A genetically encoded, fluorescent indicator for cyclic AMP in living cells.
Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.
Zaccolo
M
M
Department of Experimental Biomedical Sciences, University of Padova, 35121 Padova, Italy.
De Giorgi
F
F
Cho
C Y
CY
Feng
L
L
Knapp
T
T
Negulescu
P A
PA
Taylor
S S
SS
Tsien
R Y
RY
Pozzan
T
T
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D020732:Cytogenetic Analysis; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods*; D016296:Mutagenesis / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics
D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D017362:Cyclic AMP Response Element-Binding Protein; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005576:Colforsin; D003994:Bucladesine; D011433:Propranolol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D001618:beta-Lactamases; D007545:Isoproterenol; D009638:Norepinephrine