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. 1992 Mar 1;89(5):1656–1660. doi: 10.1073/pnas.89.5.1656

A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

P J Padfield 1, W E Balch 1, J D Jamieson 1
PMCID: PMC48511  PMID: 1371881

Abstract

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.

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Selected References

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  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Bacon R. A., Salminen A., Ruohola H., Novick P., Ferro-Novick S. The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol. 1989 Sep;109(3):1015–1022. doi: 10.1083/jcb.109.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker D., Wuestehube L., Schekman R., Botstein D., Segev N. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):355–359. doi: 10.1073/pnas.87.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  5. Barrowman M. M., Cockcroft S., Gomperts B. D. Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature. 1986 Feb 6;319(6053):504–507. doi: 10.1038/319504a0. [DOI] [PubMed] [Google Scholar]
  6. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  7. Bruzzone R., Halban P. A., Gjinovci A., Trimble E. R. A new, rapid, method for preparation of dispersed pancreatic acini. Biochem J. 1985 Mar 1;226(2):621–624. doi: 10.1042/bj2260621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burgoyne R. D., Morgan A. Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule. FEBS Lett. 1989 Mar 13;245(1-2):122–126. doi: 10.1016/0014-5793(89)80204-2. [DOI] [PubMed] [Google Scholar]
  9. Calés C., Hancock J. F., Marshall C. J., Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  10. Cockcroft S., Howell T. W., Gomperts B. D. Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis. J Cell Biol. 1987 Dec;105(6 Pt 1):2745–2750. doi: 10.1083/jcb.105.6.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dexter D., Rubins J. B., Manning E. C., Khachatrian L., Dickey B. F. Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules. J Immunol. 1990 Sep 15;145(6):1845–1850. [PubMed] [Google Scholar]
  12. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  13. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fischer von Mollard G., Südhof T. C., Jahn R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 1991 Jan 3;349(6304):79–81. doi: 10.1038/349079a0. [DOI] [PubMed] [Google Scholar]
  15. Gibbs J. B., Marshall M. S. The ras oncogene--an important regulatory element in lower eucaryotic organisms. Microbiol Rev. 1989 Jun;53(2):171–185. doi: 10.1128/mr.53.2.171-185.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gomperts B. D. GE: a GTP-binding protein mediating exocytosis. Annu Rev Physiol. 1990;52:591–606. doi: 10.1146/annurev.ph.52.030190.003111. [DOI] [PubMed] [Google Scholar]
  17. Gomperts B. D., Tatham P. E. GTP-binding proteins in the control of exocytosis. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):983–992. doi: 10.1101/sqb.1988.053.01.113. [DOI] [PubMed] [Google Scholar]
  18. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  19. Hersey S. J., Perez A. Permeable cell models in stimulus-secretion coupling. Annu Rev Physiol. 1990;52:345–361. doi: 10.1146/annurev.ph.52.030190.002021. [DOI] [PubMed] [Google Scholar]
  20. Kitagawa M., Williams J. A., De Lisle R. C. Amylase release from streptolysin O-permeabilized pancreatic acini. Am J Physiol. 1990 Aug;259(2 Pt 1):G157–G164. doi: 10.1152/ajpgi.1990.259.2.G157. [DOI] [PubMed] [Google Scholar]
  21. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Michaeli T., Field J., Ballester R., O'Neill K., Wigler M. Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. EMBO J. 1989 Oct;8(10):3039–3044. doi: 10.1002/j.1460-2075.1989.tb08454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  24. Mizoguchi A., Kim S., Ueda T., Kikuchi A., Yorifuji H., Hirokawa N., Takai Y. Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain. J Biol Chem. 1990 Jul 15;265(20):11872–11879. [PubMed] [Google Scholar]
  25. Nakano A., Brada D., Schekman R. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J Cell Biol. 1988 Sep;107(3):851–863. doi: 10.1083/jcb.107.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Padfield P. J., Ding T. G., Jamieson J. D. Ca2(+)-dependent amylase secretion from pancreatic acinar cells occurs without activation of phospholipase C linked G-proteins. Biochem Biophys Res Commun. 1991 Jan 31;174(2):536–541. doi: 10.1016/0006-291x(91)91450-q. [DOI] [PubMed] [Google Scholar]
  28. Padfield P. J., Jamieson J. D. Low molecular weight GTP-binding proteins associated with zymogen granule membranes from rat pancreas. Biochem Biophys Res Commun. 1991 Jan 31;174(2):600–605. doi: 10.1016/0006-291x(91)91459-p. [DOI] [PubMed] [Google Scholar]
  29. Plutner H., Schwaninger R., Pind S., Balch W. E. Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J. 1990 Aug;9(8):2375–2383. doi: 10.1002/j.1460-2075.1990.tb07412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Santos E., Nebreda A. R. Structural and functional properties of ras proteins. FASEB J. 1989 Aug;3(10):2151–2163. doi: 10.1096/fasebj.3.10.2666231. [DOI] [PubMed] [Google Scholar]
  31. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  32. Stearns T., Willingham M. C., Botstein D., Kahn R. A. ADP-ribosylation factor is functionally and physically associated with the Golgi complex. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1238–1242. doi: 10.1073/pnas.87.3.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stone J. C., Vass W. C., Willumsen B. M., Lowy D. R. p21-ras effector domain mutants constructed by "cassette" mutagenesis. Mol Cell Biol. 1988 Aug;8(8):3565–3569. doi: 10.1128/mcb.8.8.3565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  35. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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