Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Forskolin as a Tool for Examining Adenylyl Cyclase Expression, Regulation, and G Protein Signaling

Abstract

1. As initially shown by Seamon and Daly, the diterpene forskolin directly activates adenylyl cyclase (AC) and raises cyclic AMP levels in a wide variety of cell types. In this review, we discuss several aspects of forskolin action that are often unappreciated. These include the utility of labeled forskolin as a means to quantitate the number of AC molecules; results of those types of studies, coupled with efforts to increase AC expression, document that such expression stoichiometrically limits cyclic AMP formation by hormones and neurotransmitters.

2. Response to forskolin is also strongly influenced by the activation of AC by the heterotrimeric G-protein, Gs. Gs-promoted enhancement of AC activity in response to forskolin occurs not only when cells are incubated with exogenously administered agonists that activate G-protein-coupled receptors but also by agonists that can be endogenously released by cells.

3. Such agonists, which include ATP and prostaglandins, serve as autocrine/paracrine regulators of cellular levels of cyclic AMP under “basal” conditions and also in response to forskolin and to agonists that promote release of such regulators.

4. The ability of forskolin to prominently activate cyclic AMP generation has proved valuable for understanding stoichiometry of the multiple components involved in “basal” cyclic AMP formation, in enzymologic studies of AC as well as in defining responses to cyclic AMP in cells within and outside the nervous system.

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

References

  1. Alousi, A. A., Jasper, J. R., Insel, P. A., and Motulsky, H. J. (1991). Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J. 5:2300–2303.

  2. Appel, N. M., Robbins, J. D., De Souza, E. B., and Seamon, K. B. (1992). [125I]-Labeled forskolin analogs which discriminate adenylyl cyclase and a glucose transporter: Pharmacological characterization and localization of binding sites in rat brain by in vitro receptor autoradiography. J. Pharmacol. Exp. Ther. 263:1415–1423.

  3. Barber, R. (1988). Forskolin binding to intact S49 lymphoma cells. Second Messengers Phosphoproteins 12:59–71.

  4. Beavo, J. A., and Brunton, L. L. (2002). Cyclic nucleotide research–Still expanding after half a century. Nat. Rev. Mol. Cell. Biol. 3:710–718.

  5. Bender, J. L., and Neer, E. J. (1983). Properties of the adenylate cyclase catalytic unit from caudate nucleus. J. Biol. Chem. 258:2432–2439.

  6. Cassel, D., and Selinger, Z. (1977). Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. U.S.A. 74:3307–3311.

  7. Cerione, R. A., Sibley, D. R., Codina, J., Benovic, J. L., Winslow, J., Neer, E. J., Birnbaumer, L., Caron, M. G., and Lefkowitz, R. J. (1984). Reconstitution of a hormone-sensitive adenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit. J. Biol. Chem. 259:9979–9982.

  8. Daly, J. W. (1984). Forskolin, adenylate cyclase, and cell physiology: An overview. Adv. Cyclic Nucleotide Prot. Phosph. Res. 17:81–89.

  9. Daly, J. W., Padgett, W., and Seamon, K. B. (1982). Activation of cyclic AMP-generating systems in brain membranes and slices by the diterpene forskolin: Augmentation of receptor-mediated responses. J. Neurochem. 38:532–544.

  10. Darfler, F. J., Mahan, L. C., Koachman, A. M., and Insel, P. A. (1982). Stimulation of forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J. Biol. Chem. 257:11901–11907.

  11. Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997). Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase. J. Biol. Chem. 272:22272–22277.

  12. Dessauer, C. W., Tesmer, J. J., Sprang, S. R., and Gilman, A. G. (1998). Identification of a Gi alpha binding site on type V adenylyl cyclase. J. Biol. Chem. 273:25831–25839.

  13. Fagan, K. A., Smith, K. E., and Cooper, D. M. (2000). Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains. J. Biol. Chem. 275:26530–26537.

  14. Gao, M., Ping, P., Post, S., Insel, P. A., Tang, R., and Hammond, H. K. (1998). Increased expression of adenylyl cyclase type VI proportionately increases beta-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes. Proc. Nat. Acadv Sci. U.S.A. 95:1038–1043.

  15. Green, D. A., and Clark, R. B. (1982a). Direct evidence for the role of the coupling proteins in forskolin activation of adenylate cyclase. J. Cyclic Nucleotide Res. 8:337–346.

  16. Green, D. A., and Clark, R. B. (1982bs). Direct evidence for the role of the coupling proteins in forskolin activation of adenylate cyclase. J. Cyclic Nucleotide Res. 8:337–346.

  17. Hanoune, J., and Defer, N. (2001). Regulation and role of adenylyl cyclase isoforms. Annu. Rev. Pharmacol. Toxicol. 41:145–174.

  18. Jasper, J. R., Michel, M. C., and Insel, P. A. (1988). Molecular mechanism of beta-adrenergic receptor blockers with intrinsic sympathomimetic activity. FASEB J. 2:2891–2894.

  19. Jasper, J. R., Post, S. R., Desai, K. H., Insel, P. A., and Bernstein, D. (1995). Colchicine and cytochalasin B enhance cyclic AMP accumulation via postreceptor actions. J. Pharmacol. Exp. Ther. 274:937–942.

  20. Laurenza, A., Robbins, J. D. and Seamon, K. B. (1992). Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: Synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase. Mol. Pharmacol. 41:360–368.

  21. Laurenza, A., and Seamon, K. B. (1991). High-affinity binding sites for [3H]forskolin. Methods Enzymol. 195:52–65.

  22. Lazarowski, E. R., Homolya, L., Boucher, R. C., and Harden, T. K. (1997). Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J. Biol. Chem. 272:24348–24354.

  23. Lazarowski, E. R., and Harden, T. K. (1999). Quantitation of extracellular UTP using a sensitive enzymatic assay. Br. J. Pharmacol. 127:1272–1278.

  24. Leiber, D., Jasper, J. R., Alousi, A. A., Martin, J., Bernstein, D., and Insel, P. A. (1993). Alteration in Gs-mediated signal transduction in S49 lymphoma cells treated with inhibitors of microtubules. J. Biol. Chem. 268:3833–3837.

  25. MacEwan, D. J., Kim, G. D., and Milligan, G. (1996). Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108–15 cells transfected to co-express adenylate cyclase type II and the beta 2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity. Biochem. J. 318:1033–1039.

  26. May, D. C., Ross, E. M., Gilman, A. G., and Smigel, M. D. (1985). Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins. J. Biol. Chem. 260:15829–15833.

  27. Milligan, G. (1996). The stoichiometry of expression of protein components of the stimulatory adenylyl cyclase cascade and the regulation of information transfer. Cell. Signall. 8:87–95.

  28. Morris, D. I., Robbins, J. D., Ruoho, A. E., Sutkowski, E. M., and Seamon, K. B. (1991a). Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. J Biol. Chem. 266:13377–13384.

  29. Morris, D. I., Speicher, L. A., Ruoho, A. E., Tew, K. D., and Seamon, K. B. (1991b). Interaction of forskolin with the P-glycoprotein multidrug transporter. Biochemistry 30:8371–8379.

  30. Ono, K., Fozzard, H. A., and Hanck, D. A. (1995). A direct effect of forskolin on sodium channel bursting. Pflugers Arch. 429:561–569.

  31. Ostrom, R. S., Gregorian, C., and Insel, P. A. (2000a). Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J. Biol. Chem. 275:11735–11739.

  32. Ostrom, R. S., Liu, X., Head, B. P., Gregorian, C., Seasholtz, T. M., and Insel, P. A. (2002). Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: Expression in caveolin-rich and non-caveolin domains. Mol. Pharmacol. 62:983–992.

  33. Ostrom, R. S., Post, S. R., and Insel, P. A. (2000b). Stoichiometry and compartmentation in G protein-coupled receptor signaling: Implications for therapeutic interventions involving Gs. J. Pharmacol. Exp. Ther. 294:407–412.

  34. Ostrom, R. S., Violin, J. D., Coleman, S., and Insel, P. A. (2000c). Selective enhancement of beta-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: Colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Mol. Pharmacol. 57:1075–1079.

  35. Pfeuffer, E., and Pfeuffer, T. (1989). Affinity labeling of forskolin-binding proteins. Comparison between glucose carrier and adenylate cyclase. FEBS Lett. 248:13–17.

  36. Post, S. R., Aguila-Buhain, O., and Insel, P. A. (1996). A key role for protein kinase A in homologous desensitization of the beta 2-adrenergic receptor pathway in S49 lymphoma cells. J. Biol. Chem. 271:895–900.

  37. Post, S. R., Hilal-Dandan, R., Urasawa, K., Brunton, L. L., and Insel, P. A. (1995). Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem. J. 311:75–80.

  38. Robbins, J. D., Appel, N. M., Laurenza, A., Simpson, I. A., De Souza, E. B., and Seamon, K. B. (1992). Differential identification and localization of adenylyl cyclase and glucose transporter in brain using iodinated derivatives of forskolin. Brain Res. 581:148–152.

  39. Rybin, V. O., Xu, X., Lisanti, M. P., and Steinberg, S. F. (2000). Differential targeting of beta-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J. Biol. Chem. 275:41447–41457.

  40. Schwencke, C., Okumura, S., Yamamoto, M., Geng, Y. J., and Ishikawa, Y. (1999). Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane. J. Cell. Biochem. 75:64–72.

  41. Seamon, K. B., Daly, J. W., Metzger, H., de Souza, N. J., and Reden, J. (1983). Structure–activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives. J. Med. Chem. 26:436–439.

  42. Seamon, K. B., Padgett, W., and Daly, J. W. (1981). Forskolin: Unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. U.S.A. 78:3363–3367.

  43. Seamon, K. B., Vaillancourt, R., Edwards, M., and Daly, J. W. (1984). Binding of [3H]forskolin to rat brain membranes. Proc. Natl. Acad. Sci. U.S.A. 81:5081–5085.

  44. Shanahan, M. F., Morris, D. P., and Edwards, B. M. (1987). [3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter. J. Biol. Chem. 262:5978–5984.

  45. Sievert, M. K., Pilli, G., Liu, Y., Sutkowski, E. M., Seamon, K. B., and Ruoho, A. E. (2002). Photoaffinity labeling of adenylyl cyclase. Methods Enzymol. 345:188–197.

  46. Steinberg, S. F., and Brunton, L. L. (2001). Compartmentation of g protein-coupled signaling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. 41:751–773.

  47. Sternweis, P. C., and Gilman, A. G. (1982). Aluminum: A requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc. Natl. Acad. Sci. U.S.A. 79:4888–4891.

  48. Sutkowski, E. M., Tang, W. J., Broome, C. W., Robbins, J. D., and Seamon, K. B. (1994). Regulation of forskolin interactions with type I, II, V and VI adenylyl cyclases by Gs alpha. Biochemistry 33:12852–12859.

  49. Tesmer, J. J., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997). Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTP gammas. Science 278:1907–1916.

  50. Torres, B., Zambon, A. C., and Insel, P. A. (2002). P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D(1) cells. A mechanism for nucleotide-mediated autocrine-paracrine regulation. J. Biol. Chem. 277:7761–7765.

  51. Wadzinski, B. E., Shanahan, M. F., and Ruoho, A. E. (1987). Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label. J. Biol. Chem. 262:17683–17689.

  52. Zhang, G., Liu, Y., Ruoho, A. E., and Hurley, J. H. (1997). Structure of the adenylyl cyclase catalytic core. Nature 386:247–253.

  53. Zheng, B., Ma, Y. C., Ostrom, R. S., Lavoie, C., Gill, G. N., Insel, P. A., Huang, X. Y., and Farquhar, M. G. (2001). RGS-PX1, a GAP for G alpha s and sorting nexin in vesicular trafficking. Science 294:1939–1942.

  54. Zhong, H., Guerrero, S. W., Esbenshade, T. A., and Minneman, K. P. (1996). Inducible expression of beta 1-and beta 2-adrenergic receptors in rat C6 glioma cells: Functional interactions between closely related subtypes. Mol. Pharmacol. 50:175–184.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Insel, P.A., Ostrom, R.S. Forskolin as a Tool for Examining Adenylyl Cyclase Expression, Regulation, and G Protein Signaling. Cell Mol Neurobiol 23, 305–314 (2003). https://doi.org/10.1023/A:1023684503883

Download citation

  • forskolin
  • adenylyl cyclase
  • G-protein-coupled receptors
  • ATP release
  • P2Y receptors
  • stoichiometry
  • Gs