Cellular and Molecular Neurobiology

, Volume 23, Issue 3, pp 305–314 | Cite as

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

  • Paul A. Insel
  • Rennolds S. Ostrom


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.

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


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  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.Google Scholar
  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.Google Scholar
  3. Barber, R. (1988). Forskolin binding to intact S49 lymphoma cells. Second Messengers Phosphoproteins 12:59–71.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  8. Daly, J. W. (1984). Forskolin, adenylate cyclase, and cell physiology: An overview. Adv. Cyclic Nucleotide Prot. Phosph. Res. 17:81–89.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  17. Hanoune, J., and Defer, N. (2001). Regulation and role of adenylyl cyclase isoforms. Annu. Rev. Pharmacol. Toxicol. 41:145–174.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  21. Laurenza, A., and Seamon, K. B. (1991). High-affinity binding sites for [3H]forskolin. Methods Enzymol. 195:52–65.Google Scholar
  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.Google Scholar
  23. Lazarowski, E. R., and Harden, T. K. (1999). Quantitation of extracellular UTP using a sensitive enzymatic assay. Br. J. Pharmacol. 127:1272–1278.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  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.Google Scholar
  52. Zhang, G., Liu, Y., Ruoho, A. E., and Hurley, J. H. (1997). Structure of the adenylyl cyclase catalytic core. Nature 386:247–253.Google Scholar
  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.Google Scholar
  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.Google Scholar

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Paul A. Insel
    • 1
  • Rennolds S. Ostrom
    • 1
  1. 1.Department of Pharmacology, School of MedicineUniversity of CaliforniaSan Diego, La JollaUSA

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