Co-Localization by Immunofluorescence of the α Subunit(S) of Gi with Cytoplasmic Structures

  • Jean M. Lewis
  • Marilyn J. Woolkalis
  • George L. Gerton
  • David R. Manning
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)

Abstract

GTP-binding proteins established in signal transduction (G proteins) are hetemtrimers consisting of α, β, and γ subunits. From the initial discovery Gs, the family of G proteins has expanded in both number and scope of action. Arrangements of G proteins within the plasma membrane and among subcellular structures, however, remain largely uncharacterized. Compartmentation within the plasma membrane may indeed provide the means by which agonists can operate selectively on particular enzymes or channels (Brass et al., 1988; Neer et al., 1988). G proteins have also been detected within the cytosol (Bokoch et al., 1988; Rotrosen et al., 1988; Spicher et al., 1988) and in granule membranes (Toutant et al., 1987; Rotrosen et al., 1988). While cytoplasmic populations of G proteins may simply represent internal stores bound for recruitment to the plasma membrane, there is evidence that they may perform more active roles critical to normal cell function. The existence of guanine nucleotide-sensitivity, for example, has been demonstrated in transport of protein within the Golgi apparatus (Melançon et al., 19871, degranulation (Cockroft et al., 1987; Lindau and Nusse,1987), release of Ca++ from endoplasmic reticulum (Nasmith and Grinstein, 1987), and perhaps translocation of nascent proteins (Audigier et al., 1988; Connolly and Gilmore, 1986; Hoffman and Gilmore, 1988). In parallel to these findings, a recent study has shown that a 41-kDa, G-like protein co-fractionates with rough endoplasmic reticulum (Audigier et al, 1988). Clearly, knowledge of the distributions of G proteins within the cell will be critical to our understanding of G protein function.

Keywords

Lymphoma Tyrosine MgCl2 Fractionation Cytosol 

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References

  1. Audigier, Y., Nigam, S.K., and Blobel, G., 1988, Identification of a G protein in rough endoplasmic reticulum of canine pancreas, J. Biol. Chem., 263:16352.PubMedGoogle Scholar
  2. Beckers, C.J.M., and Balch, W.E., 1989, Calcium and GTP: Essential components in vesicular trafficking between the endoplasmic reticulum and golgi apparatus, J. Cell Biol., 108:1245.PubMedCrossRefGoogle Scholar
  3. Bokoch, G.M., Bickford, K., and Bohl, B.P., 1988, Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate, Gn, J. Cell Biol., 106:1927.PubMedCrossRefGoogle Scholar
  4. Brabet, P., Dumuis, A., Sebben, M., Pantaloni, C., Bockaert, J., and Homburger, V., 1988, Immunocytochemical localization of the guanine nucleotide-binding protein G. in primary cultures of neuronal and glial cells, J. Neuroscience. 8:701.Google Scholar
  5. Brass, L.F., Woolkalis, M.J., and Manning, D.R., 1988, Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase, J. Biol. Chem.. 263:5348.PubMedGoogle Scholar
  6. Cockroft, S., Howell, T.W., and Gomperts, B.D., 1987, 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., 105:2745.CrossRefGoogle Scholar
  7. Connelley, T., and Gilmore, R., 1986, Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein, J. Cell Biol., 103:2253.CrossRefGoogle Scholar
  8. Cortes, R., Hokfelt, T., Schalling, M., Goldstein, M., Goldsmith, P., Spiegel, A., Unson, C., and Walsh, J., 1988, Antiserum raised against residues 159–168 of the guanine nueleotide-binding protein Gi3-α reacts with ependymal cells and some neurons in the rat brain containing cholecystokinin- or cholecystokinin- and tyrosine 3-hydroxylase-like immunoreactivities, Proc. Natl. Acad. Sci., U.S.A., 85:9351.PubMedCrossRefGoogle Scholar
  9. Garty, N., Galiani, D., Aharonheim, A., Ho, Y.-K., Phillips, D.M., Dekel, N., and Salomon, Y., 1988, G-proteins in mammalian gametes: an immunocytochemical study, J. Cell Science. 91:21.PubMedGoogle Scholar
  10. Gawler, D., Milligan, G., Spiegel, A.M., Unson, C.G., and Houslay, M.D., 1987, Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes, Nature, 327:229.PubMedCrossRefGoogle Scholar
  11. Goodner, C., Sweet, I.R., and Harrison, H.C., 1988, Rapid reduction and return of surface insulin receptors after exposure to brief pulses of insulin in perfused rat hepatocytes, Diabetes. 37:1316.PubMedCrossRefGoogle Scholar
  12. Grunwald, G., Gierschik, P., Nirenberg, M., and Spiegel, A., 1986, Detection of α- transducin in retinal rods but not cones, Science. 231:856.PubMedCrossRefGoogle Scholar
  13. Hoffman, K., and Gilmore, R., 1988, Guanosine triphosphate promotes the post- translational integration of opsin into the endoplasmic reticulum membrane, J. Biol. Chem., 263:4381.PubMedGoogle Scholar
  14. Lerea, C.L., Somers, D.E., Hurley, J.B., Block, I.B., and Bunt-Milam, A.H., 1986, Identification of specific transducin α subunits in retinal rod and cone photoreceptors, Science. 234:77.PubMedCrossRefGoogle Scholar
  15. Lindau, M., and Nusse, O., 1987, Pertussis toxin does not affect the time course of exocytosis in mast cells stimulated by intracellular application of GTP-γ-S, FEBS Letts., 222:317.CrossRefGoogle Scholar
  16. Melançon, P., Glick, B.S., Malhotra, V., Weidman, P.J., Serafíni, T., Gleason, M.L., Orci, L., and Rothman, J.E., 1987, Involvement of GTP-binding “G” proteins in transport through the Golgi stack, Cell. 51:1053.PubMedCrossRefGoogle Scholar
  17. Nakano, A., Brada, D., and Schekman, R., 1988, A membrane glycoprotein, Secl2p, required for protein transport from the endoplasmic reticulum to the golgi apparatus in yeast, J. Cell Biol., 107:851.PubMedCrossRefGoogle Scholar
  18. Nasmith, P., and Grinstein, S., 1987, Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils, J. Biol. Chem., 262:13558.PubMedGoogle Scholar
  19. Neer, E.J., and Clapham, D.E., 1988, Roles of G protein subunits in transmembrane signalling, Nature. 333:129.PubMedCrossRefGoogle Scholar
  20. Pfeffer, S.R., and Rothman, J.E., 1987, Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi, Ann. Rev. Biochem., 56:829.PubMedCrossRefGoogle Scholar
  21. Ransnas, L.A., and Insel, P.A., 1988, Quantitation of the guanine nucleotide binding regulatory protein Gs in S49 cell membranes using antipeptide antibodies to αs, J. Biol. Chem., 263:9482.PubMedGoogle Scholar
  22. Rotrosen, D., Gallin, J.I., Spiegel, A.M., and Malech, H.L., 1988, Subcellular localization of G in human neutrophils, J. Biol. Chem., 263:10958.PubMedGoogle Scholar
  23. Segev, N., Mulholland, J., and Botstein, D., 1988, The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery, Cell, 52:915.PubMedCrossRefGoogle Scholar
  24. Spicher, K., Hinsch, K.D., Gausepohl, H., Frank, R., Rosenthal, W., and Schulz, G., 1988, Immunochemical detection of the alpha-subunit of the G-protein, Gz, in membranes and cytosols of mammalian cells, Biochem. Biophys. Res. Commnn., 157:883.CrossRefGoogle Scholar
  25. Toutant, M., Aunis, D., Bockaert, J., Homburger, V., and Rouot, B., 1987, Presence of three pertussis toxin substrates and the G immunoreactivity in both plasma and granule membranes of chromaffin cells, FEBS Lett., 215:339.PubMedCrossRefGoogle Scholar
  26. Wang, C., 1985, Insulin-stimulated glucose uptake in rat diaphragm during postnatal development: Lack of correlation with the number of insulin receptors and of intracellular glucose transporters, Proc. Natl. Acad. Sci., U.S.A., 82:3621.PubMedCrossRefGoogle Scholar
  27. Wardzala, L.J., Simpson, I.A., Rechler, M.M., and Cushman, S.W., 1984, Potential mechanism of stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell: apparent redistribution of receptors recycling between a large intracellular pool and the plasma membrane, J.Biol. Chem., 259:8378.PubMedGoogle Scholar
  28. Watkins, D.C., Northrup, J.K., and Malbon, C.C., 1987, Regulation of G-proteins in differentiation, J. Biol. Chem., 262:10651.PubMedGoogle Scholar
  29. Woolkalis, M.J., Nakada, M.T., and Manning, D.R., 1986, Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus, J. Biol. Chem., 261:3408.PubMedGoogle Scholar
  30. Worley, P.F., Baraban, J.M., Van Dop, C., and Neer, E.J., 1986, Go, a guanine nucleotide-binding protein: Immunohistochemical localization in rat brain resembles distribution of second messenger systems, Proc. Natl. Acad. Sci., U.S.A., 83:4561.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Jean M. Lewis
    • 1
  • Marilyn J. Woolkalis
    • 1
  • George L. Gerton
    • 2
  • David R. Manning
    • 1
  1. 1.Department of PharmacologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA
  2. 2.Department of Obstetrics and GynecologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA

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