# Patent

## Title
Camp reporters and high throughput assays

## Abstract

cAMP reporters useful for obtaining measurements of cAMP levels with high spatial and temporal resolution and in high throughput assays.

## Background
This application claims the benefit of and is a continuation-in-part of PCT/US2005/030099 filed Aug. 23, 2005, which claims the benefit of provisional applications Ser. No. 60/603,623 filed Aug. 23, 2004 and Ser. No. 60/681,923 filed May 17, 2005. This application also claims the benefit of Ser. No. 60/730,583 filed Oct. 27, 2005. Each of these applications is incorporated by reference in its entirety herein.


This invention was made using funds from NIH grants DK073368, GM066170 and GM08763 and USPHS training grant T32 CA009243. The government retains certain rights in the invention.


FIELD OF THE INVENTION
The invention relates to detection of cAMP levels.
BACKGROUND OF THE INVENTION
Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. Various methods exist for detecting and measuring intracellular cAMP, but none are ideally suited for monitoring spatial and temporal distributions of cAMP in living cells. For example, radioimmunoassay or enzyme immunoassays for measuring cAMP require destroying large amounts of cells or tissue, have very poor spatial and temporal resolution, and measure total rather than free cAMP. Use of engineered cyclic nucleotide-gated channels to detect free cAMP provides good temporal resolution and quantification but uses indirect calcium measurements or nontrivial patch-clamp techniques and lacks the flexibility of measuring cAMP changes within various subcellular compartments (Rich et al., Proc. Natl. Acad. Sci. USA 98, 13049-54, 2001; Rich et al., J. Gen. Physiol. 116, 147-61, 2000). Free cAMP can be imaged in single cells microinjected with fluorophore-labeled C and R subunits (Adams et al., Nature 349, 694-97, 1991) or in cells expressing two colors of GFP mutants fused to the C and R subunits (Zaccolo et al., Nat. Cell Biol. 2, 25-29, 2000), which dissociate from each other and lose fluorescence resonance energy transfer upon elevation of cAMP. However, the expression levels of the two fusions have to be carefully matched to allow reliable measurement. Even so, mixed tetramerization may occur between the fluorophore-attached subunits and endogenous partners, reducing the number of functional reporter molecules. Furthermore, it can be difficult to target such bimolecular reporters to different subcellular locations while maintaining appropriate stoichiometry.
There is a need in the art for sensitive cAMP reporters and methods which can be used for accurate measurements of spatial and temporal cAMP distributions in living cells.



BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1. Domain structure and comparison of FRET responses for cAMP reporters. Sandwiched between enhanced CFP (ECFP) and citrine are truncated forms of Epac2 or full length Epac1 (with or without an R522E mutation). The construct comprising full-length Epac1 generated the biggest FRET response and was designated as ICUE1.
FIGS. 2A-C. Responses of ICUE1 to changes in cellular cAMP levels. FIG. 2A, FRET response of HEK-293 cells transfected with ICUE1. The first image is a YFP-only image. Pseudocolor images depict the FRET response of the reporter to isoproterenol (ISO) stimulation at various time points. Scale bar represents 10 μm. FIG. 2B, Representative emission ratio time courses of ICUE1 and the R522E mutant stimulated with 10 μM ISO followed by 10 μM propranolol and 50 μM forskolin (FSK). FIG. 2C, Representative emission ratio time courses of ICUE1 stimulated with 10 μM ISO, 50 μM FSK, 10 μM PGE1, 300 μM 8-pCPT-2′-O-Me-cAMP, or 100 μM of DMNB-cAMP followed by UV uncaging. The flash signs indicate 5 second UV flash at two different time points.
FIGS. 3A-E. Fusions of ICUE1 targeted to various subcellular locations. FIG. 3A, Domain structures of the fusion constructs. FIG. 3B, YFP-only images showing plasma membrane and nuclear distributions of various fusions. Scale bars represent 10 μm. Merged pseudocolor images showing co-localization of nuclear localized ICUE1 with Hoechst 33342 cell-permeable dye in nucleus and mitochondria-targeted ICUE1 with MitoTracker at mitochondria. FIG. 3C, Representative emission ratio time courses for untagged (ICUE1), plasma membrane-targeted (pm ICUE1), mitochondria-targeted (MitoCOX- and MitoDAKAP1-ICUE1 ) and nuclear-localized cAMP reporters (NLS-ICUE1) stimulated with ISO (10 μM). FIG. 3D, Representative emission ratio time courses for pm ICUE1 stimulated with PGE1 (10 μM), followed by the removal of PGE1 and the addition of ISO (10 μM). FIG. 3E, Representative emission ratio time courses for NLS-ICUE1 in response to PGE1 (10 μM) and ISO (10 μM) separated by a washing step.
FIGS. 4A-C. Simultaneous imaging of cAMP reporters targeted to different subcellular locations. FIG. 4A, Cellular distribution of different fusions. FIG. 4B, Representative emission ratio time courses for the pm ICUE1 and nuclear localized PKA activity reporter (NLS-AKAR) in the same cell stimulated with ISO (10 μM). Identical results were found in four different cells. The AKAR response was plotted using normalized ratio of yellow to cyan emissions. FIG. 4C, Representative emission ratio time courses for pm ICUE1 and NLS-ICUE1 in the same cell stimulated with 10 μM ISO followed by 10 μM propranolol (n=4).
FIG. 5. Graph showing emission ratio time courses for ICUE2 and targeted versions of ICUE2. Y axis, normalized emission ratio (cyan/yellow).
FIG. 6. Graph showing emission ratio time courses for ICUE2 and ICUE3.
FIG. 7. High throughput analysis of the effect of a combination of various small molecules was performed using stable ICUE2 HEK293 cells in a 96-well plate format. The ratios of cyan emission/yellow emission were calculated before and after drug additions, and the average changes in emission ratios were plotted. Different drugs were used in the following concentrations: 1 μM isoproterenol (Iso), 10 μM H-89, 100 μM IBMX, and 50 μM forskolin (FSK). 1 cycle=90 seconds.
FIG. 8. High throughput analysis of the effect of a combination of various small molecules was performed using stable ICUE2 HEK293 cells in a 96-well plate format. The ratios of cyan emission/yellow emission were calculated before and after drug additions, and the average changes in emission ratios were plotted. Different drugs were used in the following concentrations: 1 μM isoproterenol (Iso), 10 μM H-89, 100 μM IBMX, and 50 μM forskolin (FSK). 1 cycle=60 seconds.
FIG. 9. High throughput analysis of the effect of a combination of various small molecules was performed using HEK293 cells expressing ICUE3 in a 96-well plate format. The ratios of cyan emission/yellow emission were calculated before and after drug additions, and the average changes in emission ratios were plotted. Different drugs were used in the following concentrations: 1 μM isoproterenol (Iso), 10 μM H-89, 100 μM IBMX, 50 μM forskolin (FSK), and 100 μM propranolol (PRO).
FIG. 10. Development of improved ICUE. FIG. 10A, domain structures of the second generation of the “Indicator of cAMP Using Epac,” ICUE2, as well as its Venus and cpVenus (cpV) variants. ICUE cpV L194 (R373E) contains an R373E mutation in the cAMP binding site. FIG. 10B, representative time courses of HEK-293 cells expressing ICUE cpV L194 (ICUE3, circles), ICUE2 (squares), and ICUE (R373E) (triangles), treated with 50 μM FSK.
FIG. 11. High throughput cellular assays based on ICUE3. Summary of ICUE3 responses in HEK-293 cells treated with indicated drugs. Ratios of cyan/yellow emission were calculated and plotted versus cycle numbers. Each cycle lasted 145 seconds. Error bars represent standard deviation (n=7). Different drugs were used in the following concentrations: ISO, 1 μM; H-89, 10 μM; 3-isobutyl-1-methylxanthine (IBMX), 100 μM; FSK, 50 μM; and propranolol (Pro), 10 μM.
FIG. 12. Ratios of YFP fluorescence intensities to CFP fluorescence intensities for ICUE constructs. Average values of YFP intensities were divided by the average intensities of CFP after YFP photobleaching. YFP photobleaching was achieved by irradiating without neutral density filters through a 525DF40 filter (Chroma Technologies).
FIG. 13. High-throughput screening with ICUE3. FIG. 13A, normalized emission ratio (cyan/yellow) from cells expressing ICUE treated with individual library compounds, compared to the negative control in which only buffer was added and the positive control in which isoproterenol (ISO) (250 nM) and 3-Isobutyl-1-methylxanthine (IBMX)(100 μM) was added (agonist screen). FIG. 13B, normalized emission ratio (cyan/yellow) from cells expressing ICUE first treated with individual library compounds for about 15 min then stimulated by ISO (250 nM) and IBMX (100 μM) (antagonist screen). The positive control in which only ISO and IBMX was added and the negative control in which only buffer was added are shown as labeled.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides highly sensitive reporter molecules by which temporal and spatial distribution of cAMP can be determined in living tissues. “cAMP reporters” (also referred to as “reporters”) of the invention comprise (a) a donor moiety; (b) a polypeptide linked to the donor moiety and comprising a cAMP-binding domain of an “exchange protein directly activated by cAMP” (Epac) or a cAMP-binding domain (including mutated versions) from protein kinase A, a cyclic nucleotide-gated channel, or other cAMP-responsive domain or protein (e.g., the Tandem GAF Domain of phosphodiesterases; Gross-Langenhoff et al., J. Biol. Chem. 282, 2841-46); and (c) an acceptor moiety linked to the polypeptide. In the absence of cAMP, the donor moiety and the acceptor moiety are in sufficient proximity to each other to exhibit a detectable resonance energy transfer when the donor is excited. Binding of cAMP to the cAMP-binding domain causes a conformational change which changes the distance or relative orientation between the donor and acceptor moieties and alters the resonance energy transfer between the moieties. The degree of alteration reflects cAMP levels and can be detected qualitatively or quantitatively.
cAMP reporters of the invention are useful for detecting intracellular cAMP and for assessing intracellular cAMP dynamics, although they also can be used in in vitro assays. Nucleic acid molecules encoding cAMP reporters of the invention can be delivered to cells using standard DNA transfection techniques, thereby generating cells which express high levels of the reporters. The reporters have advantages over previous methods for assessing cAMP dynamics inside cells. The reporters are unimolecular and can be readily targeted to different subcellular locations or fused to signaling components. They can be used to examine compartmentalized Epac activities and their physiological functions. For example, as described in the Examples below, a cAMP reporter targeted to plasma membrane, mitochondria, or nucleus revealed differential dynamics of cAMP signaling in response to the activation of the β-adrenergic receptor (β-AR) or the prostanoid receptor.
cAMP reporters of the invention permit simultaneous imaging of cAMP dynamics and PKA phosphorylation in single living cells using locus-specific reporters. Methods of the invention take advantage of spatial separation of subcellular events and provide unambiguous temporal correlation of these events. This methodology complements multi-color imaging (Violin et al., J. Cell Biol. 161, 899-909, 2003; DeBernardi & Brooker, Proc. Natl. Acad. Sci. USA 93, 4577-82, 1996) and is well suited for simultaneous monitoring of multiple signaling events and for evaluating the information flow within signaling cascades or crosstalk between different pathways (Zaccolo, Cir. Res. 94, 866-73, 2004).
Polypeptides
Polypeptides used in cAMP reporters of the invention comprise a cAMP-binding domain of an Epac, e.g., Epac1 or Epac2. Epac1 and Epac2 are well-characterized, and the locations of their cAMP-binding domains are known. See de Rooij et al., J. Biol. Chem. 275, 20829-36, 2000. Useful polypeptides include full-length, truncated, and mutated Epac1 or Epac2 from any species which has an Epac, such as rodents (e.g., mice, rats) and primates (e.g., humans, orangutans). The amino acid sequences of several Epac1 and Epac2 proteins are provided in SEQ ID NOS:1, 3, and 17-20. Nucleic acid sequences which encode SEQ ID NOS:1, 3, and 20 are shown in SEQ ID NOS:2, 4, and 21, respectively. The cAMP-binding domain in a cAMP reporter typically can bind cAMP; however, polypeptides comprising non-functional cAMP-binding domains are also useful, for example, for use in control reporters. The polypeptide itself preferably does not substantially emit light or transfer energy to excite the acceptor moiety.
Donor and Acceptor Moieties
As used here, a “donor moiety” is a fluorophore or a luminescent moiety. The absorption spectrum of the “acceptor moiety” overlaps the emission spectrum of the donor moiety. The acceptor moiety does not need to be fluorescent and can be a fluorophore, chromophore, or quencher. In some embodiments both the donor and acceptor moieties are fluorescent proteins. In other embodiments both the donor and acceptor moieties are luminescent moieties. In yet other embodiments, either one of the donor or acceptor moieties can be a fluorescent protein while the other moiety is a luminescent moiety. In other embodiments, the acceptor moiety is a “quencher moiety.”
When both the donor and acceptor moieties are fluorophores, resonance energy transfer is detected as “fluorescence resonance energy transfer” (FRET). If a luminescent moiety is involved, resonance energy transfer is detected as “luminescent resonance energy transfer” (LRET). LRET includes “bioluminescent resonance energy transfer” (BRET; Boute et al., Trends Pharmacol. Sci. 23, 351-54, 2002; Ayoub et al., J. Biol. Chem. 277, 21522-28, 2002). Because excitation of the donor moiety does not require exogenous illumination in an LRET method, such methods are particularly useful in live tissue and animal imaging, because penetration of the excitation light is no longer a concern. LRET methods have a high contrast and high signal-to-noise ratio; 2) no photobleaching occurs; and 3) quantification is simplified because the acceptor moiety is not directly excited.
Suitable acceptor moieties include, for example, a coumarin, a xanthene, a fluorescein, a fluorescent protein, a circularly permuted fluorescent protein, a rhodol, a rhodamine, a resorufin, a cyanine, a difluoroboradiazaindacene, a phthalocyanine, an indigo, a benzoquinone, an anthraquinone, an azo compound, a nitro compound, an indoaniline, a diphenylmethane, a triphenylmethane, and a zwitterionic azopyridinium compound.
Suitable donor moieties include, but are not limited to, a coumarin, a xanthene, a rhodol, a rhodamine, a resorufin, a cyanine dye, a bimane, an acridine, an isoindole, a dansyl dye, an aminophthalic hydrazide, an aminophthalimide, an aminonaphthalimide, an aminobenzofuran, an aminoquinoline, a dicyanohydroquinone, a semiconductor fluorescent nanocrystal, a fluorescent protein, a circularly permuted fluorescent protein, and fluorescent lanthanide chelate.
Fluorescent Proteins
In some preferred embodiments either or both of the donor and acceptor moieties is a fluorescent protein. Suitable fluorescent proteins include green fluorescent proteins (GFP), red fluorescent proteins (RFP), yellow fluorescent proteins (YFP), and cyan fluorescent proteins (CFP). Useful fluorescent proteins also include mutants and spectral variants of these proteins which retain the ability to fluoresce.
RFPs include Discosoma RFPs, such Discosoma DsRed (SEQ ID NO:9) or a mutant thereof which includes an Ile125Arg mutation, or a non-oligomerizing tandem DsRed containing, for example, two RFP monomers linked by a peptide linker. For example, a non-oligomerizing tandem RFP can contain two DsRed monomers or two mutant DsRed-I125R monomers linked by a peptide (having, for example, the amino acid sequence shown in SEQ ID NO:10).
Useful GFPs include an Aequorea GFP (e.g., SEQ ID NO:11), a Renilla GFP, a Phialidium GFP, and related fluorescent proteins for example, a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), or a spectral variant of the CFP or YFP. CFP (cyan) and YFP (yellow) are color variants of GFP. CFP and YFP contain 6 and 4 mutations, respectively. They are Tyr66Try, Phe66Leu, Ser65Thr, Asn145Ile, Met153Thr, and Val163Ala in CFP and Ser65Gly, Val168Leu, Ser72Ala, and Thr203Tyr. Spectral variants include an enhanced GFP (EGFP; SEQ ID NO:12), an enhanced CFP (ECFP; SEQ ID NO:13), an enhanced YFP (EYFP; SEQ ID NO:14), and an EYFP with V68L and Q69K mutations. Other examples of fluorescent proteins comprising mutations are Aequorea GFP with one or more mutations at amino acid residues A206, L221 or F223 of SEQ ID NO:11 (e.g., mutations A206K, L221K, F223R, Q80R); mutations L221K and F223R of ECFP (SEQ ID NO:12), and EYFP-V68L/Q69K of SEQ ID NO:11. See also US 2004/0180378; U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079; Chalfie et al., Science 263:802-805, 1994.
Other useful GFP-related fluorescent proteins include those having one or more folding mutations, and fragments of the proteins that are fluorescent, for example, an A. victoria GFP from which the two N-terminal amino acid residues have been removed. Several of these fluorescent proteins contain different aromatic amino acids within the central chromophore and fluoresce at a distinctly shorter wavelength than the wild type GFP species. For example, the engineered GFP proteins designated P4 and P4-3 contain, in addition to other mutations, the substitution Y66H; and the engineered GFP proteins designated W2 and W7 contain, in addition to other mutations, Y66W.
Folding mutations in Aequorea GFP-related fluorescent proteins improve the ability of the fluorescent proteins to fold at higher temperatures and to be more fluorescent when expressed in mammalian cells, but have little or no effect on the peak wavelengths of excitation and emission. If desired, these mutations can be combined with additional mutations that influence the spectral properties of GFP to produce proteins with altered spectral and folding properties, and, particularly, with mutations that reduce or eliminate the propensity of the fluorescent proteins to oligomerize. Folding mutations, with respect to SEQ ID NO:11, include the substitutions F64L, V68L, S72A, T44A, F99S, Y145F, N1461, M153T, M153A, V163A, I167T, S175G, S205T, and N212K.
Luminescent Moieties
Luminescent moieties useful in a cAMP reporter include lanthanides, which can be in the form of a chelate, including a lanthanide complex containing the chelate (e.g, β-diketone chelates of cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, or ytterbium). Lanthanide chelates are well known in the art. See Soini and Kojola, Clin. Chem. 29, 65, 1983; Hemmila et al., Anal. Biochem. 137, 335 1984; Lovgren et al., In: Collins & Hoh, eds., Alternative Immunoassays, Wiley, Chichester, U.K., p. 203, 1985; Hemmila, Scand. J. Clin. Lab. Invest. 48, 389, 1988; Mikola et al., Bioconjugate Chem. 6, 235, 1995; Peruski et al., J. Immunol. Methods 263, 35-41, 2002; U.S. Pat. Nos. 4,374,120; and 6,037,185. Suitable β-diketones are, for example, 2-naphthoyltrifluoroacetone (2-NTA), 1-naphthoyltrifluoroacetone (1-NTA), p-methoxybenzoyltrifluoroacetone (MO-BTA), p-fluorobenzoyltrifluoroacetone (F-BTA), benzoyltrifluoroacetone (BTA), furoyltrifluoroacetone (FTA), naphthoylfuroylmethane (NFM), dithenoylmethane (DTM), and dibenzoylmethane (DBM). See also US 20040146895.
Luminescent proteins include, but are not limited to, lux proteins (e.g., luxCDABE from Vibrio fischerii), luciferase proteins (e.g., firefly luciferase, Gaussia luciferase, Pleuromamma luciferase, and luciferase proteins of other beetles, Dinoflagellates (Gonylaulax; Pyrocystis;), Annelids (Dipocardia), Molluscs (Lativa), and Crustacea (Vargula; Cypridina), and green fluorescent proteins of bioluminescent coelenterates (e.g., Aequorea Victoria, Renilla mullerei, Renilla reniformis; see Prendergast et al., Biochemistry 17, 3448-53, 1978; Ward et al., Photochem. Photobiol. 27, 389-96, 1978; Ward et al., J. Biol. Chem. 254, 781-88, 1979; Ward et al., Photochem. Photobiol. Rev 4, 1-57, 1979; Ward et al., Biochemistry 21, 4535-40, 1982). Many of these proteins are commercially available. Firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind. Recombinantly produced firefly luciferase is available from Promega Corporation, Madison, Wis. Jellyfish aequorin and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.
The DNA sequences of the aequorin and other luciferases employed for preparation of some cAMP reporters of the invention can be derived from a variety of sources. For example, cDNA can be prepared from mRNA isolated from the species disclosed above. See Faust, et al., Biochem. 18, 1106-19, 1979; De Wet et al., Proc. Natl. Acad. Sci. USA 82, 7870-73, 1985.
Luciferase substrates (luciferins) are well known and include coelenterazine (available from Molecular Probes, Eugene, Oreg.) and ENDURENTM. These cell-permeable reagents can be directly administered to cells, as is known in the art. Luciferin compounds can be prepared according to the methods disclosed by Hori et al., Biochemistry 14, 2371-76, 1975; Hori et al., Proc. Natl. Acad. Sci. USA 74, 4285-87, 1977).
Dark Quenchers
In some embodiments the acceptor moiety is a quencher moiety, preferably a “dark quencher” (or “black hole quencher”) as is known in the art. In this case, the change in conformation which occurs upon cAMP binding eliminates quenching, resulting in an increase in energy emission from the donor moiety. “Dark quenchers” themselves do not emit photons. Use of a “dark quencher” reduces or eliminates background fluorescence or luminescence which would otherwise occur as a result of energy transfer from the donor moiety. Suitable quencher moieties include dabcyl (4-(4′-dimethylaminophenylazo)-benzoic acid), QSYTM-7 carboxylic acid, succinimidyl ester (N,N′-dimethyl-N,N′-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbon yl) piperidinylsulfone-rhodamine (a diarylrhodamine derivative from Molecular Probes, Eugene, Oreg.). Suitable quencher moieties are disclosed, for example, in US 2005/0118619; US 20050112673; and US 20040146959.
Any suitable fluorophore may be used as the donor moiety provided its spectral properties are favorable for use with the chosen dark quencher. The donor moiety can be, for example, a Cy-dye, Texas Red, a Bodipy dye, or an Alexa dye. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, a fluorescein (e.g., fluorescein, tetrachlorofluorescein, hexachlorofluorescein), rhodamine, tetramethyl-rhodamine, or other like compound. Suitable fluorescent moieties for use with dark quenchers include xanthene dyes, such as fluorescein or rhodamine dyes, including 6-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX). Suitable fluorescent reporters also include the naphthylamine dyes that have an amino group in the alpha or beta position. For example, naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).
Other suitable fluorescent moieties include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethy-loxacarbocyanine (CyA); 1H,5H,1H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinol-izin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahyd-ro-inner salt (TR or Texas Red); BODIPYTM dyes; benzoxaazoles; stilbenes; pyrenes; and the like.
Subcellular Targeting Sequences
cAMP reporters of the invention optionally can include a subcellular targeting sequence which can target a cAMP reporter to a subcellular domain such as a plasma membrane, a nuclear membrane, a cytosol, an endoplasmic reticulum, a mitochondria, a mitochondrial matrix, a chloroplast, a medial trans-Golgi cisternae, a lumen of a lysosome, or a lumen of an endosome. Many such targeting sequences are known in the art. Examples include the plasma membrane targeting sequence shown in SEQ ID NO:6, the nuclear localization signal sequence shown in SEQ ID NO:5, the mitochondrial localization sequence shown in SEQ ID NO:7, and the mitochondrial matrix targeting signal shown in SEQ ID NO:8. Targeting sequences can be linked to cAMP reporters using, for example, a tetracysteine motif such as Cys Cys Xaa Xaa Cys Cys (SEQ ID NO:15). Targeting sequences can be linked at either the N- or C-terminus of a cAMP reporter or at intermediate points in the reporter.
In some embodiments, cAMP reporters of the invention do not include those which consist of YFP which is not circularly permuted, CFP which is not circularly permuted, and any of the following polypeptides: amino acids 1-443 of SEQ ID NO:3 (a mouse Epac2), amino acids 1-149 of SEQ ID NO:3, amino acids 29-149 of SEQ ID NO:3, amino acids 285-443 of SEQ ID NO:3, amino acids 304-443 of SEQ ID NO:3, amino acids 310-443 of SEQ ID NO:3, amino acids 285-454 of SEQ ID NO:3, amino acids 285-460 of SEQ ID NO:3, and amino acids 157-316 of SEQ ID NO:1 (human Epac1).
Assembly of cAMP Reporters
cAMP reporters which are fusion proteins preferably can be expressed recombinantly, and the invention provides nucleic acid molecules for this purpose. A nucleic acid molecule encoding a cAMP reporter can comprise any nucleotide sequence which encodes the amino acid sequence of the reporter. Nucleic acid molecules of the invention include single- and double-stranded DNA (including cDNA) and mRNA. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
In some embodiments the nucleic acid molecules are expression constructs which contain the necessary elements for the transcription and translation of an inserted coding sequence encoding a cAMP reporter. Expression constructs can be used as vectors for introducing cAMP reporters into cells. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding cAMP reporters and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
Expression vectors of the invention can be expressed in a variety of host cells. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems, particularly mammalian systems, including human systems. See WO 01/98340, which is incorporated herein by reference in its entirety. The choice of vector components and appropriate host cells is well within the capabilities of those skilled in the art.
Alternatively, protein or non-protein donor and/or acceptor moieties can be linked to the polypeptide by covalent attachment. There are a variety of methods known in the art which are useful for this purpose. For example, the attachment can be direct, via a functional group on the polypeptide (e.g., amino, carboxyl and sulfhydryl groups) and a reactive group on the fluorophore. Free amino groups in the polypeptide can be reacted with fluorophores derivatized with isothiocyanate, maleic anhydride, N-hydroxysuccinimide, tetrafluorylphenyl and pentafluoryl esters. Free carboxyl groups in the polypeptide can be reacted with carbodiimides such as 1-ethyl-3-[dimethylaminopropyl]carbodiimide hydrochloride to create a reactive moiety that will react with an amine moiety on the donor or acceptor moiety. Sulfhydryl groups can be attached to donor or acceptor moieties modified with maleimide and iodoacetyl groups, although such linkages are more susceptible to reduction than linkages involving free amino groups. The polypeptide can also be linked indirectly via an intermediate linker or spacer group, using chemical groups such as those listed above.
It is also possible to produce cAMP reporters of the invention using chemical methods to synthesize the amino acid sequence of the polypeptide and, optionally, one or more fluorescent or luminescent proteins. Methods include direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of polypeptide portions of cAMP reporters can be separately synthesized and combined using chemical methods to produce a full-length reporter molecule. See WO 01/98340.
Delivery of cAMP Reporters to Cells
All cAMP reporters of the invention can be introduced into cells in vitro using reversible permeabilization techniques. See U.S. Pat. Nos. 6,127,177; 6,902,931; Russo et al., Nature Biotechnology 15, 278-82, March 1997; Santangelo et al., Nucleic Acids Res. 32, 1-9, Apr. 14, 2004.
If the cAMP reporter is a fusion protein, expression vectors comprising a cAMP reporter-encoding nucleotide sequence can be transfected into any cell in vitro in which it is desired to monitor cAMP levels or distribution. Any transfection method known in the art can be used, including, for example, including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun,” and DEAE- or calcium phosphate-mediated transfection.
Useful vectors and methods of delivering the vectors to cells in vivo are disclosed, for example, in U.S. Pat. Nos. 6,825,012; 6,878,549; 6,645,942; 6,692,737; 6,689,758; 6,669,935; and 6,821,957.
Methods of Detecting cAMP
The invention provides various methods for detecting cAMP by detecting conformational changes in a cAMP reporter. Broadly, the methods involve detecting a change in resonance energy transfer of a cAMP reporter of the invention when the reporter is subjected to a change in cAMP concentration. cAMP binding to the reporter induces a conformational change that changes resonance energy transfer from the donor moiety to the acceptor moiety.
A change in resonance energy transfer can readily be detected using methods well known in the art. See, e.g., US 2005/0118619; US 2002/0137115; US 2003/0165920; US 2003/0186229; US 2004/0137479; US 2005/0026234; US 2005/0054573; US 2005/0118619; U.S. Pat. Nos. 6,773,885; 6,803,201; 6,818,420;
Ayoub et al., 2002; Boute et al., 2002; Domin et al., Prog. Biomed. Optics and Imaging, Proc. SPIE, vol 5139, 2003, pp 238-242; Evellin et al., Methods Mol. biol. 284, 259-70, 2004; Honda et al., Proc. Natl. Acad. Sci. USA 98, 437-42, Feb. 27, 2001; Honda et al., Methods Mol. Biol. 3, 27-44, 1005; Mongillo et al., Cir. Res. 95, 67-75, Jul. 9, 2004; Mongillo et al., Methods Mol Biol. 307, 1-14, 2005; Nagai et al., Proc. Natl. Acad. Sci. USA 101, 10554-59, Jul. 20, 2004; Nikolaev et al., J. Biol. Chem. 279, 37215-18, 2004; Polit et al., Eur. J. Biochem. 270, 1413-23, 2003; Ponsioen et al., EMBO Rep. 5, 1176-80, 2004; Santangelo et al., Nucl. Acids Res. 32, 1-9, e-published Apr. 14, 2004; and Warrier et al., Am. J. Physiol. Cell Phiol. 289, C455-61, August 2005. Properties which can be detected as resonance energy transfer (RET) measurements include a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, anisotropy, a polarization of emitted light, resonance energy transfer, and a quenching of emission at a wavelength. Fluorescence activated cell sorting (FACS) also can be used (see below).
cAMP reporters of the invention can be used in cell-free systems, in isolated cells (for example, in primary cell culture or a cell line) or in cells in situ (e.g., in an isolated tissue sample, an isolated whole organ, or in a mammal). Subcellular distribution of cAMP or changes in cAMP concentration can be detected, for example, as described in Example 2, below. Absolute cAMP levels can be detected by obtaining a RET measurement in the assay system and comparing it to a standard curve obtained in vitro.
In some embodiments, steady-state RET measurements are first obtained and then measurements are taken after addition of a test compound to the assay system. Test compounds can be used, for example, to increase cAMP concentration to make it easier to detect cAMP in a particular subcellular compartment or to monitor the effect of the test compound on cAMP concentration (e.g., in drug-screening methods). Test compounds can be pharmacologic agents already known in the art to affect cAMP levels or can be compounds previously unknown to have such an activity. Compounds known to affect cAMP levels include, for example, β-adrenergic receptor agonists (e.g., norepinephrine, epinephrine, isoproterenol, sulfonterol, metaproterenol, SB-251023), β-adrenergic receptor antagonists (e.g., propranolol, butoxamine, practolol, alprenolol, pindolol, nadolol, metaprolol, SR-59230A), direct or indirect activators of adenylate cyclase (e.g., forskolin, prostalglandin E1), cAMP analogs (e.g., 8-(4-chloro-phenylthio)-2′-O-methyl adenosine 3′,5′-monophosphate; N6,2′-O-dibutyryl cyclic adenosine 3′,5′monophosphate (Bt2cAMP)), and photolytic release agents (e.g., P-(4,5-dimethoxy-2-nitrobenzyl) adenosine 3′,5′-monophosphate, and phosphodiesterase inhibitors such as 3-isobutyl-1-methylxanthine).
Test compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
High Throughput Assays
Because of their expanded dynamic range, cAMP reporters of the invention can be used advantageously in high throughput analysis of potential pharmacological agonists and antagonists. High throughput assays have sufficient signal amplitude (up to a 35% change in the case of stable cell lines) and tight error bars (see FIG. 9) to distinguish readily the effects of a series of drugs and drug-combinations. The high throughput assay format complements single-cell imaging approaches and can be used to investigate the complexity of cAMP signaling. High throughput assays can be used to screen test compounds to determine their effect on cAMP production and degradation. The methods also are useful for defining previously unknown mechanisms for established drugs. New drug candidates that target G protein coupled receptors, G proteins (Gs, Gi), phosphodiesterases, and adenylyl cyclase can be identified. The methods can also be used diagnostically.
A typical assay employs a 96-well plate format, although other formats can be used (e.g., 192- or 384-well plates). Embodiments of high throughput assays are described in Examples 9 and 12. High throughput assays which use cells expressing a cAMP reporter of the invention are particularly useful. Cellular assays systems negate the need to determine appropriate cofactors for an intended target prior to screening. Cellular assays also ensure that the active compounds already possess adequate solubility, membrane permeability, stability, and the ability to act within the biological complexity of the cell, so that false hits with incompatible properties can be eliminated in the early screening stages.
Fluorescence activated cell sorting (FACS) is well-suited for use with high throughput methods of the invention. For example, emission ratios of cyan-to-yellow for individual cells are detected during the first sorting; not all cells will have the same emission ratio, and a distribution for the whole population will be plotted. The cells can be contacted with a test compound, and emission ratios of individual cells can be detected again during the second sorting. The difference in emission ratios, typically presented as a shift in the distribution, reflects the changes in cAMP concentration.
In some embodiments, steady-state RET measurements are first obtained and then measurements are taken after addition of a test compound to the assay system. Test compounds can be used, for example, to increase cAMP concentrations activity or to monitor the effect of the test compound on cAMP concentrations (e.g., in drug-screening methods). Test compounds can be pharmacologic agents already known in the art to affect cAMP levels or can be compounds previously unknown to have such an activity. Compounds known to affect cAMP levels include, for example, β-adrenergic receptor agonists (e.g., norepinephrine, epinephrine, isoproterenol, sulfonterol, metaproterenol, SB-251023), β-adrenergic receptor antagonists (e.g., propranolol, butoxamine, practolol, alprenolol, pindolol, nadolol, metoprolol, SR-59230A), direct or indirect activators of adenylate cyclase (e.g., forskolin, prostalglandin E1), cAMP analogs (e.g., 8-(4-chloro-phenylthio)-2′-O-methyl adenosine 3′,5′-monophosphate; N6,2′-O-dibutyryl cyclic adenosine 3′,5′monophosphate (Bt2cAMP)), and photolytic release agents (e.g., P-(4,5-dimethoxy-2-nitrobenzyl) adenosine 3′,5′-monophosphate, and phosphodiesterase inhibitors such as 3-isobutyl-1-methylxanthine).
Test compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
Kits
The invention provides kits comprising one or more cAMP reporters of the invention.
The kits also may provide all or a subset of the reagents that are required for practicing the invention. The kits may comprise written instructions, in paper or electronic form, or a reference to an on-line set of instructions. The instructions may contain data against which the results determined using the kit can be compared. Containers which hold the components of any given kit can vary. The kits may be divided into compartments or contain separate vessels for each component. The components may be mixed together or may be separated. Optional components of the kit include means for collecting, processing, and/or storing test samples.
All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1
Preparation and Function of cAMP Reporters
We generated a number of proteins by fusing the amino terminus of various Epac truncations to ECFP and the carboxyl terminus to citrine, an improved version of YFP (FIG. 1). Full length Epac1 (1-881, SEQ ID NO:1) and truncated forms of Epac2, T316-A501 (amino acids 316-501 of SEQ ID NO:20) and P350-A501 (amino acids 350-501 of SEQ ID NO:20), were created by PCR using Epac1 (de Rooij et al., Nature 396, 474-77, 1998.) or Epac2 (SEQ ID NO:20; Ozaki et al., Nature Cell Biology 2, 805-11, 2000) as the templates. In one construct, ECFP and citrine were fused together with a domain (amino acids P350-A501 of SEQ ID NO:20) containing the second cyclic nucleotide monophosphate-binding domain from Epac2 and a C-terminal lid (the α-helix that stabilizes the cAMP-binding site) (FIG. 1). Mutation R522E was incorporated by the QUICKCHANGE® method (Stratagene). Enhanced cyan fluorescent protein (ECFP) and citrine were fused to the N and C terminal ends of the individual gene constructs (FIG. 1). The constructs were first generated in pRSET B (Invitrogen) and subcloned into pcDNA3 (Invitrogen) behind a Kozak sequence for mammalian expression.
For nuclear targeting, the nuclear localization signal PKKKRKVEDA (SEQ ID NO:5) was added to the C terminus. Localization to the mitochondrial matrix was achieved by fusing the first 12 amino acids of subunit IV of human cytochrome oxidase c and a four-residue linker (SEQ ID NO:8) to the N terminal of the construct. For plasma membrane targeting of ICUE1, the sequence KKKKKSKTKCVIM (SEQ ID NO:6) was inserted at the. C terminus. The signal sequence MAIQLRSLFPLALPGMLALLGWWWFFSRKK (SEQ ID NO:7) was inserted at the N terminus for targeting ICUE to mitochondria.
EXAMPLE 2
Cell Culture and Imaging
Cell Culture. HEK-293, HeLa and PC12 cells were plated onto sterilized glass coverslips in 35 mm dishes and grown to 50-90% confluency in DMEM (10% FBS at 37° C., 5% CO2). Cells were then transfected with FuGENE-6 transfection reagent (Roche) or calcium phosphate and allowed to grow for 12-24 hours before imaging. Colocalization studies were performed by incubating transfected HEK-293 cells with MitoTracker Red 580 or Hoechst 33342 cell-permeable dyes (Molecular Probe) for staining mitochondria or nucleic acids, respectively.
Imaging. Cells were washed twice with Hanks' balanced salt solution buffer after 12- to 24-h incubation at 37° C. culture medium. Cells were maintained in buffer in the dark at room temperature with addition of isoproterenol (Aldrich), forskolin (Calbiochem), Prostaglandin E1 (PGE1) (Sigma), and 8-(4-chloro-phenylthio)-2′-O-methyl adenosine 3′,5′-monophosphate (8-pCPT-2′-O-Me-cAMP) (Axxora Biolog) as indicated. Cells were also treated with P-(4,5-dimethoxy-2-nitrobenzyl) adenosine 3′,5′-monophosphate (DMNB-cAMP) (Molecular Probe). Uncaging of cAMP was performed as previously described (Zhang et al., Proc. Natl. Acad. Sci. USA 98, 14997-15002, 2001.).
Cells were imaged on a Zeiss Axiovert 200M microscope with a cooled charge-coupled device camera MicroMAX BFT512 (Roper Scientific) controlled by METAFLUOR 6.2 software (Universal Imaging). Dual-emission ratio imaging used a 420DF20 excitation filter, a 450DRLP dichroic mirror and two emission filters (475DF40 for ECFP, 535DF25 for citrine) alternated by a filter changer Lambda 10-2 (Sutter Instruments). Exposure time was 100-500 ms and images were taken every 8-30 s. Fluorescent images were background-corrected by subtracting autofluorescence intensities of untransfected cells (or background with no cells) from the emission intensities of fluorescent cells expressing reporters. The ratios of cyan to yellow emissions were then calculated at different time points and normalized by dividing all ratios by the emission ratio just prior to stimulation therefore setting the basal emission ratio to 1.
FRET efficiency was determined by acceptor photobleaching as reported (Miyawaki & Tsien, Methods Enzymol. 327, 472-500, 2000). Briefly, citrine was photobleached at the end of the experiment by intense illumination with a 525DF40 filter. ECFP fluorescence intensities before (Fda) and after citrine photobleaching (Fd) and the equation E=1−(Fda/Fd) were then used to calculate the FRET efficiency.
EXAMPLE 3
Function of cAMP Reporters
A cAMP reporter in which ECFP and citrine were fused together with a domain (P350-A501; amino acids 350-501 of SEQ ID NO:20) containing the second cyclic nucleotide monophosphate-binding domain from Epac2 and a C-terminal lid was expressed in HEK-293 cells. This reporter showed variable ratios of cyan to yellow emissions which are inversely correlated with expression level of the protein. This concentration dependence indicates intermolecular FRET between different reporter molecules that may occur due to oligomerization or aggregation (Zacharias et al., Science 296, 913-16, 2002). Upon cAMP elevations, this protein did not show a cAMP-dependent FRET change. We incorporated a larger portion of Epac2 sequence N-terminal to the binding domain (T316-A501; amino acids 316-501 of SEQ ID NO:20) (FIG. 1) and obtained a construct that showed more homogeneous emission ratios and a 5% increase in emission ratio of cyan to yellow upon cAMP elevations.
To improve the dynamic range of the response and to develop a reporter for Epac activation, we sandwiched the full-length Epac1 between ECFP and citrine (FIG. 1). When this reporter (designated as ICUE1 ) was transfected in HEK-293 cells, the fluorescence was uniformly distributed in the cytosolic compartment in 60% of the cells (FIG. 2A, leftmost image). In the remaining 40% of the cells, the protein was localized to perinuclear region or mitochondria, consistent with our previous observation using full-length Epac1 fused to YFP (Qiao et al., J. Biol. Chem. 277, 26581-86, 2002). A similar expression pattern was also observed in HeLa and PC12 cells.
Stimulation of endogenous β-adrenergic receptor (β-AR) with isoproterenol generated a FRET decrease in HEK293 cells expressing ICUE1, resulting in an increase in the ratio of cyan to yellow emissions (FIGS. 2A and 2B). The change in emission ratios was detectable within several seconds and reached a plateau of 16.8%±1.0 (average value±S.E.M, n=8) signal increase within 1.5-3 min. This FRET change consisted of reciprocal decreases in yellow and increases in cyan emission and the FRET efficiencies were measured by acceptor photobleaching to be 29%±3 and 21%±1 (n=3), respectively, before and after isoproterenol stimulation. The presence of propranolol, a β-adrenergic-receptor antagonist, prevented the isoproterenol-stimulated response.
We next tested if the FRET response was reversible. Addition of 10 μM propranolol after the isoproterenol-stimulated response reached the plateau resulted in an initial decrease in emission ratio of cyan to yellow in 2-3 minutes, and a full recovery over 6-8 minutes. Removal of isoproterenol had the same effect. Finally, a second rise in emission ratio was induced by addition of forskolin to activate adenylate cyclase (AC) and elevate cAMP. The change in emission ratio reached a plateau in 3-5 minutes (FIG. 2B).
To verify the FRET response is due to cAMP binding, we generated a variant of the reporter that carries a point mutation in the cAMP binding domain. Arginine 279 in Epac1 is a conserved residue that contributes to cAMP binding (Rehmann et al., Nat. Struc. Biol. 10, 26-32, 2003). EpacR279E is defective in cAMP binding (de Rooij et al., Nature 396, 474-77, 1998; Mei et al., J. Biol. Chem. 277, 11497-504, 2002), and mutation of the same Arginine to Glutamate (R522E) in the reporter completely abolished the FRET change induced by isoproterenol and forskolin (FIG. 2B, n=7).
Different means of elevating intracellular cAMP revealed different kinetics for the FRET response (FIG. 2C). As shown in FIGS. 2B and 2C, activation of β-AR with a selective agonist such as isoproterenol (10 μM) induced decreases in FRET in 1.5-3 minutes, while stimulation of adenylate cyclase required slightly longer treatment with forskolin (3-5 minutes) to produce a maximal increase in emission ratios (19.6%±1.0 response, n=8) (FIG. 2C). Addition of 10 μM prostaglandin E1 (PGE1) to increase cAMP levels (Rich et al., Proc. Natl. Acad. Sci. USA 98, 10349-54, 2001) induced a FRET response (13.3%±0.9, n=7) within 3-5 minutes, noticeably delayed compared to the response induced by isoproterenol (FIG. 2C). A newly characterized analogue of cAMP, 8-pCPT-2′-O-Me-cAMP, specifically activates Epac but not PKA (Enserink et al., Nat. Cell Biol. 4, 901-06, 2002). This cAMP analog, when administrated at 300 μM, required 10-15 minutes to produce a half-maximal increase in emission ratios (t1/2) (FIG. 2C, 12.8%±0.9, n=6).
The fastest intracellular responses were generated by photolytic release (“uncaging”) of cAMP from a membrane-permeant ester, DMNB-cAMP (Nerbonne et al., Nature 310, 74-76, 1984). Cells expressing ICUE1 were first incubated with 100 μM DMNB-cAMP for 3 minutes and were exposed to UV to uncage the cAMP intracellularly. Flash of 5 seconds acutely increased the emission ratio by 4.7%±0.7 (n=8) in just 15-30 seconds (FIG. 2C). The response was then quickly reversed due to the degradation of uncaged cAMP by phosphodiesterase (PDE). The slower time courses of the other responses are presumably due to rate-limiting steps in activating adenylate cyclase and accumulating sufficient cAMP, rather than the kinetics of cAMP binding to Epac1, or the FRET response of the reporter.
EXAMPLE 4
cAMP Dynamics within Subcellular Compartments
To directly monitor cAMP dynamics at different subcellular locations inside cells, we prepared several fusions of ICUE1 to various specific targeting motifs (FIG. 3A). To localize the reporter to the plasma membrane, we fused the plasma membrane-targeting signal of small guanosine triphosphatase K-ras4B (Roy et al., Biochem. 39, 8298-307, 2000) to the C terminus of ICUE 1. This targeting motif combined a farnesylated cysteine residue with a strongly polybasic sequence and effectively targeted the reporter to the plasma membrane (FIG. 3B).
As shown in FIG. 3C, plasma membrane targeted ICUE1 generated a FRET response of 18.3%±1.2 (n=8) upon stimulation with isoproterenol. The response time (t1/2=24.9 s±2.8, n=8) was shortened by 40% compared to the time course for the cytoplasmically-distributed ICUE1 (t1/2=40.5 s±3.3, n=8), while both plasma membrane-targeted and cytoplasmically-distributed ICUE1 generated rapid responses upon whole-cell cAMP uncaging. These results indicate that this delay in response of untargeted ICUE1 is not due to the intrinsic kinetic properties of the localized reporters, but most likely due to restricted release of cAMP from the plasma membrane to cytosol (Rich et al., J. Gen. Physiol. 116, 147-61, 2000).
EXAMPLE 5
cAMP Dynamics and Epac Activation in Mitochondria
Epac localizes to mitochondria in a subpopulation of cells, but monitoring of cAMP accumulation at mitochondria has not been possible with previous methods. To examine the cAMP dynamics and Epac activation at this subcellular location, we fused two different mitochondria targeting motifs to ICUE1 (FIG. 3A). The first MitoCOX-ICUE1 was generated by fusing the targeting sequence of subunit IV of cytochrome c oxidase (COX) to the N-terminus of ICUE1. This COX sequence delivers fused proteins to the mitochondrial matrix (Hurt et al., EMBO J. 4, 2061-68). As shown in FIG. 3B, MitoCOX-ICUE1 was partially targeted to mitochondria (Filippin et al., J. Biol. Chem. 278, 39224-34, 2003), showing partial colocalization with a cell permeable mitochondrial dye, MitoTracker.
Activation of β-AR by isoproterenol generated a FRET response (19.0%±1.6, n=5) in the punctate mitochondria structure within 2-3 minutes (t1/2=40.4 s±7.3, n=5), indicating that cAMP can enter mitochondria and accumulate in the matrix. In a second mitochondria-targeted ICUE1 (MitoDAKAP1-ICUE1), a mitochondria targeting motif taken from the N-terminal sequence of DAKAP1a (Ma & Taylor, J. Biol. Chem. 277, 27328-36, 2002) effectively targeted ICUE1 to mitochondria (FIG. 3B), where the isoproterenol stimulated FRET response (14.5%±1.5, t1/2=42.4 s±2.5, n=6) is similar to the cytosolic response (FIG. 3C).
When fused to a nuclear localization signal (NLS), ICUE1 was appropriately targeted to the nucleus (FIG. 3B), where its response to isoproterenol stimulation was smaller (5.6%±0.5, n=12) than the 16.8%±1.0 FRET change for cytoplasmically-distributed ICUE1 (FIG. 3C). Stimulation with bicarbonate to activate endogenous soluble adenylate cyclase in HEK-293 cells did not generate a cAMP-dependent response in the nucleus, possibly due to limited copy numbers of soluble AC in this cell type or the sensitivity of the detection. Interestingly, the isoproterenol-stimulated response in the nucleus is not delayed (t1/2=38.5 s±3.5, n=12) compared to that from untargeted ICUE1. This indicates that the available pool of cAMP in the nucleus, while possibly smaller, is not kinetically crippled due to the fast diffusion of cAMP from cytosol to nucleus.
To test if activation of different receptors leads to production of different pools of cAMP, we compared the cAMP responses induced by PGE1 and isoproterenol at different subcellular sites. At the plasma membrane, addition of PGE1 generated a 12.6%±0.9 (n=8) emission ratio increase within 2-3 minutes. Surprisingly, sustained stimulation with 10 μM PGE1 did not produce a transient response as observed previously using cyclic nucleotide-gated ion channels (Rich et al., Proc. Natl. Acad. Sci. USA 98, 13049-54, 2001). In contrast, the response at the plasma membrane is sustained until removal of PGE1 (FIG. 3D). Variable cellular PDE activities may be responsible for this discrepancy.
After removal of PGE1, a second response of similar amplitude was induced by stimulation with isoproterenol. Both untargeted and mitochondria targeted ICUE1 showed similar responses to PGE1 and isoproterenol. In the nucleus, PGE1 also stimulated a small response (3.4%±0.4, n=13), noticeably a few percentages smaller than that induced by isoproterenol in the same cell (FIG. 3E).
EXAMPLE 6
Simultaneous Imaging of cAMP Dynamics and PKA Phosphorylation
Soluble AC and regulatory and catalytic subunits of protein kinase A (PKA) coexist in the nucleus of mammalian cells (Zippin et al., J. Cell Biol. 164, 527-34, 2004). The activation of bicarbonate-responsive soluble AC in the nucleus led to a rapid increase in PKA-dependent phosphorylation, which was detectable within two minutes. The immediate presence of a nuclear pool of cAMP following β-AR activation raised the question whether this pool of cAMP could produce functional PKA responses in the nucleus. Here, we took advantage of the targeted cAMP indicators and a PKA activity reporter, AKAR (Zhang et al., Proc. Natl. Acad. Sci. USA 98, 14997-5002, 2001), to examine the temporal correlation of cAMP dynamics and PKA activation within single living cells.
We co-expressed the plasma membrane-targeted ICUE1 and nuclear-localized AKAR in HEK-293 cells (FIG. 4A). An immediate increase in emission ratios of cyan to yellow occurred at the plasma membrane upon stimulation with isoproterenol, indicating an acute rise in cAMP. The emission ratio in the nucleus did not increase in the same time frame. After a delay of 5-10 minutes, in the presence of the sustained cAMP response a gradual increase in ratio of yellow to cyan emissions occurred and reached a plateau in 20-30 minutes. Untargeted AKAR generated acute responses in 2-3 minutes in the cytosol of HEK-293 cells upon stimulation with isoproterenol. This is consistent with acute cytosolic AKAR responses upon cAMP elevations we previously reported (Zhang et al., 2001). Therefore, the delay in response indicates that PKA phosphorylation in the nucleus does not occur immediately following cAMP production (FIG. 4B). This delayed nuclear response of PKA phosphorylation is consistent with a slow diffusional translocation of the catalytic (C) subunit of PKA into the nucleus following the dissociation of catalytic and regulatory subunits in the cytoplasm upon cAMP elevation (Harootunian et al., Mol. Cell Biol. 4, 993-1002, 1993; Meinkoth et al., Proc. Natl. Acad. Sci. USA 87, 9595-99, 1990).
As a control experiment, we also recorded cAMP responses from single cells co-expressing both plasma membrane-targeted and nuclear-localized ICUE1 for direct comparison of the cAMP dynamics at the plasma membrane and in the nucleus. As shown in FIG. 4C, isoproterenol stimulated an acute cAMP response at the plasma membrane followed by a response in the nucleus, which reached the plateau in 2-3 minutes. This is consistent with data obtained from separate cells expressing either targeted reporter indicating that ICUE reporter molecules do not notably perturb cAMP distribution throughout the cell. This acute nuclear cAMP response is in sharp contrast to the delayed response of PKA phosphorylation in the nucleus, which requires 20-30 minutes to reach the maximum. Thus, the presence of this nuclear pool of cAMP immediately following cAMP production is not sufficient to generate a detectable phosphorylation of AKAR by PKA within the nucleus. This lack of immediate PKA response could be due to either the absence of the PKA holoenzyme in the nucleus or insufficient activation of soluble AC-coupled PKA by this pool of cAMP. In this case, the slow diffusion of the C subunit rather than the fast diffusion of cAMP as the rate-limiting step may provide the temporal control of β-AR-stimulated PKA-dependent phosphorylation in the nucleus.
EXAMPLE 7
Preparation and Function of a cAMP Reporter Comprising a Truncated Epac1 (ICUE2)
In about 40% of transfected cells, untargeted ICUE1 localizes to mitochondria and perinuclear region, which has been documented as the subcellular localization of endogenous Epac1. To create a more uniformly expressed reporter, we deleted the disheveled, Eg1-10, and pleckstrin homology (DEP) domain (amino acids 1-148) which is responsible for this localization. The truncation was generated by PCR amplification of ICUE1 in pRSETB bacterial vector starting from glycine 149 of Epac1 through to the end of citrine using the forward primer shown in SEQ ID NO:15:










5′-CGCGGTACCCCCGTGGGAACTCATGAGATGG-3′







and a pRSETB reverse primer. The PCR fragment was then ligated into pcDNA3 mammalian vector containing ECFP. As a result, the truncated reporter, ICUE2, is more diffusible, showing no specific subcellular targeting.

Imaging with ICUE2 in HEK-293 cells revealed a 40-50% increase in cyan/yellow emission ratio upon stimulation of cAMP production with forskolin, compared to a 15-30% response generated by ICUE1. Maximum FRET response was reached in 1.5-3 minutes upon stimulation with isoproterenol, which is on the same time scale as the ICUE1 response. Targeted versions of ICUE2 exhibited the increased dynamic range in cyan/yellow emission ratio as well, therefore improving the signal-to-noise ratio.
ICUE2 also responded to lower concentrations of isoproterenol. We observed FRET responses upon the addition of 0.1 μM, 1 μM, as well as 10 μM isoproterenol, which was the lowest concentration of isoproterenol that generated a FRET response of ICUE 1. The ICUE2 response reverses in an average of 9 minutes once it reaches maximum without addition of β-AR antagonist, propranolol, or washing out of agonist.
EXAMPLE 8
Preparation and Function of a cAMP Reporter Comprising a Circularly Permuted Acceptor Moiety (ICUE3)
The dynamic range of ICUE2 was increased by replacing citrine with a circularly permuted YFP, cpVenus L194 to form a cAMP reporter termed ICUE3. Circular permutation introduces new N and C termini to a protein and can improve the dynamic range of FRET-based reporters by altering the relative orientation of fluorescent proteins (Nagai et al., 2004).
Upon stimulation with the adenylate cyclase activator forskolin, ICUE3 produced a maximum 100% increase in cyan/yellow emissions ratio in HEK-293 cells. Like ICUE2, the ICUE3 response is also reversible when cells are stimulated by the β-adrenergic receptor agonist, isoproterenol.
EXAMPLE 9
High Throughput Assay Using ICUE2
HEK293 cells which stably express ICUE2 (“ICUE2 stable HEK293 cells”) were grown to confluency in 10 cm dishes in DMEM, 10% FBS, 1% penicillin/streptomycin, and 250 μg/ml G418. HEK-293 cells were transfected with cDNA encoding ICUE2 using calcium phosphate at 40% confluency and allowed to grow for 40 hours. Cells were removed from the dish using 0.05% trypsin, spun down, and replaced with fresh media. ICUE2 stable HEK293 cells were counted on a hemocytometer using trypan blue stain. One hundred fifty thousand (150,000) cells were plated per well on a 96-well clear bottom assay plate in a total of 0.2 ml of media per plate and allowed to double once. Media was removed, cells were washed with Hanks buffered salt solution (HBSS) and then covered with 0.05 ml of HBSS per well. In a separate sterile plate, appropriate drugs were added to each well containing HBSS for a total volume of 0.05 ml.
Plate reading used a 420DF20 excitation filter and two emission filters (470DF40 for cyan and 535DF25 for yellow). A baseline was established in three cycles, each consisting of a full plate reading of yellow intensity, followed by a reading of cyan intensity. Cells were then treated with isoproterenol (ISO; Aldrich), forskolin (Calbiochem), H89, propranolol, and 3-isobutyl-1-methylxanthine (IBMX) as indicated. Readings were taken in six or twenty additional cycles. Each cycle lasted 60 to 145 sec. Baseline FRET ratios were calculated as the average ratio of yellow/cyan for each well before the addition of drug. FRET change was calculated as the percent increase of FRET ratio over baseline for each well during a given cycle. The FRET changes of all wells having undergone the same drug treatment were then averaged within each cycle, and that cycle was correlated with time.
ICUE2 stable HEK293 cells were treated with an array of drugs as shown in FIGS. 7 and 8 (also see Table 1). Addition of 1 μM ISO induced transient responses, consistent with single-cell imaging data. Addition of 100 μM IBMX, an inhibitor of phosphodiesterases (PDEs), increased the amplitude and sustained the responses, indicating that PDEs play an important role in switching off cAMP signaling.
When cells were treated with 50 μM FSK, sustained responses were observed for cAMP accumulation. Treatment of cells with both 50 μM FSK and 10 μM H89 increased the amplitude of ICUE2 signal, giving an emission ratio change of 35%. It was previously shown that protein kinase A (PKA) can activate some PDE isoforms. Further studies can be carried out to elucidate PDE roles in controlling intracellular cAMP levels and PKA activities.
HEK293 cells transiently transfected with ICUE3 showed similar results, although with slightly smaller responses of up to 25% change in emission ratio (FIG. 9 and Table 2). The ISO-stimulated responses were inhibited by the presence of 10 mM propranolol (PRO), a general β-adrenergic receptor antagonist. As a negative control, 10 μM PRO had no effect on the FSK response of ICUE3. As another negative control, addition of the buffer with no drugs generated minimal changes in emission ratios.
This example demonstrates that the cAMP reporter ICUE2 and ICUE3 can be used in high throughput analysis of pharmacological agonists and antagonists. An assay using a 96-well format has sufficient signal amplitude (up to 35% change in the case of stable cell lines) and tight error bars (FIG. 9) to distinguish the effects of a series of drugs and drug combinations. Thanks to the reversibility of the reporters, transient responses can be distinguished from sustained responses.
EXAMPLE 10
Cell Culture and Imaging of cAMP Reporter-Expressing Cells
We incorporated cpV N144, cpV E172, cpV K156, and cpV L194 (ICUE3) into ICUE2 using PCR amplification (FIG. 10). Within the cpVenus (cpV) gene, the first residue is a methionine followed by residue P196 in the case of cpV L194, which is named by the residue at the new C-terminus. ICUE3 R373E was generated by the QUICKCHANGE® method (Stratagene). All constructs were initially generated in pRSET B (Invitrogen) then subcloned to pcDNA3 (Invitrogen) behind a Kozak sequence for mammalian expression.
Cell culture and imaging. HEK293 cells were plated onto sterilized glass coverslips in 35 mm dishes and grown to  ̃50% confluency in DMEM containing 10% FBS at 37° C. with 5% CO2. Cells were transfected with calcium phosphate and allowed to grow for 12-24 hours before imaging. After washing once with HBSS, cells were maintained in buffer in the dark at 20-25° C. ISO, FSK, H89, Thapsigargin (Sigma), and phorbol dibutyrate (PDBu, Sigma) were added as indicated. Cells were imaged with a Zeiss Axiovert 200M microscope with a 40×/1.3NA oil-immersion objective lens and cooled CCD camera as described in Ananthanarayanan et al., Proc. Natl. Acad. Sci. USA 102, 15081-86, 2005. Briefly, dual emission ratio imaging used a 420DF20 excitation filter, a 450DRLP dichroic mirror, and two emission filters (475DF40 for cyan and 535DF25 for yellow). The ratio of cyan-to-yellow emissions were then calculated at different time points and normalized by dividing all ratios by the emission ratio before stimulation, setting basal emission ratio as 1. FRET efficiency was determined by acceptor photobleaching as reported in Miyawaki & Tsien, Methods Enzymol 327, 472-500, 2000.
Live cell plate reading. HEK293 cells were transfected with cDNA encoding ICUE reporters using calcium phosphate at 40% confluency and allowed to grow for 40 hours. Cells were then trypsinized and plated in a Costar 3603 96-well assay plate (Coming) at a density of 150,000 cells per well. After incubation for another 24 hours, cells were washed once with HBSS and left in 150 μl HBSS at 20-25° C. Fluorescence reading was taken on a FLUOstar OPTIMA fluorescence microplate reader (BMG Labtechnologies Inc.) using a 420DF20 excitation filter and two emission filters (470DF40 for cyan and 525DF25 for yellow). A baseline was established in three cycles, each consisting of a full plate reading of yellow intensity, followed by a reading of cyan intensity. Each cycle lasted between 64 and 145 seconds. Cells were then treated with ISO, FSK, H89, PRO, or IBMX as indicated in FIG. 10. Readings were taken in additional cycles. FRET change was calculated as the percent increase of emission ratios (cyan-to-yellow) over baseline for each well during a given cycle. To assess the efficacy of the assay, several statistical parameters were calculated, including Z factor, coefficient of variation, and signal-to-noise ratio (Zhang et al., J. Biomol. Screen. 4, 67-73, 1999).
HEK293 cells expressing the four constructs individually (cpV N144, cpV E172, cpV K156, and cpV L194) were stimulated with FSK, and average emission ratio changes were calculated. In the case of cpV N144, low yellow fluorescence intensity was observed, yielding a reporter with a reduced dynamic range (FIG. 10A). The cpV E172 and cpV K156 did not significantly change the dynamic range, showing an average response of 65.3%±4.50% (n=6) and 45.0%±10.9% (n=14), respectively. On the other hand, incorporation of cpV L194 showed a more dramatic effect. Upon stimulation with FSK, HEK293 cells expressing ICUE cpV L194 generated an emission ratio change of 76-93% with an average of 84.5%±7.50% (n=6) (FIG. 10B). Thus, this approach of using cpV variants to create variations in the relative spatial orientation of YFP with respect to CFP proved effective for multiple FRET reporters. This improved indicator of cAMP, named ICUE3, maintained the specificity for cAMP, as a loss-of-function mutation in the cAMP binding site (de Rooij et al., Nature 396, 474-77, 1998) abolished the FRET response of ICUE3 (FIG. 10B). When Cypet and Ypt were used as the FRET pair, the resulting ICUE construct generated emission ratio changes of up to 110%; experimental variations and reduced fluorescence intensities in HEK-293 cells suggested that additional optimization can be carried out.
EXAMPLE 11
Cell-Based Assay Using ICUE3
HEK293 cells expressing ICUE3 were treated with an array of drugs as shown in FIG. 11. Addition of 1 μM ISO induced transient responses, consistent with single cell imaging data, with a maximal response of 8.10%±1.4% (n=7). Such responses can be inhibited by the presence of 10 μM PRO. Addition of 100 μM IBMX sustained the response and increased its amplitude.
When cells were treated with 50 μM FSK, a sustained response was observed for cAMP accumulation (emission ratio change of 17.9%±3.6%; n=7) The presence of 10 μM H89 did not diminish the response of ICUE3. Stimulation by FSK in the presence of H89 appeared to increase the response amplitude of ICUE3, similar to the effect caused by the combination of 50 μM FSK and 100 μM IBMX. As a negative control, 10 μM PRO had no effect on the FSK-stimulated response of ICUE3. Addition of buffer without drugs generated minimal changes in emission ratios.
EXAMPLE 12
High Throughput Cell-Based Assay Using ICUE3
Live cell clinical compound screen. HEK-293 cells expressing ICUE3 were trypsinized and plated in a Costar 3603 96-well plate (Corning) at a density of 150,000 cells per well. After incubation for 24 hours, cells were washed once with HBSS and left in 190 μL of HBSS at 20-25° C. Fluorescence readings were taken as described above with each cycle lasting 90 sec. Following baseline acquisition, cells in each experimental well were treated with a compound from the Johns Hopkins Clinical Compound Library to a final concentration of 10 μM. Control cells were treated with 10 μL of 10% fetal bovine serum (FBS) in a solution of phosphate buffered saline (PBS) at pH 7.4, the solution used to dissolve library compounds. Readings were taken for 10 cycles spanning a time of approximately 15 minutes, after which cells in experimental wells and positive controls were treated with 0.25 μM ISO plus 100 μM IBMX, while negative control cells received 0.5% (v/v) DMSO in HBSS. Ten final cycles were then performed. FRET responses were calculated as described above. Negative control (10% FBS, 0.5% DMSO) and positive control (10% FBS, 0.25 μM ISO) curves were generated. Agonist hits were defined as compounds eliciting responses that are larger than six times of standard deviation above the baseline or 50% of the positive control. Antagonist hits were defined as compounds that decreased the ISO stimulated response by 50% or by six times of the standard deviation. Hits identified based on only one extreme outlier data point in the entire time course were considered false positives. From the plate-reading data, individual channels (yellow and cyan) of all hits were also examined for abnormal changes upon addition of library compounds. An example of detection of a compound with inherent yellow fluorescence is an increase in yellow emission upon excitation with little or no change in cyan emission. Fluorescence or colorimetric properties of compounds were further verified by comparing to literature data or through direct fluorescence measurement. Such hits were labeled as false positives.
Fluorescence microscopy-based secondary screen. A fluorescence microscopy-based secondary screen was used to examine the remaining hits. HEK-293 cells expressing ICUE were followed via fluorescence microscopy before and after treatment with these compounds at a final concentration of 10 μM. Compounds that caused cell rounding, blebbing, lift-up, or severe shrinkage were considered cytotoxic and labeled as false positives. Compounds that did not affect emission ratio dynamics either before or after treatment with isoproterenol (agonist and antagonist secondary screen respectively) were also labeled as false positives. Those that did antagonize or agonize in both primary and secondary screens were labeled as true positives.







TABLE 1





Average percent FRET response ± standard deviation for all cycles (cycle 1-20) for


the high throughput analysis shown in FIG. 8. 1 cycle = 60 seconds.





















1
2
3
4
5





 1 μM Iso
 8.22% ± 5.97%
 20.2% ± 1.69%
21.59% ± 2.65%
22.44% ± 2.42%
21.47% ± 2.92%


 1 μM Iso + 10 μM H89
10.79% ± 5.97%
19.28%% ± 5.88%
20.72% ± 4.61%
19.28% ± 5.06%
18.65% ± 5.26%


 1 μM Iso + 100 μM IBMX
14.13% ± 6.98%
20.97% ± 7.35%
27.76% ± 4.41%
28.57% ± 3.83%
24.63% ± 9.07%


50 μM FsK
 0.36% ± 7.51%
 2.67% ± 4.53%
 8.71% ± 6.66%
15.29% ± 3.37%
15.23% ± 6.55%


50 μM Fsk + 10 μM H89
10.65% ± 17.5%
12.54% ± 15.11%
16.85% ± 9.42%
22.38% ± 8.4%
24.66% ± 7.81%


50 μM Fsk + 100 μM IBMX
19.13% ± 2.68%
26.42% ± 4.29%
30.35% ± 6.55%
32.77% ± 10.52%
33.24% ± 10.84%






6
7
8
9
10





 1 μM Iso
19.64% ± 1.73
17.36% ± 1.28%
15.93% ± 2.0%
14.45% ± 3.55%
11.07% ± 2.19%


 1 μM Iso + 10 μM H89
17.29% ± 3.90%
15.62% ± 3.07%
11.73% ± 6.75%
10.20% ± 4.70%
 9.74% ± 6.29%


 1 μM Iso + 100 μM IBMX
 25.8% ± 8.08%
25.41% ± 8.86%
 26.9% ± 7.29%
27.49% ±  6.27%
 24.6% ± 8.21%


50 μM FsK
18.73% ± 3.97%
20.79% ± 4.6%
22.82% ± 4.58%
24.98% ± 3.69%
 23.4% ± 4.53%


50 μM Fsk + 10 μM H89
26.72% ± 7.05%
27.15% ± 8.66%
28.15% ± 9.89%
 28.0% ± 6.89%
28.77% ± 8.79%


50 μM Fsk + 100 μM IBMX
34.31% ± 13.16%
33.42% ± 13.48%
33.42% ± 13.03%
34.16% ± 13.75%
34.85% ± 13.52%



11
12
13
14
15





 1 μM Iso
 6.94% ± 1.93
 2.26% ± 5.45%
 2.47% ± 6.06%
 2.27% ± 3.37%
 1.80% ± 5.84%


 1 μM Iso + 10 μM H89
 7.99% ± 4.08%
 7.73% ± 3.38%
 6.60% ± 4.19%
 5.46% ± 2.77%
 5.37% ± 3.59%


 1 μM Iso + 100 μM IBMX
27.68% ± 5.95%
25.24% ± 6.85%
25.99% ± 5.56%
24.86% ± 6.02%
25.92% ± 5.35%


50 μM FsK
24.46% ± 3.51%
25.44% ± 2.14%
24.97% ± 3.91%
 25.6% ± 4.31%
 27.0% ± 1.87%


50 μM Fsk + 10 μM H89
30.11% ± 7.77%
28.92% ± 8.35%
29.54% ± 7.01%
30.64% ± 7.81%
31.14% ± 7.65%


50 μM Fsk + 100 μM IBMX
34.16% ± 15.2%
35.09% ± 15.11%
35.44% ± 14.71%
35.43% ± 15.66%
36.44% ± 16.43%



16
17
18
19
20





 1 μM Iso
 1.05% ± 5.87%
 1.29% ± 2.72%
 1.68% ± 6.12%
 2.52% ± 3.54%
 0.93% ± 4.45%


 1 μM Iso + 10 μM H89
 3.37% ± 3.12%
 2.73% ± 6.59%
 3.98% ± 3.63
 4.01% ± 1.59%
 5.10% ± 3.20%


 1 μM Iso + 100 μM IBMX
25.77% ± 4.74%
22.86% ± 6.32%
20.58% ± 7.09%
19.45% ± 8.36%
 20.2% ± 6.64%


50 μM FsK
25.84% ± 3.57%
26.32% ± 1.66%
25.94% ± 3.05%
25.98% ± 2.13%
25.51% ± 1.67%


50 μM Fsk + 10 μM H89
31.51% ± 7.08%
32.65% ± 6.2%
30.65% ± 7.71%
31.38% ± 6.57%
32.19% ± 7.33


50 μM Fsk + 100 μM IBMX
35.93% ± 16.26%
 35.6% ± 16.65%
35.34% ± 15.77%
35.87% ± 17.04%
35.99% ± 17.19%
















TABLE 2







Average percent FRET response ± standard deviation at different time points for the high throughput analysis shown


in FIG. 9.














0 sec.
145 sec.
290 sec.
435 sec.
580 sec.
725 sec.





HBSS
2.09% ± 2.50%
2.41% ± 2.16%
1.46% ± 1.12%
1.95% ± 1.74%
2.15% ± 1.44%
2.09% ± 1.26%


 1 μM ISO + 10 μM
0.142% ± 2.46% 
 2.1% ± 2.07%
0.978% ± 1.96% 
0.928% ± 1.42% 
0.457% ± 2.11% 
0.581% ± 1.29% 


PRO
7.61% ± 3.19%
 8.1% ± 1.38%
6.54% ± 1.37%
 3.73% ± 0.911%
2.57% ± 1.33%
1.83% ± 1.06%


 1 μM ISO + 100 μM
9.73% ± 1.62%
15.4% ± 1.92%
17.9% ± 2.8% 
18.0% ± 2.22%
18.2% ± 2.33%
19.0% ± 2.2% 


IBMX








50 μM FSK
9.05% ± 3.18%
13.0% ± 3.17%
14.8% ± 3.35%
16.7% ± 3.33%
16.8% ± 3.27%
17.9% ± 3.63%


50 μM FSK + 10 μM
8.52% ± 3.25%
15.6% ± 3.15%
15.6% ± 2.2% 
18.1% ± 3.05%
17.3% ± 2.33%
18.2% ± 2.21%


PRO








50 μM FSK + 100 μM
14.0% ± 3.65%
19.9% ± 7.12%
20.7% ± 2.47%
21.7% ± 2.37%
23.0% ± 2.31%
24.1% ± 2.71%


IBMX








50 μM FSK + 10 μM
14.5% ± 2.0% 
18.5% ± 2.56%
20.5% ± 2.9% 
21.9% ± 2.83%
22.1% ± 2.46%
23.2% ± 2.43%


H89

## Claims

1. A cAMP reporter comprising:
(a) a donor moiety;
(b) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1, which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety;
(c) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited;
wherein each of the donor and acceptor moieties is a protein.

2. The cAMP reporter of claim 1 wherein at least one of the donor and acceptor moieties is a fluorescent protein.

3. The cAMP reporter of claim 1 wherein each of the donor and the acceptor moieties is a fluorescent protein.

4. The cAMP reporter of claim 1 wherein at least one of the donor and acceptor moieties is a luminescent protein.

5. The cAMP reporter of claim 1 wherein the acceptor moiety is selected from the group consisting of a fluorescent protein, a circularly permuted fluorescent protein, and a luminescent protein.

6. The cAMP reporter of claim 1 wherein the donor moiety is selected from the group consisting of a fluorescent protein, a circularly permuted fluorescent protein, and a luminescent protein.

7. The cAMP reporter of claim 1 wherein the donor moiety is a fluorescent protein selected from the group consisting of a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), and fluorescent mutants thereof.

8. The protein of claim 1 wherein the acceptor moiety is a luminescent protein.

9. The protein of claim 1 wherein the acceptor moiety is a luminescent protein selected from the group consisting of an aequorin, an obelin, a lux protein, a luciferase protein, a phycobiliprotein, a pholasin, and a green fluorescent protein.

10. The cAMP reporter of claim 1 further comprising a subcellular targeting sequence fused to the N-terminus of the donor moiety or the C-terminus of the acceptor moiety.

11. The cAMP reporter of claim 10 wherein the subcellular targeting sequence targets the reporter to a subcellular location selected from the group consisting of a plasma membrane, a nuclear membrane, an endoplasmic reticulum, a mitochondria, a mitochondrial matrix, a chloroplast, a medial trans-Golgi cisternae, a lumen of a lysosome, and a lumen of an endosome.

12. The cAMP reporter of claim 10 which comprises a subcellular targeting sequence selected from the group consisting of a plasma membrane targeting sequence comprising SEQ ID NO: 6, a nuclear localization signal sequence comprising SEQ ID NO: 5, a mitochondrial localization sequence comprising SEQ ID NO: 7, and a mitochondrial matrix targeting signal comprising SEQ ID NO: 8.

13. A cAMP reporter comprising:
(a) a donor moiety;
(b) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1, which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety;
(c) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited,

wherein the donor moiety is enhanced cyan fluorescent protein and the acceptor moiety is cpVenus L194.

14. A nucleic acid molecule which encodes a cAMP reporter comprising:
(a) a donor moiety;
(b) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1, which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety;
(c) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited;
wherein each of the donor and acceptor moieties is a protein.

15. A nucleic acid molecule which encodes a cAMP reporter comprising:
(a) a donor moiety;
(b) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1, which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety;
(c) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited,

wherein the donor moiety is enhanced cyan fluorescent protein and the acceptor moiety is cpVenus L194.

16. A host cell comprising the nucleic acid molecule of claim 14.

17. A method for detecting a change in cAMP concentration, comprising:
detecting a first resonance energy transfer of a cAMP reporter at a first time point, wherein the cAMP reporter comprises
(a) a donor moiety;
(b) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1,

which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety; and
(c) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited, wherein each of the donor and acceptor moieties is a protein;

detecting a second resonance energy transfer of the cAMP reporter at a second time point; and comparing the first and the second resonance energy transfers,
wherein a difference between the first and the second resonance energy transfers reflects a change in cAMP concentration between the first and second time points.

18. The method of claim 17 wherein the first and second resonance energy transfers are detected by determining a property selected from the group consisting of a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, anisotropy, a polarization of emitted light, resonance energy transfer, and a quenching of emission at a wavelength.

19. The method of claim 17 wherein the cAMP reporter is in a cell-free system.

20. The method of claim 17 wherein the cAMP reporter is in a cell.

21. The method of claim 20 wherein the cell is in vivo.

22. The method of claim 20 wherein the cell is in vitro.

23. The method of claim 20 wherein the cell is in a tissue sample.

24. The method of claim 20 wherein the cell is in a whole organ.

25. The method of claim 20 wherein the cell is in a well of a multi-well plate.

26. The method of claim 25 wherein each of a plurality of wells of the multi-well plate comprises a cell which comprises the cAMP reporter.

27. The method of claim 17 further comprising determining the second resonance energy transfer in the presence of a test compound.

28. The method of claim 20 wherein the first and second resonance energy transfers are detected using fluorescence activated cell sorting.

29. The method of claim 26 further comprising contacting each well of the plurality with a different test compound and determining second resonance energy transfers in the presence of the different test compounds.

30. A kit, comprising:
(a) a cAMP reporter comprising:
(1) a donor moiety;
(2) a polypeptide consisting of amino acids 149-881 of SEQ ID NO:1, which comprises a cAMP-binding domain of an exchange protein directly activated by cAMP (Epac), wherein the polypeptide is linked to the donor moiety;
(3) an acceptor moiety linked to the polypeptide, wherein the donor moiety and the acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited, wherein each of the donor and acceptor moieties is a protein; and

(b) instructions for the method of claim 17.

31. The nucleic acid molecule of claim 14 wherein at least one of the donor and acceptor moieties is a fluorescent protein.

32. The nucleic acid molecule of claim 14 wherein each of the donor and the acceptor moieties is a fluorescent protein.

33. The nucleic acid molecule of claim 14 wherein at least one of the donor and acceptor moieties is a luminescent protein.

34. The nucleic acid molecule of claim 14 wherein the acceptor moiety is a circularly permuted fluorescent protein.

35. The nucleic acid molecule of claim 14 wherein the donor moiety is a circularly permuted fluorescent protein.

36. The nucleic acid molecule of claim 14 wherein the donor moiety is a fluorescent protein selected from the group consisting of a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), and fluorescent mutants thereof.

37. The nucleic acid molecule of claim 14 wherein the acceptor moiety is a luminescent protein selected from the group consisting of an aequorin, an obelin, a lux protein, a luciferase protein, a phycobiliprotein, a pholasin, and a green fluorescent protein.

38. The nucleic acid molecule of claim 14 wherein the cAMP reporter further comprises a subcellular targeting sequence fused to the 5′-terminus of the donor moiety or the 3′-terminus of the acceptor moiety.

39. The nucleic acid molecule of claim 38 wherein the subcellular targeting sequence targets the reporter to a subcellular location selected from the group consisting of a plasma membrane, a nuclear membrane, an endoplasmic reticulum, a mitochondria, a mitochondrial matrix, a chloroplast, a medial trans-Golgi cisternae, a lumen of a lysosome, and a lumen of an endosome.

40. The nucleic acid molecule of claim 38 wherein the cAMP reporter further comprises a subcellular targeting sequence selected from the group consisting of a plasma membrane targeting sequence comprising SEQ ID NO:6, a nuclear localization signal sequence comprising SEQ ID NO:5, a mitochondrial localization sequence comprising SEQ ID NO:7, and a mitochondrial matrix targeting signal comprising SEQ ID NO:8.

41. The nucleic acid molecule of claim 14 which is in an expression vector.

42. A method of producing a cAMP reporter, comprising:
culturing a host cell comprising an expression vector which comprises the nucleic acid molecule of claim 14 under conditions which permit expression of the cAMP reporter from the nucleic acid molecule; and
recovering the cAMP reporter.

43. A host cell comprising the nucleic acid molecule of claim 15.

44. The cAMP reporter of claim 1 wherein the acceptor moiety is citrine.

45. The cAMP reporter of claim 1 wherein the donor moiety is enhanced cyan fluorescent protein.

46. The cAMP reporter of claim 1 wherein the donor moiety is a cyan fluorescent protein.

47. The cAMP reporter of claim 1 wherein the acceptor moiety is a yellow fluorescent protein.

48. The cAMP reporter of claim 1 wherein the donor moiety is a cyan fluorescent protein and the acceptor moiety is a yellow fluorescent protein.

49. The cAMP reporter of claim 1 wherein the donor moiety is an enhanced cyan fluorescent protein and the acceptor moiety is a yellow fluorescent protein.

50. The cAMP reporter of claim 1 wherein the donor moiety is a cyan fluorescent protein and the acceptor moiety is citrine.

51. The cAMP reporter of claim 1 wherein the donor moiety is a cyan fluorescent protein and the acceptor moiety is cpVenus L194.