Signaling

M. Berrera, G. Dodoni, S. Monterisi, V. Pertegato, I. Zamparo, and M. Zaccolo(*)

Contents

1 Introduction

2 Cyclic AMP Signaling and Specificity of Response

3 Detection of cAMP: Accumulation Assays versus Real-Time Monitoring

4 cAMP Measurement in Living Cells: CNG Channels

5 cAMP Measurements in Living Cells Using FRET-Based Technology . . .

290 290 293

5.1 PKA-Based Sensors

5.2 Epac-Based Sensors

5.3 Sensors Based on Single cAMP-Binding Domains

6 Conclusions

References

Abstract The study of cAMP signaling has received a renewed impulse since the recognition that a key aspect of this pathway is the tight spatial control of signal propagation. The study of the mechanism that regulates cAMP signaling in space and time has prompted the development of new methodological approaches to detect cAMP in intact cells. Over the last decades, techniques to assess cAMP concentration with high spatial and temporal resolution in living cells have been elaborated that are based on fluorescent molecules and the phenomenon of fluorescence resonance energy transfer (FRET). A FRET-based indicator of cAMP concentration is typically a protein, including two fluorophores that are linked to a cAMP-binding domain. Binding of cAMP causes a change in the protein conformation and, as a consequence, in the distance between the fluorophores, thus altering the energy transfer between them. Several FRET indicators have been developed, differing in their affinity for cAMP, kinetic features and intracellular targeting. Such indicators enable the measurement of cAMP fluctuations as they happen in the complex intracellular environment and are proving to be effective tools to dissect compartmentalized cAMP signaling.

M. Zaccolo

Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, via Orus 2, 35100 Padova, Italy and Faculty of Biomedical and Life Sciences, University of Glasgow, University Avenue, G12 8QQ, Glasgow, UK [email protected]

E. Klussmann, J. Scott (eds.) Protein-Protein Interactions as New Drug Targets. 285

Handbook of Experimental Pharmacology 186, © Springer-Verlag Berlin Heidelberg 2008

Abbreviations: AC, Adenylyl cyclase; AKAP, A-kinase anchoring protein; cAMP, 3'-5'-Cyclic adenosine monophosphate; C, Catalytic subunit of PKA; CFP, Cyan fluorescent protein; CNG, Cyclic-nucleotide-gated channel; DD, dimerization/docking domain; DEP, Disheveled, Egl-10 and Pleckstrin homology domain; Epac, Exchange protein directly activated by cAMP; FRET, Fluorescence resonance energy transfer; GEF, Guanine nucleotide exchange factor; GFP, Green fluorescent protein; HCN, Hyperpolarization-activated cyclic nucleotide modulated channel; IBMX, 3-Isobutyl-1-methylxanthine; IS, Inhibitory site of PKA; mp, Myristoylation and palmitoylation sequence; nls, Nuclear localization sequence; NE, Norepinephrine; PDE, Phosphodiesterase; PGE1, Prostaglandin E 1; PKA, Protein kinase A; R, Regulatory subunit of PKA; Rol, Rolipram; YFP, Yellow fluorescent protein

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