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G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis

Nature Neuroscience volume 8, pages 421–425 (2005)Cite this article

Abstract

The activation of G protein–coupled receptors (GPCRs) can result in an inhibition of Ca2+-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca2+ influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca2+ entry. Here we characterize direct inhibitory actions of G protein βγ (Gβγ) on Ca2+-triggered vesicle exocytosis in permeable PC12 cells. Gβγ inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gβγ bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca2+-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gβγ interactions with the Ca2+-dependent fusion machinery.

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Figure 1: Gβγ inhibits Ca2+-triggered vesicle exocytosis in permeable PC12 cells.
Figure 2: Gβγ inhibition is exerted at a late stage of Ca2+-triggered vesicle exocytosis using a previously unknown effector.
Figure 3: Gβγ binding to SNARE complexes and inhibition of secretion are attenuated by BoNT A cleavage of SNAP25.
Figure 4: Gβγ binding site on SNARE complexes overlaps with Ca2+-dependent synaptotagmin binding site.

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References

  1. Sharp, G.W. Mechanisms of inhibition of insulin release. Am. J. Physiol. 271, C1781–C1799 (1996).

    Article  CAS  Google Scholar 

  2. Lang, J. Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion. Eur. J. Biochem. 259, 3–17 (1999).

    Article  CAS  Google Scholar 

  3. Knight, D.E. & Baker, P.F. Guanine nucleotides and Ca2+-dependent exocytosis. FEBS Lett. 189, 345–349 (1985).

    Article  CAS  Google Scholar 

  4. Ahnert-Hilger, G., Brautigam, M. & Gratzl, M. Ca2+-stimulated catecholamine release from alpha-toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G proteins. Biochemistry 26, 7842–7848 (1987).

    Article  CAS  Google Scholar 

  5. Luini, A. & DeMatteis, M.A. Evidence that receptor-linked G protein inhibits exocytosis by a post-second messenger mechanism in AtT-20 cells. J. Neurochem. 54, 30–38 (1990).

    Article  CAS  Google Scholar 

  6. Ohara-Imaizumi, M. et al. Regulatory role of the GTP-binding protein Go in the mechanism of exocytosis in adrenal chromaffin cells. J. Neurochem. 58, 2275–2284 (1992).

    Article  CAS  Google Scholar 

  7. Lang, J. et al. Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and Go or by the expression of their active Gα subunits. EMBO J. 14, 3635–3644 (1995).

    Article  CAS  Google Scholar 

  8. Man-Son-Hing, H., Zoran, M.J., Lukowiak, K. & Haydon, P.G. A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels. Nature 341, 237–239 (1989).

    Article  CAS  Google Scholar 

  9. Silinsky, E.M. & Solsona, C.S. Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog. J. Physiol. (Lond.) 457, 315–328 (1992).

    Article  CAS  Google Scholar 

  10. Miller, R.J. Presynaptic receptors. Annu. Rev. Pharmacol. Toxicol. 38, 201–227 (1998).

    Article  CAS  Google Scholar 

  11. Blackmer, T. et al. G protein βγ subunit-mediated presynaptic inhibition: regulation of exocytic fusion downstream of Ca2+ entry. Science 292, 293–297 (2001).

    Article  CAS  Google Scholar 

  12. Walent, J.H., Porter, B.W. & Martin, T.F.J. A novel 145kD brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells. Cell 70, 765–775 (1992).

    Article  CAS  Google Scholar 

  13. Gaudet, R., Bohm, A. & Sigler, P.B. Crystal structure at 2.4A resolution of the complex of transducin βγ and its regulator, phosducin. Cell 87, 577–588 (1996).

    Article  CAS  Google Scholar 

  14. Muller, S., Straub, A., Schroder, S., Bauer, P.H. & Lohse, M.J. Interactions of phosducin with defined G protein βγ-subunits. J. Biol. Chem. 271, 11781–11786 (1996).

    Article  CAS  Google Scholar 

  15. Ueda, N. et al. G protein βγ subunits—simplified purification and properties of novel isoforms. J. Biol. Chem. 269, 4388–4395 (1994).

    CAS  PubMed  Google Scholar 

  16. Martin, T.F. Stages of regulated exocytosis. Trends Cell Biol. 7, 271–276 (1997).

    Article  CAS  Google Scholar 

  17. Jahn, R., Lang, T. & Sudhof, T.C. Membrane fusion. Cell 112, 519–533 (2003).

    Article  CAS  Google Scholar 

  18. Banerjee, A., Barry, V.A., DasGupta, B.R. & Martin, T.F.J. N-ethylmaleimide-sensitive factor acts at a pre-fusion ATP-dependent step in Ca2+-activated exocytosis. J. Biol. Chem. 271, 20223–20226 (1996).

    Article  CAS  Google Scholar 

  19. Hay, J.C. & Martin, T.F.J. Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins. J. Cell Biol. 119, 139–151 (1992).

    Article  CAS  Google Scholar 

  20. Clapham, D.E. & Neer, E.J. G protein βγ subunits. Annu. Rev. Pharmacol. Toxicol. 37, 167–203 (1997).

    Article  CAS  Google Scholar 

  21. Hay, J.C. et al. ATP-dependent inositide phosphorylation required for Ca2+-activated secretion. Nature 374, 173–177 (1995).

    Article  CAS  Google Scholar 

  22. Blank, J.L., Brattain, K.A. & Exton, J.H. Activation of cytosolic phosphoinositide phospholipase C by G protein βγ subunits. J. Biol. Chem. 267, 23069–23075 (1992).

    CAS  PubMed  Google Scholar 

  23. Smith, R.J. et al. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J. Pharmacol. Exp. Ther. 253, 688–697 (1990).

    CAS  PubMed  Google Scholar 

  24. Li, Y. et al. Sites for Gα binding on the G protein β subunit overlap with sites for regulation of phospholipase Cβ and adenylyl cyclase. J. Biol. Chem. 273, 16265–16272 (1998).

    Article  CAS  Google Scholar 

  25. Ford, C.E. et al. Molecular basis for interactions of G protein βγ subunits with effectors. Science 280, 1271–1273 (1998).

    Article  CAS  Google Scholar 

  26. Gerona, R.R., Larsen, E.C., Kowalchyk, J.A. & Martin, T.F.J. The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J. Biol. Chem. 275, 6328–6336 (2000).

    Article  CAS  Google Scholar 

  27. Zhang, X. et al. Ca2+-dependent synaptotagmin binding to SNAP25 is essential for Ca2+-triggered exocytosis. Neuron 34, 599–611 (2002).

    Article  CAS  Google Scholar 

  28. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    Article  CAS  Google Scholar 

  29. Wang, C.-T. et al. Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles. Science 294, 1111–1115 (2001).

    Article  CAS  Google Scholar 

  30. Grishanin, R.N. et al. CAPS acts at a pre-fusion step in dense-core vesicle exocytosis as a PIP2 binding protein. Neuron 43, 551–562 (2004).

    Article  CAS  Google Scholar 

  31. Mazzoni, M.R., Malinski, J.A. & Hamm, H.E. Structural analysis of rod GTP-binding protein Gt . J. Biol. Chem. 266, 14072–14081 (1991).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health grants to T.F.J.M. and H.E.H.

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Author notes

  1. Trillium Blackmer and Eric C Larsen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, University of Illinois at Chicago, 840 West Taylor Street, Chicago, 60607, Illinois, USA

    Trillium Blackmer & Simon Alford

  2. Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, 53706, Wisconsin, USA

    Eric C Larsen, Judith A Kowalchyk & Thomas F J Martin

  3. Department of Pharmacology, Vanderbilt University School of Medicine, 23rd Avenue South at Pierce Avenue, Nashville, 37232, Tennessee, USA

    Cheryl Bartleson, Eun-Ja Yoon, Anita M Preininger & Heidi E Hamm

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  1. Trillium Blackmer
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Correspondence to Heidi E Hamm or Thomas F J Martin.

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Blackmer, T., Larsen, E., Bartleson, C. et al. G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nat Neurosci 8, 421–425 (2005). https://doi.org/10.1038/nn1423

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