Skip to main content
SpringerLink
Log in

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

  • Original Article
  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.

The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ATPγS:

Adenosine 5′-0-(3-Thiotriphosphate)

GTPγS:

Guanosine 5′-0-(3-thiotri-phosphate)

(Sp)-cAMPS:

Adenosine 3′,5′-monophosphorothioate-Sp-isomer

dcAMP:

2′-deoxyadenosine 3′,5′-monophosphate

Hepes:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

DTT:

Dithiothreitol

buffer A:

10 mM KH2PO4/Na2HPO4, pH 6.5

buffer B:

40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7

References

  1. Konijn TM, Van de Meene JGC, Bonner JT, Barkley DS: The acrasin activity of adenosine-3′5′-cyclic phosphate. Proc Natl Acad Sci USA 58: 1152–1154, 1967

    Google Scholar 

  2. Devreotes PN: Cyclic nucleotides and cell-cell communication in Dictyostelium discoideum. Adv Cycl Nucl Res 15: 55–96, 1983

    Google Scholar 

  3. Gerisch G: Cyclic AMP and other signals controlling cell development and differentiation in Dictyostelium. Annu Rev Biochem 56: 853–879, 1987

    Article  CAS  PubMed  Google Scholar 

  4. Van Haastert PJM, Konijn TM: Signal transduction in the cellular slime molds. Mol Cell Endrocrinol 26: 1–17, 1982

    Google Scholar 

  5. Gerisch G, Fromm H, Huesgen A, Wick U: Control of cell-contact sites by cAMP pulses in differentiating Dictyostelium cells. Nature 255: 547–549, 1975

    Google Scholar 

  6. Ross FM, Newell PC: Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism. J Gen Microbiol 127: 339–350, 1981

    Google Scholar 

  7. Van Haastert PJM, Van Lookeren Campagne MM, Ross FM: Altered cGMP-phosphodiesterase activity in chemotactic mutants of Dictyostelium discoideum. FEBS Lett 147: 149–152, 1982

    Google Scholar 

  8. Van Haastert PJM, Van der Heijden PR: Excitation, adaptation and deadaptation of the cAMP mediated cGMP response in Dictyostelium discoideum. J Cell Biol 96: 347–353, 1983

    Google Scholar 

  9. Van Haastert PJM: Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum. J Cell Biol 105: 2301–2306, 1987

    Google Scholar 

  10. Dinauer MC, Steck TL, Devreotes PN: V. Adaptation of the cAMP signaling response during cAMP stimulation. J Cell Biol 86: 554–561, 1980

    Google Scholar 

  11. Van Haastert PJM: Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. J Biol Chem 262: 7700–7704, 1987

    Google Scholar 

  12. Van Haastert PJM: Adenosine 3′,5′-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum. J Biol Chem 262: 7705–7710, 1987

    Google Scholar 

  13. Dinauer MC, Steck TL, Devreotes PN: IV Recovery of the cAMP signaling response after adaptation to cAMP. J Cell Biol 86: 545–553, 1980

    Google Scholar 

  14. Klein C: A slowly dissociating form of the cell surface cyclic adenosine 3′:5′-monophosphate receptor of Dictyostelium discoideum. J Biol Chem 254: 12573–12578, 1979

    Google Scholar 

  15. Kesbeke F, Van Haastert PJM: Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum. Biochim Biophys Acta 847: 33–39, 1985

    Google Scholar 

  16. Van Haastert PJM, De Wit RJW: Demonstration of receptor heterogeneity and negative cooperativity by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum. J Biol Chem 259: 13321–13332, 1984

    Google Scholar 

  17. Van Haastert PJM, De Wit RJW, Janssens PMW, Kesbeke F, De Goede J: G-protein mediated interconversions of cell surface cAMP receptors and their involvement in excitation and desensitization of guanylate cyclase in Dictyostelium discoideum. J Biol Chem 261: 6904–6911, 1986

    Google Scholar 

  18. Van Haastert PJM: Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum. Biochem Biophys Res Commun 124: 597–604, 1984

    Google Scholar 

  19. Janssens PMW, Arents JC, Van Haastert PJM, Van Driel R: Forms of the chemotactic adenosine 3′,5′-cyclic phosphate receptor in isolated Dictyostelium discoideum membranes and interconversions induced by guanine nucleotides. Biochemistry 25: 1314–1320, 1986

    Google Scholar 

  20. De Wit RJW, Snaar-Jagalska BE: Folate and cAMP modulate GTP binding to isolated membranes of Dictyostelium discoideum. Functional coupling between cell surface receptors and G-protein. Biochem Biophys Res Commun 129: 11–17, 1985

    Google Scholar 

  21. Theibert A, Devreotes PN: Surface receptor-mediated activation of adenylate cyclase in wild-type cells and aggregation deficient mutants. J Biol Chem 261: 15121–15125, 1986

    Google Scholar 

  22. Van Haastert PJM, Snaar-Jagalska BE, Janssens PMW: The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes. Eur J Biochem 162: 251–258, 1987

    Google Scholar 

  23. Rodbell M: The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284: 17–22, 1980

    Google Scholar 

  24. Gilman AG: G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615–649, 1987

    Google Scholar 

  25. Katada T, Bokoch GM, Smigel MD, Ui M, Gilman AG: The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes. J Biol Chem 259: 3586–3595, 1984

    Google Scholar 

  26. Ui M: Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol Sci 5: 277–279, 1984

    Google Scholar 

  27. Jakobs KH, Aktories K, Schultz G: Mechanism of pertussis toxin action on the adenylate cyclase system. Inhibition of the turn-on reaction of the inhibitory regulatory site. Eur J Biochem 140: 177–181, 1984

    Google Scholar 

  28. Van Dop C, Yamanaka G, Steinberg F, Sekura RD, Manclark LR, Stryer L, Bourne HR: ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J Biol Chem 259: 23–26, 1984

    Google Scholar 

  29. Manning DR, Gilman AG: The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins. J Biol Chem 258: 7059–7063, 1983

    Google Scholar 

  30. Sternweis PC, Robishaw JD: Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem 259: 13806–13813, 1984

    Google Scholar 

  31. Neer EJ, Lok JM, Wolf LG: Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem 259: 14222–14229, 1984

    Google Scholar 

  32. Malbon CC, Mangano TJ, Watkins DC: Heart contains two substrates (MΓ = 40,000 and 41,000) for pertussis toxin-catalyzed ADP-ribosylation that co-purify with Nε. Biochem Biophys Res Commun 128: 809–815, 1985

    Google Scholar 

  33. Stadel JM, Nambi P, Shorr RGL, Sawyer DR, Caron MG, Lefkowitz RJ: Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the β-adrenergic receptor. Proc Natl Acad Sci USA 80: 3173–3177, 1983

    Google Scholar 

  34. Benovic JL, Pike LJ, Corione RA, Staniszewski C, Yoshimasa T, Codina J, Caron MG, Lefkowitz RJ: Phosphorylation of the mammalian β-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. J Biol Chem 260: 7094–7101, 1985

    Google Scholar 

  35. Bouvier M, Hausdorff WP, De Blasi A, O'Dowd BF, Kobilka BK, Caron MG, Lefkowitz RJ: Removal of phosphorylation sites from the β2-adrenergic receptor delays onset of agonist-promoted desensitization. Nature 333: 370–373, 1988

    Google Scholar 

  36. Katada T, Gilman AG, Watanabe Y, Bauer S, Jakobs KH: Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur J Biochem 151: 431–437, 1985

    Google Scholar 

  37. Klein P, Theibert A, Fontana D, Devreotes PN: Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum. J Biol Chem 260: 1757–1764, 1985

    Google Scholar 

  38. Klein C, Lubs-Haukeness J, Simons S: cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum. J Cell Biol 100: 715–720, 1985

    Google Scholar 

  39. Devreotes PN, Sherring JA: Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in Dictyostelium. J Biol Chem 260: 6378–6384, 1985

    Google Scholar 

  40. Klein P, Vaughan R, Borleis J, Devreotes PN: The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript and developmental regulation of expression. J Biol Chem 262: 358–364, 1987

    Google Scholar 

  41. Banga HS, Walker RK, Winberry LK, Rittenhouse SE: Pertussis toxin can activate human platelets. J Biol Chem 262: 14871–14874, 1987

    Google Scholar 

  42. Konijn TM: Microbiological assay of 3′,5′-AMP. Experientia (Basel) 26: 367–369, 1970

    Google Scholar 

  43. Van Haastert PJM: A method for studying cAMP relay in Dictyostelium discoideum: the effect of temperature on cAMP-relay. J Gen Microbiol 130: 2559–2564, 1984

    Google Scholar 

  44. Snaar-Jagalska BE, Devreotes PN, Van Haastert PJM: Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy. J Biol Chem 263: 897–901, 1987

    Google Scholar 

  45. Das OP, Henderson E: A novel technique for gentle lysis of eucaryotic cells. Isolation of plasma membranes from Dictyostelium discoideum. Bioch Biophys Acta 736: 45–56, 1983

    Google Scholar 

  46. Van Haastert PJM: Alteration of receptor/G-protein interaction by putative endogenous protein kinase activity in Dictyostelium discoideum membranes. J Biol Chem 262: 3239–3243, 1987

    Google Scholar 

  47. Kopf GS, Woolkalis MJ, Gerton GL: Evidence for a guanine nucleotidebinding regulatory protein in invertebrate and mammalian sperm. J Biol Chem 261: 7327–7331, 1986

    Google Scholar 

  48. Khachatrian L, Hewlett A, Klein C: Guanine nucleotide inhibition of adenylate cyclase in Dictyostelium discoideum. J Cyclic Nucleotide Prot Phosph Res 10: 179–188, 1985

    Google Scholar 

  49. Rosenthal W, Binder T, Schultz G: NADP efficiently inhibits endogenous but not pertussis toxin-catalyzed covalent modification of membrane proteins incubated with NAD. FEBS Lett 211: 137–143, 1987

    Google Scholar 

  50. Moss J, Stanley SJ, Watkins PA, Burns DL, Manclark CR, Koslow HR, Hewlett EL: Stimulation of the thiol-dependend ADP-ribosyltransferase and NAD glycohydrolase activity of Bordetella pertussis toxin by adenine nucleotides, phospholipids and detergents. Biochemistry 25: 2720–2725, 1986

    Google Scholar 

  51. Lad PM, Olson CV, Smiley PA: Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to pertussis toxin. Proc Natl Acad Sci USA 82: 869–873, 1985

    Google Scholar 

  52. Backlund PS Jr, Meade BD, Manclark CR, Cantoni GL, Aksamit RR: Pertussis toxin inhibition of chemotaxis and ADP-ribosylation of a membrane protein in a human-mouse hybrid cell line. Proc Natl Acad Sci USA 82: 2637–2641, 1985

    Google Scholar 

  53. Snaar-Jagalska BE, Jakobs KH, Van Haastert PJM: Agonist-stimulated high affinity GTP-ase in Dictyostelium discoideum. FEBS Lett 236: 139–144, 1988

    Google Scholar 

  54. Pupillo M, Kumagai A, Pitt G, Firtel RA, Devreotes PN: Multiple α subunits of G proteins in Dictyostelium. Proc Natl Acad Sci USA, in press.

  55. Kumagai A, Pupillo M, Gundersen R, Miake-Lye R, Devreotes PN, Firtel RA: Regulation and function of Gα protein subunits in Dictyostelium. Cell 57: 265–275, 1989

    Google Scholar 

Download references

Author information

Author notes

  1. B. Ewa Snaar-Jagalska & Peter J. M. Van Haastert

    Present address: Department of Biochemistry, University of Groningen, Nijenborgh 16, 9747 AG, Groningen, The Netherlands

Authors and Affiliations

  1. Cell Biology and Genetics Unit, Zoological Laboratory, University of Leiden, Kaiserstraat 63, 2311 GP, Leiden, The Netherlands

    B. Ewa Snaar-Jagalska & Peter J. M. Van Haastert

Authors
  1. B. Ewa Snaar-Jagalska
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter J. M. Van Haastert
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ewa Snaar-Jagalska, B., Van Haastert, P.J.M. Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum . Mol Cell Biochem 92, 177–189 (1990). https://doi.org/10.1007/BF00218135

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00218135

Key words

Search

Navigation