Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jun 15;14(12):2723–2730. doi: 10.1002/j.1460-2075.1995.tb07273.x

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

R Regazzi 1, C B Wollheim 1, J Lang 1, J M Theler 1, O Rossetto 1, C Montecucco 1, K Sadoul 1, U Weller 1, M Palmer 1, B Thorens 1
PMCID: PMC398391  PMID: 7796801

Abstract

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.

Full text

PDF
2723

Images in this article

2724Image
on p.2724
2726Image
on p.2726

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahnert-Hilger G., Weller U., Dauzenroth M. E., Habermann E., Gratzl M. The tetanus toxin light chain inhibits exocytosis. FEBS Lett. 1989 Jan 2;242(2):245–248. doi: 10.1016/0014-5793(89)80478-8. [DOI] [PubMed] [Google Scholar]
  2. Archer B. T., 3rd, Ozçelik T., Jahn R., Francke U., Südhof T. C. Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2. J Biol Chem. 1990 Oct 5;265(28):17267–17273. [PubMed] [Google Scholar]
  3. Aruffo A., Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8573–8577. doi: 10.1073/pnas.84.23.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asfari M., Janjic D., Meda P., Li G., Halban P. A., Wollheim C. B. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992 Jan;130(1):167–178. doi: 10.1210/endo.130.1.1370150. [DOI] [PubMed] [Google Scholar]
  5. Baumert M., Maycox P. R., Navone F., De Camilli P., Jahn R. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J. 1989 Feb;8(2):379–384. doi: 10.1002/j.1460-2075.1989.tb03388.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bennett M. K., García-Arrarás J. E., Elferink L. A., Peterson K., Fleming A. M., Hazuka C. D., Scheller R. H. The syntaxin family of vesicular transport receptors. Cell. 1993 Sep 10;74(5):863–873. doi: 10.1016/0092-8674(93)90466-4. [DOI] [PubMed] [Google Scholar]
  7. Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
  8. Bittner M. A., Holz R. W. Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components. J Biol Chem. 1992 Aug 15;267(23):16219–16225. [PubMed] [Google Scholar]
  9. Bittner M. A., Holz R. W. Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway. Cell Mol Neurobiol. 1993 Dec;13(6):649–664. doi: 10.1007/BF00711564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Blasi J., Chapman E. R., Link E., Binz T., Yamasaki S., De Camilli P., Südhof T. C., Niemann H., Jahn R. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature. 1993 Sep 9;365(6442):160–163. doi: 10.1038/365160a0. [DOI] [PubMed] [Google Scholar]
  11. Blasi J., Chapman E. R., Yamasaki S., Binz T., Niemann H., Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 1993 Dec;12(12):4821–4828. doi: 10.1002/j.1460-2075.1993.tb06171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Block M. R., Glick B. S., Wilcox C. A., Wieland F. T., Rothman J. E. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7852–7856. doi: 10.1073/pnas.85.21.7852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Braun J. E., Fritz B. A., Wong S. M., Lowe A. W. Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas. J Biol Chem. 1994 Feb 18;269(7):5328–5335. [PubMed] [Google Scholar]
  14. Cain C. C., Trimble W. S., Lienhard G. E. Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes. J Biol Chem. 1992 Jun 15;267(17):11681–11684. [PubMed] [Google Scholar]
  15. Chilcote T. J., Galli T., Mundigl O., Edelmann L., McPherson P. S., Takei K., De Camilli P. Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells. J Cell Biol. 1995 Apr;129(1):219–231. doi: 10.1083/jcb.129.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Clary D. O., Griff I. C., Rothman J. E. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell. 1990 May 18;61(4):709–721. doi: 10.1016/0092-8674(90)90482-t. [DOI] [PubMed] [Google Scholar]
  17. Clary D. O., Rothman J. E. Purification of three related peripheral membrane proteins needed for vesicular transport. J Biol Chem. 1990 Jun 15;265(17):10109–10117. [PubMed] [Google Scholar]
  18. Elferink L. A., Trimble W. S., Scheller R. H. Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system. J Biol Chem. 1989 Jul 5;264(19):11061–11064. [PubMed] [Google Scholar]
  19. Ferro-Novick S., Jahn R. Vesicle fusion from yeast to man. Nature. 1994 Jul 21;370(6486):191–193. doi: 10.1038/370191a0. [DOI] [PubMed] [Google Scholar]
  20. Gaisano H. Y., Sheu L., Foskett J. K., Trimble W. S. Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. J Biol Chem. 1994 Jun 24;269(25):17062–17066. [PubMed] [Google Scholar]
  21. Galli T., Chilcote T., Mundigl O., Binz T., Niemann H., De Camilli P. Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells. J Cell Biol. 1994 Jun;125(5):1015–1024. doi: 10.1083/jcb.125.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Glick B. S., Rothman J. E. Possible role for fatty acyl-coenzyme A in intracellular protein transport. Nature. 1987 Mar 19;326(6110):309–312. doi: 10.1038/326309a0. [DOI] [PubMed] [Google Scholar]
  23. Hayashi T., McMahon H., Yamasaki S., Binz T., Hata Y., Südhof T. C., Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 1994 Nov 1;13(21):5051–5061. doi: 10.1002/j.1460-2075.1994.tb06834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunt J. M., Bommert K., Charlton M. P., Kistner A., Habermann E., Augustine G. J., Betz H. A post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron. 1994 Jun;12(6):1269–1279. doi: 10.1016/0896-6273(94)90443-x. [DOI] [PubMed] [Google Scholar]
  25. Ishihara H., Asano T., Tsukuda K., Katagiri H., Inukai K., Anai M., Kikuchi M., Yazaki Y., Miyazaki J. I., Oka Y. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia. 1993 Nov;36(11):1139–1145. doi: 10.1007/BF00401058. [DOI] [PubMed] [Google Scholar]
  26. Jacobsson G., Bean A. J., Scheller R. H., Juntti-Berggren L., Deeney J. T., Berggren P. O., Meister B. Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12487–12491. doi: 10.1073/pnas.91.26.12487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jonas J. C., Li G., Palmer M., Weller U., Wollheim C. B. Dynamics of Ca2+ and guanosine 5'-[gamma-thio]triphosphate action on insulin secretion from alpha-toxin-permeabilized HIT-T15 cells. Biochem J. 1994 Jul 15;301(Pt 2):523–529. doi: 10.1042/bj3010523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li G., Regazzi R., Balch W. E., Wollheim C. B. Stimulation of insulin release from permeabilized HIT-T15 cells by a synthetic peptide corresponding to the effector domain of the small GTP-binding protein rab3. FEBS Lett. 1993 Jul 26;327(2):145–149. doi: 10.1016/0014-5793(93)80159-r. [DOI] [PubMed] [Google Scholar]
  29. Malhotra V., Orci L., Glick B. S., Block M. R., Rothman J. E. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack. Cell. 1988 Jul 15;54(2):221–227. doi: 10.1016/0092-8674(88)90554-5. [DOI] [PubMed] [Google Scholar]
  30. McMahon H. T., Ushkaryov Y. A., Edelmann L., Link E., Binz T., Niemann H., Jahn R., Südhof T. C. Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature. 1993 Jul 22;364(6435):346–349. doi: 10.1038/364346a0. [DOI] [PubMed] [Google Scholar]
  31. Montecucco C., Schiavo G. Mechanism of action of tetanus and botulinum neurotoxins. Mol Microbiol. 1994 Jul;13(1):1–8. doi: 10.1111/j.1365-2958.1994.tb00396.x. [DOI] [PubMed] [Google Scholar]
  32. Montecucco C., Schiavo G. Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci. 1993 Sep;18(9):324–327. doi: 10.1016/0968-0004(93)90065-u. [DOI] [PubMed] [Google Scholar]
  33. Orci L., Malhotra V., Amherdt M., Serafini T., Rothman J. E. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell. 1989 Feb 10;56(3):357–368. doi: 10.1016/0092-8674(89)90239-0. [DOI] [PubMed] [Google Scholar]
  34. Protopopov V., Govindan B., Novick P., Gerst J. E. Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae. Cell. 1993 Sep 10;74(5):855–861. doi: 10.1016/0092-8674(93)90465-3. [DOI] [PubMed] [Google Scholar]
  35. Ralston E., Beushausen S., Ploug T. Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues. J Biol Chem. 1994 Jun 3;269(22):15403–15406. [PubMed] [Google Scholar]
  36. Reetz A., Solimena M., Matteoli M., Folli F., Takei K., De Camilli P. GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 1991 May;10(5):1275–1284. doi: 10.1002/j.1460-2075.1991.tb08069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Regazzi R., Li G. D., Deshusses J., Wollheim C. B. Stimulus-response coupling in insulin-secreting HIT cells. Effects of secretagogues on cytosolic Ca2+, diacylglycerol, and protein kinase C activity. J Biol Chem. 1990 Sep 5;265(25):15003–15009. [PubMed] [Google Scholar]
  38. Regazzi R., Li G., Ullrich S., Jaggi C., Wollheim C. B. Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes. J Biol Chem. 1989 Jun 15;264(17):9939–9944. [PubMed] [Google Scholar]
  39. Rorsman P., Berggren P. O., Bokvist K., Ericson H., Möhler H., Ostenson C. G., Smith P. A. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels. Nature. 1989 Sep 21;341(6239):233–236. doi: 10.1038/341233a0. [DOI] [PubMed] [Google Scholar]
  40. Rothman J. E., Warren G. Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr Biol. 1994 Mar 1;4(3):220–233. doi: 10.1016/s0960-9822(00)00051-8. [DOI] [PubMed] [Google Scholar]
  41. Sadoul K., Lang J., Montecucco C., Weller U., Regazzi R., Catsicas S., Wollheim C. B., Halban P. A. SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol. 1995 Mar;128(6):1019–1028. doi: 10.1083/jcb.128.6.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schiavo G., Benfenati F., Poulain B., Rossetto O., Polverino de Laureto P., DasGupta B. R., Montecucco C. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992 Oct 29;359(6398):832–835. doi: 10.1038/359832a0. [DOI] [PubMed] [Google Scholar]
  44. Schiavo G., Rossetto O., Santucci A., DasGupta B. R., Montecucco C. Botulinum neurotoxins are zinc proteins. J Biol Chem. 1992 Nov 25;267(33):23479–23483. [PubMed] [Google Scholar]
  45. Sorenson R. L., Garry D. G., Brelje T. C. Structural and functional considerations of GABA in islets of Langerhans. Beta-cells and nerves. Diabetes. 1991 Nov;40(11):1365–1374. doi: 10.2337/diab.40.11.1365. [DOI] [PubMed] [Google Scholar]
  46. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  47. Südhof T. C., De Camilli P., Niemann H., Jahn R. Membrane fusion machinery: insights from synaptic proteins. Cell. 1993 Oct 8;75(1):1–4. [PubMed] [Google Scholar]
  48. Takizawa P. A., Malhotra V. Coatomers and SNAREs in promoting membrane traffic. Cell. 1993 Nov 19;75(4):593–596. doi: 10.1016/0092-8674(93)90477-8. [DOI] [PubMed] [Google Scholar]
  49. Thomas-Reetz A., Hell J. W., During M. J., Walch-Solimena C., Jahn R., De Camilli P. A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5317–5321. doi: 10.1073/pnas.90.11.5317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thorens B. Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8641–8645. doi: 10.1073/pnas.89.18.8641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thorens B., Sarkar H. K., Kaback H. R., Lodish H. F. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell. 1988 Oct 21;55(2):281–290. doi: 10.1016/0092-8674(88)90051-7. [DOI] [PubMed] [Google Scholar]
  52. Vallar L., Biden T. J., Wollheim C. B. Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells. J Biol Chem. 1987 Apr 15;262(11):5049–5056. [PubMed] [Google Scholar]
  53. Weller U., Dauzenroth M. E., Meyer zu Heringdorf D., Habermann E. Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties. Eur J Biochem. 1989 Jul 1;182(3):649–656. doi: 10.1111/j.1432-1033.1989.tb14874.x. [DOI] [PubMed] [Google Scholar]
  54. Weller U., Mauler F., Habermann E. Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis. Naunyn Schmiedebergs Arch Pharmacol. 1988 Aug;338(2):99–106. doi: 10.1007/BF00174855. [DOI] [PubMed] [Google Scholar]
  55. Wollheim C. B., Meda P., Halban P. A. Isolation of pancreatic islets and primary culture of the intact microorgans or of dispersed islet cells. Methods Enzymol. 1990;192:188–223. doi: 10.1016/0076-6879(90)92071-k. [DOI] [PubMed] [Google Scholar]
  56. Wollheim C. B., Pozzan T. Correlation between cytosolic free Ca2+ and insulin release in an insulin-secreting cell line. J Biol Chem. 1984 Feb 25;259(4):2262–2267. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES