A synthetic peptide of the N‐terminus of ADP‐ribosylation factor (ARF) inhibits regulated exocytosis in adrenal chromaffin cells

Abstract

We have investigated the role of ADP-~bosylation factor (ARF) in regulated exocytosis in di~tonin-~~eabil~ed adrenal chroma& cells by the use of a synthetic peptide, hARFl(Z-17), based on the N-terminus of the protein hARF1(2-17) inhibited &+-dependent but not basal exocytosis, whereas equimoiar leveis of other synthetic peptides were ineffective. The inhibitory effect of hARFl(Z-17) was dose-dependent and half-maximal at 12 ,uM. GTPyS-induced secretion in the presence of non-stimulatory CA*+ concentrations was also inhibited by hARF1(2-17).These results point to a hitherto unsuspected role for ARF in regulated exocytosis, and the potency of the hARF1(2-17) peptide suggests that ARF is essential for exocytosis in bovine adrenal chromafhn cells,

GTP-Binding Proteins and Regulated Exocytosis

“…To date, studies that address ARF function have been few. In chromaffin cells, SG-associated ARF has been implicated in exocytosis on the basis that ARF peptide inhibits calcium-dependent secretion (Morgan and Burgoyne, 1993;Galas et al, 1997) and GTPyS-stimulated PLD activity and exocytosis (Fensome et al, 1996). An ARF-like protein, ARL 184, was also shown to potentiate acetylcholine release in stably transfected PC12 cells (Icard-Liepkalns et al, 1997).…”

“…Important studies in this regard were reported by Vitale et al (1993) (Oberhauser et al, 1992) and pancreatRapl ic acini (Piiper et al, 1995), and in chromaffin cells (Senyshyn et al, 1992). Also, in permeabilized chromaffin cells, the synthetic peptide hARF (2-17) (Morgan and Burgoyne, 1993) and myristoylated and nonmyristoylated ARF6 (2-13), synthetic peptides corresponding to residues 2-13 of ARF6, produced a dose-dependent inhibition of calcium-dependent catecholamine release (Galas et al, 1997). In some studies, due to reported non-specific effects of peptides, the validity of the data obtained has been debated.…”

Regulated Exocytosis in Chromaffin Cells

“…MyrARF6 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) produced a dose-dependent inhibition in Ca 2ϩ -dependent catecholamine secretion with a mean inhibitory dose, ID 50 , of approximately 40 M (Fig. 3).…”

“…For example, rab3 isoforms which are specifically localized on the membrane of secretory vesicles (7,8), have been proposed to serve as a regulator of exocytosis in chromaffin cells (9) and in anterior pituitary cells (10). Other investigations have postulated the participation of ARF in exocytosis (11,12).…”

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

“…Specific members of this superfamily have been considered as mediators of these GTP effects on exocytosis, including heterotrimeric G proteins (5-9) and low molecular weight ras-like proteins such as Rab (10)(11)(12)(13)(14)(15) and ARF (16). The current "fusion machine" hypothesis (17)(18)(19) envisions a core complex formed between plasma membrane syntaxin and SNAP-25 and the synaptic vesicle protein synaptobrevin/VAMP (20), with vesicular synaptotagmin putatively identified as a low-affinity calcium sensor that interacts with regulatory syntaxin 1 (21,22).…”