Abstract
The second messenger cyclic guanosine 5′-monophosphate (cGMP) plays a key role in the control and regulation of a steadily increasing number of diverse physiological processes. As the appreciation of the importance of understanding the cGMP signaling pathway has grown, so has the awareness of the limited techniques with which to study the rapid intracellular cGMP kinetics. We have previously demonstrated the construction of cygnets, cGMP indicators using energy transfer comprised of cyan and yellow variants of green fluorescent protein flanked by conformationally sensitive cGMP receptor portion taken from the cGMP-dependent protein kinase (7). Here, we report that cGMP binds to Cygnet-2.1, utilizing ECFP and Citrine, with an apparent equilibrium-binding constant of 600 nM causing a total fluorescence intensity ratio change of 45%. In contrast, cAMP could elicit a maximal 10% change in fluorescence resonance energy transfer (FRET) ratio, demonstrating an approx 500-fold selectivity for cGMP. When expressed in vascular smooth muscle cells, cygnets demonstrated even cytosolic distribution and nuclear exclusion. Cultured rat aortic smooth muscle cells, which exhibit a noncontractile, synthetic phenotype typically seen in response to atherosclerosis or vascular injury, responded to natriuretic peptide (BNP)-mediated activation of the particulate guanylyl cyclase. In conclusion, cygnets have facilitated the temporal resolution and evaluation of the contributions of cyclases and phosphodiesterases in determining overall cGMP accumulation, and the visualization of novel spatial dynamics that will contribute to more fully understanding the role of cGMP in the mediation of smooth muscle relaxation.
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References
Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.
Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.
Miyawaki, A., Griesbeck, O., Heim, R., and Tsien, R. Y. (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140.
Zaccolo, M. and Pozzan, T. (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711–1715.
Zaccolo, M., De Giorgi, F., Cho, C. Y., Feng, L., Knapp, T., Negulescu, P. A., Taylor, S. S., Tsien, R. Y., and Pozzan, T. (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell. Biol. 2, 25–29.
Honda, A., Adams, S. R., Sawyer, C. L., Lev-Ram, V., Tsien, R. Y., and Dostmann, W. R. G. (2001) Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc. Natl. Acad. Sci. USA 98, 2437–2442
Sawyer, C. L., Honda, A., and Dostmann, W. R. G. (2003) Cygnets: spatial and temporal analysis of intracellular cGMP. Proc. West. Pharmacol. Soc. 46, 28–31.
Ruth, P., Landgraf, W., Keilbach, A., May, B., Egleme, C., and Hofmann, F. (1991) The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini. Eur. J. Biochem. 202, 1339–1344.
Zhao, J., Trewhella, J., Corbin, J., Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997) Progressive cyclic nucleotide-induced conformational changes in the cGMP-dependent protein kinase studied by small angle X-ray scattering in solution. J. Biol. Chem. 272, 31,929–31,936.
Wall, M. E., Francis, S. H., Corbin, J. D., Grimes, K., Richie-Jannetta, R., Kotera, J., Macdonald, B. A., Gibson, R. R., and Trewhella, J. (2003) Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 100, 2380–2385.
Whitford, W. G. and Mertz, L. M. (1996) Multiplicity of bacoloviral infection and recombinant protein production in sf9 cells. Focus 18, 3: 75–76.
Luckow, V. A., Lee, C. S., Barry, G. F., and Olins, P. O. (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579.
Wernert, W., Flockerzi, V., and Hofmann, F. (1989), The cDNA of the two isoforms of bovine cGMP-dependent protein kinase. FEBS Lett. 251, 191–196.
Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., and Tsien, R. Y. (2001) Reducing the environmental sensitivity of yellow fluorescent protein: mechanism and applications. J. Biol. Chem. 276, 29,188–29,194.
Feil, R., Kellermann, J., and Hofmann, F. (1995) Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516. Biochemistry 34, 13,152–13,158.
Feil, R., Bigl, M., Ruth, P., and Hofmann, F. (1993) Expression of cGMP-dependent protein kinase in Escherichia coli. Mol. Cell. Biochem. 127–128, 71–80.
Keilbach, A., Ruth, P., and Hofmann, F. (1992) Detection of cGMP dependent protein kinase isozymes by specific antibodies. Eur. J. Biochem. 208, 467–473.
Dostmann, W. R. G., Nickl, C., Thiel, S., Tsigelny, I., Frank, R., and Tegge, W. (1999) Delineation of selective cyclic GMP-dependent protein kinase I substrate and inhibitor peptides based on combinatorial peptide libraries on paper. Pharmacol. Ther. 82, 373–387.
Heil, W. G., Landgraf, W., and Hofmann, F. (1987) A catalytically active fragment of cGMP-dependent protein kinase: occupation of its cGMP-binding sites does not affect its phosphotransferase activity. Eur. J. Biochem. 168, 117–121.
Ishii, K., Sheng, H., Warner, T. D., Forstermann, U., and Murad, F. (1991) A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblasts. Am. J. Physiol. 261, H598–H603.
Cornwell, T. L. and Lincoln, T. M. (1989) Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells: reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J. Biol. Chem. 264, 1146–1155.
Travo, P., Barret, G., and Burnstock, G. (1980) Differences in proliferation of primary cultures of vascular smooth muscle cells taken from male and female rats. Blood Vessels 17, 110–116.
Korshunov, V. A. and Berk, B. C. (2003) Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening. Arterioscl. Thromb. Vasc. Biol. 23, 2185–2191.
Acknowledgments
We thank Dr. F. Hofmann for providing the bovine cGMP-dependent protein kinase type Iα cDNA, and Dr. R. Y. Tsien for providing the yellow cameleon-2 plasmid and citrine cDNA. This work was supported by National Science Foundation Grant MCB-9983097, the Lake Champlain Cancer Research Organization, the Totman Medical Research Trust, and American Heart Association Grant 9920260T.
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Honda, A., Sawyer, C.L., Cawley, S.M., Dostmann, W.R.G. (2005). Cygnets. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:027
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DOI: https://doi.org/10.1385/1-59259-839-0:027
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