RAB3A EFFECTOR DOMAIN PEPTIDES INDUCE INSULIN EXOCYTOSIS VIA A SPECIFIC INTERACTION WITH A CYTOSOLIC PROTEIN DOUBLET

A key protein involved in the regulated exocytotic mechanism in neuroe ndocrine cells is the GTP-binding protein, Rab3A. Rab3A is thought to mediate exocytosis by an interaction of its effector domain with a put ative effector protein. we demonstrate here that Rab3A effector domain peptides specifically stimulated insulin exocytosis in electroporated insulin-secreting cells (K-0.5 activation, 6-8 mu M) in a Ca2+-indepe ndent manner, although in the presence of Ca2+ insulin exocytosis was further potentiated. By using a I-125-radiolabeled photoactivated cros s-linking Rab3A effector domain peptide, we identified a cytosolic pro tein doublet (REEF-1 and REEP-2), which specifically interacted with t he Rab3A effector domain. Competitive inhibition studies revealed this protein-protein interaction to be at a concentration equivalent to th at required for Rab3A effector domain peptides to trigger insulin exoc ytosis (K-i, 6-8 mu M) Furthermore, under basal secretory conditions R EEF-1 and -2 were membrane associated, but upon stimulation of exocyto sis they were released into a cytosolic fraction. Our results suggest that REEF-I and -2 are part of the regulated exocytotic machinery, and their dissociation upon stimulation of hormone release (likely from a protein complex) may be essential to the mechanism that triggers regu lated exocytosis in pancreatic beta-cells.