Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1989 Jun 15;260(3):837–841. doi: 10.1042/bj2600837

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

G Milligan 1, C G Unson 1
PMCID: PMC1138752  PMID: 2504150

Abstract

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

Full text

PDF
837

Images in this article

838Fig. 1.
on p.838
839Fig. 2.
on p.839
839Fig. 3.
on p.839

Selected References

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

  1. Backlund P. S., Jr, Aksamit R. R., Unson C. G., Goldsmith P., Spiegel A. M., Milligan G. Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line. Biochemistry. 1988 Mar 22;27(6):2040–2046. doi: 10.1021/bi00406a034. [DOI] [PubMed] [Google Scholar]
  2. Bray P., Carter A., Simons C., Guo V., Puckett C., Kamholz J., Spiegel A., Nirenberg M. Human cDNA clones for four species of G alpha s signal transduction protein. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8893–8897. doi: 10.1073/pnas.83.23.8893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buss J. E., Mumby S. M., Casey P. J., Gilman A. G., Sefton B. M. Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7493–7497. doi: 10.1073/pnas.84.21.7493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eide B., Gierschik P., Milligan G., Mullaney I., Unson C., Goldsmith P., Spiegel A. GTP-binding proteins in brain and neutrophil are tethered to the plasma membrane via their amino termini. Biochem Biophys Res Commun. 1987 Nov 13;148(3):1398–1405. doi: 10.1016/s0006-291x(87)80287-5. [DOI] [PubMed] [Google Scholar]
  5. Gawler D., Milligan G., Spiegel A. M., Unson C. G., Houslay M. D. Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes. Nature. 1987 May 21;327(6119):229–232. doi: 10.1038/327229a0. [DOI] [PubMed] [Google Scholar]
  6. Gierschik P., Milligan G., Pines M., Goldsmith P., Codina J., Klee W., Spiegel A. Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2258–2262. doi: 10.1073/pnas.83.7.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  8. Goldsmith P., Gierschik P., Milligan G., Unson C. G., Vinitsky R., Malech H. L., Spiegel A. M. Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain. J Biol Chem. 1987 Oct 25;262(30):14683–14688. [PubMed] [Google Scholar]
  9. Hancock J. F., Marshall C. J., McKay I. A., Gardner S., Houslay M. D., Hall A., Wakelam M. J. Mutant but not normal p21 ras elevates inositol phospholipid breakdown in two different cell systems. Oncogene. 1988 Aug;3(2):187–193. [PubMed] [Google Scholar]
  10. Kahn R. A., Goddard C., Newkirk M. Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase. J Biol Chem. 1988 Jun 15;263(17):8282–8287. [PubMed] [Google Scholar]
  11. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Magee A. I., Gutierrez L., McKay I. A., Marshall C. J., Hall A. Dynamic fatty acylation of p21N-ras. EMBO J. 1987 Nov;6(11):3353–3357. doi: 10.1002/j.1460-2075.1987.tb02656.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McArdle H., Mullaney I., Magee A., Unson C., Milligan G. GTP analogues cause release of the alpha subunit of the GTP binding protein, GO, from the plasma membrane of NG108-15 cells. Biochem Biophys Res Commun. 1988 Apr 15;152(1):243–251. doi: 10.1016/s0006-291x(88)80706-x. [DOI] [PubMed] [Google Scholar]
  15. McKenzie F. R., Kelly E. C., Unson C. G., Spiegel A. M., Milligan G. Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. Biochem J. 1988 Feb 1;249(3):653–659. doi: 10.1042/bj2490653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Milligan G. Foetal calf serum enhances cholera toxin-catalysed ADP-ribosylation of the pertussis toxin-sensitive guanine nucleotide binding protein, Gi2, in rat glioma C6BU1 cells. Cell Signal. 1989;1(1):65–74. doi: 10.1016/0898-6568(89)90021-1. [DOI] [PubMed] [Google Scholar]
  17. Milligan G. Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells. Biochem J. 1987 Jul 15;245(2):501–505. doi: 10.1042/bj2450501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Milligan G., Klee W. A. The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase. J Biol Chem. 1985 Feb 25;260(4):2057–2063. [PubMed] [Google Scholar]
  19. Milligan G., Mullaney I., Unson C. G., Marshall L., Spiegel A. M., McArdle H. GTP analogues promote release of the alpha subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells. Biochem J. 1988 Sep 1;254(2):391–396. doi: 10.1042/bj2540391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Milligan G., Simonds W. F., Streaty R. A., Tocque B., Klee W. A. Functional control of the delta-opiate receptor by the inhibitory guanine nucleotide-binding protein. Biochem Soc Trans. 1985 Dec;13(6):1110–1113. doi: 10.1042/bst0131110. [DOI] [PubMed] [Google Scholar]
  21. Mumby S. M., Kahn R. A., Manning D. R., Gilman A. G. Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1986 Jan;83(2):265–269. doi: 10.1073/pnas.83.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Brien R. M., Houslay M. D., Milligan G., Siddle K. The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go. FEBS Lett. 1987 Feb 23;212(2):281–288. doi: 10.1016/0014-5793(87)81361-3. [DOI] [PubMed] [Google Scholar]
  23. Schultz A. M., Tsai S. C., Kung H. F., Oroszlan S., Moss J., Vaughan M. Hydroxylamine-stable covalent linkage of myristic acid in G0 alpha, a guanine nucleotide-binding protein of bovine brain. Biochem Biophys Res Commun. 1987 Aug 14;146(3):1234–1239. doi: 10.1016/0006-291x(87)90780-7. [DOI] [PubMed] [Google Scholar]
  24. Spiegel A. M. Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol. 1987 Jan;49(1):1–16. doi: 10.1016/0303-7207(87)90058-x. [DOI] [PubMed] [Google Scholar]
  25. Sternweis P. C., Robishaw J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984 Nov 25;259(22):13806–13813. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES