michel R Popoff,Dominique Aunis,Marie-France Bader

doi:10.1074/jbc.272.33.20564

Abstract

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles and its activation by mastoparan inhibits the ATP-dependent priming of exocytosis. The effectors by which Go controls exocytosis are currently unknown. The subplasmalemmal actin network is one candidate, since it modulates secretion by controlling the movement of secretory granules to the plasma membrane. In streptolysin-O-permeabilized chromaffin cells, activation of exocytosis produces disassembly of cortical actin filaments. Mastoparan blocks the calcium-evoked disruption of cortical actin, and this effect is specifically inhibited by antibodies against Galphao and by a synthetic peptide corresponding to the COOH-terminal domain of Galphao. Disruption of actin filaments with cytochalasin E and Clostridium perfringens iota toxin partially reverses the mastoparan-induced inhibition of secretion. Furthermore, the effects of mastoparan on cortical actin and exocytosis are greatly reduced in cells treated with Clostridium botulinum C3 exoenzyme, which specifically inactivates the small G protein Rho. We propose that the control exerted by the granule-associated Go on exocytosis may be related to effects on the cortical actin network through a sequence of events which eventually involves the participation of Rho.

Highlights

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events
Studies on diverse secretory cell types have highlighted the potential roles of heterotrimeric G proteins in intracellular membrane trafficking events [1,2,3]. a and bg subunits of Gi and Go proteins have been found associated with the membrane of secretory granules in various neuroendocrine cells (4 – 6), suggesting a role in Ca21-regulated exocytosis
In permeabilized cells incubated in Ca21free medium (Fig. 1A), rhodamine-phalloidin fluorescence was most intense at the cell periphery forming a continuous and homogeneous cortical ring, in agreement with the fact that in chromaffin cells the majority of actin filaments are concentrated in the subplasmalemmal region (14 –16)

Summary

Introduction

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In streptolysin-O-permeabilized chromaffin cells, activation of exocytosis produces disassembly of cortical actin filaments. The aim of the present work was to assess whether the cortical actin network represents a possible effector by which the granule-bound Go controls the exocytotic pathway in chromaffin cells. Using streptolysin-O (SLO)1-permeabilized cells, we show that the introduction of mastoparan into the cytosol inhibits the disruption of the subplasmalemmal actin network in calcium-stimulated cells. This effect can be selectively reversed by affinity-purified antibodies prepared against Gao and by a syn-. The mastoparan-induced inhibition of secretion can be partially reversed by agents known to affect the assembly of actin and by the Clostridium botulinum C3 ADP-

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