Piiper A, Stryjek-Kaminska D, Illenberger D, Klengel R, Schmidt J M, Gierschik P, Zeuzem S
II. Medical Department, University of Frankfurt, 60590 Frankfurt a. M., Federal Republic of Germany.
Biochem J. 1997 Sep 15;326 ( Pt 3)(Pt 3):669-74. doi: 10.1042/bj3260669.
We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-beta1, -beta3, -gamma1 and -delta1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-beta1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-beta1 but not PLC-beta3, -gamma1 or -delta1. P1 also activated recombinant PLC-beta1, indicating direct activation of PLC-beta1 by Rab3 effector-domain peptides. To investigate further the structure-function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue)[KVKTVYRH (EC50 of 1 nM to stimulate amylase release) approximately TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-beta1 isoenzyme.
我们最近发现，低分子量GTP结合蛋白Rab3效应结构域的合成肽可刺激多种通透细胞中肌醇1,4,5-三磷酸的产生。为了研究肽诱导的磷脂酶C（PLC）激活机制，并确定这些肽靶向的PLC同工酶，在检测对Rab3B/D效应结构域肽（VSTVGIDFKVKTVYRH，肽P1）的反应中PLC活性之前，将分离的胰腺腺泡膜和胞质溶胶与抗PLC抗体预孵育。蛋白质印迹分析显示膜和胞质部分中存在PLC-β1、-β3、-γ1和-δ1。P1刺激膜和胞质部分中的PLC活性。抗（PLC-β1）抗体几乎完全抑制了两个亚细胞部分中P1诱导的PLC活性。此外，P1诱导的洋地黄皂苷通透的胰腺腺泡中淀粉酶释放也受到抑制。其他免疫中和抗PLC抗体没有作用，这表明P1激活PLC-β1而不是PLC-β3、-γ1或-δ1。P1还激活了重组PLC-β1，表明Rab3效应结构域肽直接激活PLC-β1。为了进一步研究肽的结构-功能关系，测试了P1的截短肽在分离的胰腺腺泡膜中激活PLC以及刺激洋地黄皂苷通透的胰腺腺泡中淀粉酶释放的能力。含有BXBXXXB（B）基序（其中B代表碱性残基，X代表任何残基）[KVKTVYRH（刺激淀粉酶释放的EC50为1 nM）≈TVGIDFKVKTVYRH > TVGIDFKVKTVYR]的肽是淀粉酶释放和PLC活性的有效刺激剂，而C末端（VSTVGIDF）、两个C末端碱性氨基酸残基（VSTVGIDFKVKTVY和KVKTVY）的缺失或BXB基序的破坏（VKTVYR）导致肽无活性。总之，本研究结果表明，含有BXBXXXB基序的短肽是激活PLC-β1同工酶的有前景的药物制剂。
