scholarly journals Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Albrecht PIIPER ◽  
Danuta STRYJEK-KAMINSKA ◽  
Daria ILLENBERGER ◽  
Rolf KLENGEL ◽  
Jürgen M. SCHMIDT ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Synthetic Peptides ◽  
Basic Residue ◽  
Pharmacological Agents ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Domain Peptide

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-β1, -β3, -γ1 and -δ1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-β1) 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-β1 but not PLC-β3, -γ1 or -δ1. P1 also activated recombinant PLC-β1, indicating direct activation of PLC-β1 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) ≈ 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-β1 isoenzyme.

Effect of a Rab3A effector domain-related peptide, CCK, and EGF in permeabilized pancreatic acini

1994 ◽  
Vol 267 (3) ◽  
pp. G350-G356
Author(s):  
S. Zeuzem ◽  
D. Stryjek-Kaminska ◽  
W. F. Caspary ◽  
J. Stein ◽  
A. Piiper
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Small Molecular Weight ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Domain Peptide

We report here that a synthetic peptide of the effector domain of the small-molecular-weight GTP-binding protein Rab3A (EDRab3AL) is a potent stimulator of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase secretion in digitonin-permeabilized pancreatic acini. Moreover, the Rab3A effector domain peptide caused phosphatidylinositol 4,5-bisphosphate breakdown, indicating that the observed increase in Ins(1,4,5)P3 is due to stimulation of a phosphoinositide-specific phospholipase C (PLC). The dose-response curve for EDRab3AL-induced amylase release was biphasic, showing a maximum at 0.3 nM EDRab3AL and a decline at higher peptide concentrations. By contrast, the dose-response curve for EDRab3AL-induced Ins(1,4,5)P3 production was monophasic, showing stimulation with increasing EDRab3AL concentrations. A peptide of the effector domain of Rab1A, EDRab1AL, had no effect, indicating that the response to EDRab3AL is specific. Cholecystokinin octapeptide (CCK-8) and EDRab3AL had additive effects on the acinar Ins(1,4,5)P3 level. Epidermal growth factor (EGF), which has recently been shown to inhibit CCK-8-induced Ins(1,4,5)P3 production in pancreatic acinar cells, also decreased EDRab3AL-induced Ins(1,4,5)P3 production. These results suggest that EDRab3AL and CCK-8 act on the same EGF-inhibitable PLC by independent mechanisms. CCK-8 increased and EGF decreased amylase release in response to submaximal EDRab3AL concentrations. By contrast, at supramaximal EDRab3AL concentrations EGF increased and CCK-8 decreased EDRab3AL-stimulated amylase release. EDRab3AL had no effect in intact acini, indicating that the site of action of EDRab3AL is intracellular. We conclude that EDRab3AL regulates phosphoinositide-specific PLC activity and thereby amylase secretion in an analogous fashion to CCK-8, but from within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals 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

1995 ◽  
Vol 309 (2) ◽  
pp. 621-627 ◽  
Author(s):  
A Piiper ◽  
D Stryjek-Kaminska ◽  
R Jahn ◽  
S Zeuzem
Keyword(s):  
Pancreatic Acinar Cells ◽  
Maximal Response ◽  
Cross Linking ◽  
Amylase Secretion ◽  
Potential Candidate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Inositol 1,4,5 Trisphosphate ◽  
Domain Peptide

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.

Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling

1992 ◽  
Vol 263 (6) ◽  
pp. C1172-C1180 ◽  
Author(s):  
A. C. Wagner ◽  
M. J. Wishart ◽  
D. I. Yule ◽  
J. A. Williams
Keyword(s):  
Okadaic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Receptor Activation ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Late Step ◽  
Stimulus Secretion Coupling ◽  
The Right

Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 microM. Phosphatase 2B is significantly inhibited only at concentrations > 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 microM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 microM okadaic acid; and 3) Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of 32P-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.

scholarly journals Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

1992 ◽  
Vol 285 (2) ◽  
pp. 597-601 ◽  
Author(s):  
A J O'Sullivan ◽  
J D Jamieson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peptide Inhibitor ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Absolute Requirement ◽  
Messenger System

The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.

Tyrphostins inhibit secretagogue-induced 1,4,5-IP3 production and amylase release in pancreatic acini

1994 ◽  
Vol 266 (3) ◽  
pp. G363-G371
Author(s):  
A. Piiper ◽  
D. Stryjek-Kaminska ◽  
J. Stein ◽  
W. F. Caspary ◽  
S. Zeuzem
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Specific Cell ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cholecystokinin Octapeptide ◽  
Permeabilized Cells

We examined the role of protein tyrosine kinase inhibitors (tyrphostins) in secretagogue-induced inositol 1,4,5-trisphosphate (1,4,5-IP3) production and amylase secretion in rat pancreatic acinar cells. The data show that various specific cell-permeant tyrphostins (methyl 2,5-dihydroxycinnamate, tyrphostin 25, and genistein) inhibited the cholecystokinin octapeptide-, carbachol-, and bombesin-induced 1,4,5-IP3 production and amylase release. In digitonin-permeabilized cells, tyrphostins decreased 1,4,5-IP3 accumulation and amylase release generated by directly stimulating G proteins with the weakly hydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate). Tyrphostins had no effect on vasoactive intestinal peptide-induced amylase secretion. In isolated pancreatic acinar membranes, cholecystokinin octapeptide caused a rapid increase in tyrosine phosphorylation of a synthetic peptide containing the 12-amino acid sequence around a tyrosine phosphorylation site in pp6osrc. These results provide evidence that tyrosine kinases are involved in the activation of phospholipase C by G protein-coupled receptors in pancreatic acinar cells.

Actions of Rab3 effector domain peptides in chief cells from guinea pig stomach

1995 ◽  
Vol 269 (3) ◽  
pp. G400-G407 ◽  
Author(s):  
G. Singh ◽  
R. D. Raffaniello ◽  
J. Eng ◽  
J. P. Raufman
Keyword(s):  
Binding Proteins ◽  
Secretory Response ◽  
Effector Domain ◽  
Chief Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Pepsinogen Secretion ◽  
Guanine Nucleotide Binding ◽  
Domain Peptide ◽  
Guinea Pig Stomach

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca2+]) < 1 nM], a maximal concentration (15 microM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 microM Rab3AL peptide was combined with increasing calcium ([Ca2+] < 1 nM to 3 microM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 microM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Sign in / Sign up

Export Citation Format

Share Document