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Protein-Protein Interactions as New Drug Targets pp 285–298Cite as

A Toolkit for Real-Time Detection of cAMP: Insights into Compartmentalized Signaling

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Part of the book series: Handbook of Experimental Pharmacology ((HEP,volume 186))

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.

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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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Author information

Authors and Affiliations

  1. Dvn Biochemistry and Molecular Biology, IBLS, Wolfson Link Building, University Avenue, Glasgow, G12 8QQ, Scotland, UK

    M. Berrera

  2. Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, via Orus 2, 35100, Padova, Italy

    G. Dodoni, S. Monterisi, V. Pertegato, I. Zamparo & M. Zaccolo

  3. Faculty of Biomedical and Life Sciences, University of Glasgow, University Avenue, G12 8QQ, Glasgow, UK

    M. Zaccolo

Authors
  1. M. Berrera
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  2. G. Dodoni
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  3. S. Monterisi
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  4. V. Pertegato
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  5. I. Zamparo
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  6. M. Zaccolo
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Editor information

Editors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Enno Klussmann

  2. The Vollum Institute, Oregon Health & Science University, Medical Research Bldg, Rm 322D 3181 SW Sam Jackson Park Road, Portland, OR, 97239-3098, USA

    John Scott Ph.D.

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© 2008 Springer-Verlag Berlin Heidelberg

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Berrera, M., Dodoni, G., Monterisi, S., Pertegato, V., Zamparo, I., Zaccolo, M. (2008). A Toolkit for Real-Time Detection of cAMP: Insights into Compartmentalized Signaling. In: Klussmann, E., Scott, J. (eds) Protein-Protein Interactions as New Drug Targets. Handbook of Experimental Pharmacology, vol 186. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-72843-6_12

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