STIMULATION OF INOSITOL 1,4,5-TRISPHOSPHATE PRODUCTION BY PEPTIDES CORRESPONDING TO THE EFFECTOR DOMAIN OF DIFFERENT RAB3 ISOFORMS AND CROSS-LINKING OF AN EFFECTOR DOMAIN PEPTIDE TARGET

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3A(AL), stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P-3 production and amylase secretion with an order of potency Rab3B/D > Rab3A(AL) > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC(50)) were 3, 0.2 and 3 nM for Ins(1,4,5)P-3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1A(AL), and a scrambled peptide of Rab3A(AL) were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P-3 production and amylase release in intact acini. Cross-linking of I-125-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3A(AL) had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B- or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to I-125-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.