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 neuroendocrine cells is the GTP-binding protein, Rab3A. Rab3A is thought to mediate exocytosis by an interaction of its effector domain with a putative effector protein. We demonstrate here that Rab3A effector domain peptides specifically stimulated insulin exocytosis in electroporated insulin-secreting cells (K0.5 activation, 6-8 microM) in a Ca(2+)-independent manner, although in the presence of Ca2+ insulin exocytosis was further potentiated. By using a 125I-radiolabeled photoactivated cross-linking Rab3A effector domain peptide, we identified a cytosolic protein doublet (REEP-1 and REEP-2), which specifically interacted with the Rab3A effector domain. Competitive inhibition studies revealed this protein-protein interaction to be at a concentration equivalent to that required for Rab3A effector domain peptides to trigger insulin exocytosis (Ki, 6-8 microM). Furthermore, under basal secretory conditions REEP-1 and -2 were membrane-associated, but upon stimulation of exocytosis they were released into a cytosolic fraction. Our results suggest that REEP-1 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 regulated exocytosis in pancreatic beta-cells.
E. P. Joslin Research Laboratory, Joslin Diabetes Center, Brigham and Women's Hospital, Boston, Massachusetts 02215.
S
S
Olszewski
J T
JT
Deeney
G T
GT
Schuppin
K P
KP
Williams
B E
BE
Corkey
C J
CJ
Rhodes
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D018274:Electroporation; D005089:Exocytosis / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D020692:rab3 GTP-Binding Proteins
D007328:Insulin; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins