ADP-ribosylation factor6 regulates both [3H]-noradrenaline and [14C]-glutamate exocytosis through phosphatidylinositol 4,5-bisphosphate.
GTP phosphohydrolase (cell regulating) (EC 3.6.1.47, ADP-ribosylation factor6, ARF6) has been shown to play an important role in different steps of membrane trafficking. It also regulates chromaffin granule exocytosis through phosphatidylcholine phosphatidohydrolase (EC 3.1.4.14, PLD) activation. In this study, the role of ARF6 in neurotransmitter release from both dense-core granules (DCGs) and synaptic vesicles (SVs) in rat brain cortex nerve endings was investigated. We observed that synaptosomal ARF6 is largely particulate but moves to a less easily pelleted compartment upon nerve ending stimulation. We also found that direct inhibition of ARF6 by a specific antibody or interference with ARF6 downstream effects by a myristoylated N-terminal ARF6 peptide both significantly decreased both [3H]-noradrenaline and [14C]-glutamate exocytosis. Addition of phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2) partially or completely restored exocytosis. These findings suggest that ARF6 plays important regulatory roles for both DCG and SV exocytosis by activating PLD and ATP:1-phosphatidyl-1D-myo-inositol 4-phosphate 5-phosphotransferase (EC 2.7.1.68, PI4P-5K) to enhance PIP2 synthesis and nerve ending membrane trafficking.
Zheng
Qian
Q
Department of Chemistry, Texas Christian University, Fort Worth, TX 76129, USA.
Bobich
Joseph A
JA
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000187:drug effects*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D009411:Nerve Endings / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D019138:Antibodies, Blocking; D019269:Phosphatidylinositol 4,5-Diphosphate; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D018698:Glutamic Acid; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat; D002118:Calcium; D009638:Norepinephrine