Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Feb 2;17(3):648–657. doi: 10.1093/emboj/17.3.648

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

J Lang 1, Y Ushkaryov 1, A Grasso 1, C B Wollheim 1
PMCID: PMC1170414  PMID: 9450990

Abstract

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

Full Text

The Full Text of this article is available as a PDF (541.7 KB).

Selected References

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

  1. Adam-Vizi V., Deri Z., Bors P., Tretter L. Lack of involvement of [Ca2+]i in the external Ca(2+)-independent release of acetylcholine evoked by veratridine, ouabain and alpha-latrotoxin: possible role of [Na+]i. J Physiol Paris. 1993;87(1):43–50. doi: 10.1016/0928-4257(93)90023-m. [DOI] [PubMed] [Google Scholar]
  2. Ahnert-Hilger G., Stecher B., Beyer C., Gratzl M. Exocytotic membrane fusion as studied in toxin-permeabilized cells. Methods Enzymol. 1993;221:139–149. doi: 10.1016/0076-6879(93)21013-x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Barnett D. W., Liu J., Misler S. Single-cell measurements of quantal secretion induced by alpha-latrotoxin from rat adrenal chromaffin cells: dependence on extracellular Ca2+. Pflugers Arch. 1996 Oct;432(6):1039–1046. doi: 10.1007/s004240050232. [DOI] [PubMed] [Google Scholar]
  5. Bokvist K., Eliasson L., Ammälä C., Renström E., Rorsman P. Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. EMBO J. 1995 Jan 3;14(1):50–57. doi: 10.1002/j.1460-2075.1995.tb06974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Capogna M., Gähwiler B. H., Thompson S. M. Calcium-independent actions of alpha-latrotoxin on spontaneous and evoked synaptic transmission in the hippocampus. J Neurophysiol. 1996 Nov;76(5):3149–3158. doi: 10.1152/jn.1996.76.5.3149. [DOI] [PubMed] [Google Scholar]
  7. Capogna M., Gähwiler B. H., Thompson S. M. Presynaptic inhibition of calcium-dependent and -independent release elicited with ionomycin, gadolinium, and alpha-latrotoxin in the hippocampus. J Neurophysiol. 1996 May;75(5):2017–2028. doi: 10.1152/jn.1996.75.5.2017. [DOI] [PubMed] [Google Scholar]
  8. Cattaneo A., Grasso A. A functional domain on the alpha-latrotoxin molecule, distinct from the binding site, involved in catecholamine secretion from PC12 cells: identification with monoclonal antibodies. Biochemistry. 1986 May 6;25(9):2730–2736. doi: 10.1021/bi00357a068. [DOI] [PubMed] [Google Scholar]
  9. Davletov B. A., Krasnoperov V., Hata Y., Petrenko A. G., Südhof T. C. High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha. J Biol Chem. 1995 Oct 13;270(41):23903–23905. doi: 10.1074/jbc.270.41.23903. [DOI] [PubMed] [Google Scholar]
  10. Davletov B. A., Shamotienko O. G., Lelianova V. G., Grishin E. V., Ushkaryov Y. A. Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein. J Biol Chem. 1996 Sep 20;271(38):23239–23245. doi: 10.1074/jbc.271.38.23239. [DOI] [PubMed] [Google Scholar]
  11. De Potter W. P., Partoens P., Schoups A., Llona I., Coen E. P. Noradrenergic neurons release both noradrenaline and neuropeptide Y from a single pool: the large dense cored vesicles. Synapse. 1997 Jan;25(1):44–55. doi: 10.1002/(SICI)1098-2396(199701)25:1<44::AID-SYN6>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  12. Detimary P., Van den Berghe G., Henquin J. C. Concentration dependence and time course of the effects of glucose on adenine and guanine nucleotides in mouse pancreatic islets. J Biol Chem. 1996 Aug 23;271(34):20559–20565. doi: 10.1074/jbc.271.34.20559. [DOI] [PubMed] [Google Scholar]
  13. Eliasson L., Renström E., Ding W. G., Proks P., Rorsman P. Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells. J Physiol. 1997 Sep 1;503(Pt 2):399–412. doi: 10.1111/j.1469-7793.1997.399bh.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gasparini S., Kiyatkin N., Drevet P., Boulain J. C., Tacnet F., Ripoche P., Forest E., Grishin E., Ménez A. The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones. J Biol Chem. 1994 Aug 5;269(31):19803–19809. [PubMed] [Google Scholar]
  15. Geppert M., Goda Y., Hammer R. E., Li C., Rosahl T. W., Stevens C. F., Südhof T. C. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 1994 Nov 18;79(4):717–727. doi: 10.1016/0092-8674(94)90556-8. [DOI] [PubMed] [Google Scholar]
  16. Gillis K. D., Misler S. Enhancers of cytosolic cAMP augment depolarization-induced exocytosis from pancreatic B-cells: evidence for effects distal to Ca2+ entry. Pflugers Arch. 1993 Jul;424(2):195–197. doi: 10.1007/BF00374612. [DOI] [PubMed] [Google Scholar]
  17. Grasso A., Alemà S., Rufini S., Senni M. I. Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line. Nature. 1980 Feb 21;283(5749):774–776. doi: 10.1038/283774a0. [DOI] [PubMed] [Google Scholar]
  18. Grasso A., Mastrogiacomo A. Alpha-latrotoxin: preparation and effects on calcium fluxes. FEMS Microbiol Immunol. 1992 Sep;5(1-3):131–137. doi: 10.1111/j.1574-6968.1992.tb05895.x. [DOI] [PubMed] [Google Scholar]
  19. Grasso A., Mercanti-Ciotti M. T. The secretion of amino acid transmitters from cerebellar primary cultures probed by alpha-latrotoxin. Neuroscience. 1993 Jun;54(3):595–604. doi: 10.1016/0306-4522(93)90231-4. [DOI] [PubMed] [Google Scholar]
  20. Hata Y., Davletov B., Petrenko A. G., Jahn R., Südhof T. C. Interaction of synaptotagmin with the cytoplasmic domains of neurexins. Neuron. 1993 Feb;10(2):307–315. doi: 10.1016/0896-6273(93)90320-q. [DOI] [PubMed] [Google Scholar]
  21. Heidelberger R., Heinemann C., Neher E., Matthews G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature. 1994 Oct 6;371(6497):513–515. doi: 10.1038/371513a0. [DOI] [PubMed] [Google Scholar]
  22. Hurlbut W. P., Chieregatti E., Valtorta F., Haimann C. Alpha-latrotoxin channels in neuroblastoma cells. J Membr Biol. 1994 Feb;138(1):91–102. doi: 10.1007/BF00211072. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kiraly-Borri C. E., Morgan A., Burgoyne R. D., Weller U., Wollheim C. B., Lang J. Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin. Biochem J. 1996 Feb 15;314(Pt 1):199–203. doi: 10.1042/bj3140199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krasnoperov V. G., Beavis R., Chepurny O. G., Little A. R., Plotnikov A. N., Petrenko A. G. The calcium-independent receptor of alpha-latrotoxin is not a neurexin. Biochem Biophys Res Commun. 1996 Oct 23;227(3):868–875. doi: 10.1006/bbrc.1996.1598. [DOI] [PubMed] [Google Scholar]
  27. Krasnoperov V. G., Bittner M. A., Beavis R., Kuang Y., Salnikow K. V., Chepurny O. G., Little A. R., Plotnikov A. N., Wu D., Holz R. W. alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron. 1997 Jun;18(6):925–937. doi: 10.1016/s0896-6273(00)80332-3. [DOI] [PubMed] [Google Scholar]
  28. Lad R. P., Smith M. A., Hilt D. C. Molecular cloning and regional distribution of rat brain cyclophilin. Brain Res Mol Brain Res. 1991 Feb;9(3):239–244. doi: 10.1016/0169-328x(91)90007-k. [DOI] [PubMed] [Google Scholar]
  29. Lang J., Boulay F., Li G., Wollheim C. B. Conserved transducer coupling but different effector linkage upon expression of the myeloid fMet-Leu-Phe receptor in insulin secreting cells. EMBO J. 1993 Jul;12(7):2671–2679. doi: 10.1002/j.1460-2075.1993.tb05928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lang J., Fukuda M., Zhang H., Mikoshiba K., Wollheim C. B. The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium. EMBO J. 1997 Oct 1;16(19):5837–5846. doi: 10.1093/emboj/16.19.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lang J., Nishimoto I., Okamoto T., Regazzi R., Kiraly C., Weller U., Wollheim C. B. Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits. EMBO J. 1995 Aug 1;14(15):3635–3644. doi: 10.1002/j.1460-2075.1995.tb00033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lelianova V. G., Davletov B. A., Sterling A., Rahman M. A., Grishin E. V., Totty N. F., Ushkaryov Y. A. Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors. J Biol Chem. 1997 Aug 22;272(34):21504–21508. doi: 10.1074/jbc.272.34.21504. [DOI] [PubMed] [Google Scholar]
  33. Littleton J. T., Bellen H. J. Synaptotagmin controls and modulates synaptic-vesicle fusion in a Ca(2+)-dependent manner. Trends Neurosci. 1995 Apr;18(4):177–183. doi: 10.1016/0166-2236(95)93898-8. [DOI] [PubMed] [Google Scholar]
  34. Maechler P., Kennedy E. D., Pozzan T., Wollheim C. B. Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells. EMBO J. 1997 Jul 1;16(13):3833–3841. doi: 10.1093/emboj/16.13.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Martín F., Moya F., Gutierrez L. M., Reig J. A., Soria B. Role of syntaxin in mouse pancreatic beta cells. Diabetologia. 1995 Jul;38(7):860–863. doi: 10.1007/s001250050364. [DOI] [PubMed] [Google Scholar]
  36. Meldolesi J., Huttner W. B., Tsien R. Y., Pozzan T. Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin. Proc Natl Acad Sci U S A. 1984 Jan;81(2):620–624. doi: 10.1073/pnas.81.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Meldolesi J., Madeddu L., Torda M., Gatti G., Niutta E. The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release. Neuroscience. 1983 Nov;10(3):997–1009. doi: 10.1016/0306-4522(83)90238-5. [DOI] [PubMed] [Google Scholar]
  38. Meldolesi J. Studies on alpha-latrotoxin receptors in rat brain synaptosomes: correlation between toxin binding and stimulation of transmitter release. J Neurochem. 1982 Jun;38(6):1559–1569. doi: 10.1111/j.1471-4159.1982.tb06633.x. [DOI] [PubMed] [Google Scholar]
  39. Misler S., Falke L. C. Dependence on multivalent cations of quantal release of transmitter induced by black widow spider venom. Am J Physiol. 1987 Sep;253(3 Pt 1):C469–C476. doi: 10.1152/ajpcell.1987.253.3.C469. [DOI] [PubMed] [Google Scholar]
  40. Nicholls D. G., Rugolo M., Scott I. G., Meldolesi J. alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7924–7928. doi: 10.1073/pnas.79.24.7924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pashkov V., Grico N., Tsurupa G., Storchak L., Shatursky O., Himmerlreich N., Grishin E. Monoclonal antibodies can uncouple the main alpha-latrotoxin effects: toxin-induced Ca2+ influx and stimulated neurotransmitter release. Neuroscience. 1993 Oct;56(3):695–701. doi: 10.1016/0306-4522(93)90366-n. [DOI] [PubMed] [Google Scholar]
  42. Pescatori M., Bradbury A., Bouet F., Gargano N., Mastrogiacomo A., Grasso A. The cloning of a cDNA encoding a protein (latrodectin) which co-purifies with the alpha-latrotoxin from the black widow spider Latrodectus tredecimguttatus (Theridiidae). Eur J Biochem. 1995 May 15;230(1):322–328. doi: 10.1111/j.1432-1033.1995.tb20566.x. [DOI] [PubMed] [Google Scholar]
  43. Petrenko A. G., Perin M. S., Davletov B. A., Ushkaryov Y. A., Geppert M., Südhof T. C. Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis. Nature. 1991 Sep 5;353(6339):65–68. doi: 10.1038/353065a0. [DOI] [PubMed] [Google Scholar]
  44. Proks P., Eliasson L., Ammälä C., Rorsman P., Ashcroft F. M. Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps. J Physiol. 1996 Oct 1;496(Pt 1):255–264. doi: 10.1113/jphysiol.1996.sp021682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Regazzi R., Wollheim C. B., Lang J., Theler J. M., Rossetto O., Montecucco C., Sadoul K., Weller U., Palmer M., Thorens B. 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. EMBO J. 1995 Jun 15;14(12):2723–2730. doi: 10.1002/j.1460-2075.1995.tb07273.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Robello M., Fresia M., Maga L., Grasso A., Ciani S. Permeation of divalent cations through alpha-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships. J Membr Biol. 1987;95(1):55–62. doi: 10.1007/BF01869630. [DOI] [PubMed] [Google Scholar]
  47. Rosenthal L., Meldolesi J. Alpha-latrotoxin and related toxins. Pharmacol Ther. 1989;42(1):115–134. doi: 10.1016/0163-7258(89)90024-7. [DOI] [PubMed] [Google Scholar]
  48. Rosenthal L., Zacchetti D., Madeddu L., Meldolesi J. Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(+)-dependent and Ca2(+)-independent effects mediated by the toxin. Mol Pharmacol. 1990 Dec;38(6):917–923. [PubMed] [Google Scholar]
  49. Rothman J. E. The protein machinery of vesicle budding and fusion. Protein Sci. 1996 Feb;5(2):185–194. doi: 10.1002/pro.5560050201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ryffel B., Woerly G., Greiner B., Haendler B., Mihatsch M. J., Foxwell B. M. Distribution of the cyclosporine binding protein cyclophilin in human tissues. Immunology. 1991 Mar;72(3):399–404. [PMC free article] [PubMed] [Google Scholar]
  51. Sabirov R. Z., Krasilnikov O. V., Ternovsky V. I., Merzliak P. G. Relation between ionic channel conductance and conductivity of media containing different nonelectrolytes. A novel method of pore size determination. Gen Physiol Biophys. 1993 Apr;12(2):95–111. [PubMed] [Google Scholar]
  52. 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]
  53. Sakurada M., Kanatsuka A., Saitoh T., Makino H., Yamamura K., Miyazaki J., Kikuchi M., Yoshida S. Relation between glucose-stimulated insulin secretion and intracellular calcium accumulation studied with a superfusion system of a glucose-responsive pancreatic beta-cell line MIN6. Endocrinology. 1993 Jun;132(6):2659–2665. doi: 10.1210/endo.132.6.8504766. [DOI] [PubMed] [Google Scholar]
  54. Sharp G. W. Mechanisms of inhibition of insulin release. Am J Physiol. 1996 Dec;271(6 Pt 1):C1781–C1799. doi: 10.1152/ajpcell.1996.271.6.C1781. [DOI] [PubMed] [Google Scholar]
  55. Shoji-Kasai Y., Yoshida A., Ogura A., Kuwahara R., Grasso A., Takahashi M. Synaptotagmin I is essential for Ca(2+)-independent release of neurotransmitter induced by alpha-latrotoxin. FEBS Lett. 1994 Oct 24;353(3):315–318. doi: 10.1016/0014-5793(94)01069-2. [DOI] [PubMed] [Google Scholar]
  56. Südhof T. C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. doi: 10.1038/375645a0. [DOI] [PubMed] [Google Scholar]
  57. Theler J. M., Mollard P., Guérineau N., Vacher P., Pralong W. F., Schlegel W., Wollheim C. B. Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP. J Biol Chem. 1992 Sep 5;267(25):18110–18117. [PubMed] [Google Scholar]
  58. Ushkaryov Y. A., Petrenko A. G., Geppert M., Südhof T. C. Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science. 1992 Jul 3;257(5066):50–56. doi: 10.1126/science.1621094. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. Valtorta F., Madeddu L., Meldolesi J., Ceccarelli B. Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane. J Cell Biol. 1984 Jul;99(1 Pt 1):124–132. doi: 10.1083/jcb.99.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Watanabe O., Torda M., Meldolesi J. The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: electron microscopy and cytotoxicity studies. Neuroscience. 1983 Nov;10(3):1011–1024. doi: 10.1016/0306-4522(83)90239-7. [DOI] [PubMed] [Google Scholar]
  62. Waterman S. A., Maggi C. A. Stimulation of neuropeptide release from sensory and enteric neurons in the guinea-pig by alpha-latrotoxin. Neuroscience. 1995 Dec;69(3):977–984. doi: 10.1016/0306-4522(95)00317-c. [DOI] [PubMed] [Google Scholar]
  63. Wheeler M. B., Sheu L., Ghai M., Bouquillon A., Grondin G., Weller U., Beaudoin A. R., Bennett M. K., Trimble W. S., Gaisano H. Y. Characterization of SNARE protein expression in beta cell lines and pancreatic islets. Endocrinology. 1996 Apr;137(4):1340–1348. doi: 10.1210/endo.137.4.8625909. [DOI] [PubMed] [Google Scholar]

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

RESOURCES