Piiper A, Stryjek-Kaminska D, Jahn R, Zeuzem S
Department of Internal Medicine, University of Frankfurt, a. M., Germany.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):621-7. doi: 10.1042/bj3090621.
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, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 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)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL 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)P3 production and amylase release in intact acini. Cross-linking of 125I-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 Rab3AAL 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 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.
Rab3蛋白定位于分泌小泡上，似乎参与调节性胞吐作用。我们之前已经表明，一种对应于小分子质量GTP结合蛋白Rab3A效应结构域的修饰肽Rab3AAL，可刺激洋地黄皂苷通透的胰腺腺泡中肌醇1,4,5-三磷酸[Ins(1,4,5)P3]的产生和淀粉酶的释放。使用单克隆抗体进行的实验表明，胰腺腺泡中存在的Rab3样蛋白不是Rab3A亚型。然而，由于四种已知的Rab3蛋白（A、B、C和D）的假定效应结构域仅在C末端的四个氨基酸残基上有所不同，Rab3A效应结构域肽可能模拟胰腺特异性Rab3亚型的作用。在本研究中，我们报告对应于不同Rab3亚型的肽刺激Ins(1,4,5)P3的产生和淀粉酶分泌的效力顺序为Rab3B/D > Rab3AAL > Rab3A = Rab3C。对于Rab3A、B/D和C效应结构域肽，引起半数最大反应（EC50）的浓度，对于Ins(1,4,5)P3积累分别为3、0.2和3 nM，对于淀粉酶释放分别为0.3、0.02和0.3 nM。与天然Rab3效应结构域肽相比，Rab1A效应结构域肽Rab1AAL和Rab3AAL的乱序肽在引发这些反应时效力低几个数量级。这些肽均不影响完整腺泡中Ins(1,4,5)P3的产生和淀粉酶的释放。125I-Rab3B/D肽与胰腺腺泡膜的交联显示在70至75 kDa处有一条带，最大强度在75 kDa。底物的放射性标记可被未标记的Rab3B/D肽取代，并在较小程度上被Rab3A肽取代，而Rab3AAL的乱序肽则没有作用。这些数据表明，磷脂酶C和胞吐作用可能受胰腺腺泡细胞中Rab3B或Rab3D样蛋白的调节。一种优先与125I-Rab3B/D效应结构域肽交联的75 kDa蛋白，可能是Rab3效应结构域肽的效应蛋白的潜在候选者。
