EP1791972A2 - cAMP REPORTERS - Google Patents

Abstract

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

Description

cAMP REPORTERS

[01] This application claims the benefit of and incorporates by reference co-pending provisional applications Serial No. 60/603,623 filed August 23, 2004 and Serial No. 60/681,923 filed May 17, 2005.

[02] This invention was made using funds from NIH grants GM066170 and GMO8763, The government retains certain rights in the invention,

FIELD OF THE INVENTION

[03] The invention relates to detection of cAMP levels.

BACKGROUND OF THE INVENTION

[04] 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 at,, Proc. Natl Acad. ScL USA 98, 13049-54, 2001; Rich etai, J. Gen. Physiol. 116, 147- 61, 2000). Free cAMP can be imaged in single cells microinjected with fluorophore- labeled C and R submits (Adams et al._t Nature 349, 694-9I₇ 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.

[05] There is a need in the art for sensitive cAMP reporters which can be used for accurate measurements of spatial and temporal cAMP distributions in living cells.

BRIEF DESCRIPTION OF THE FIGURES

[06] 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 Epacl (with or without an R522E mutation). The construct comprising full- length Epacl generated the biggest FRET response and was designated as ICUEl.

[07] FIGS. 2A-C. Responses of ICUEl to changes in cellular cAMP levels. FIG. 2A, FRET response of HEK-293 cells transfected with ICUEl. 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 ICUEl 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 ICUEl 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.

[08] FIGS. 3A-E. Fusions of ICUEl 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 ICUEl with Hoechst 33342 cell-permeable dye in nucleus and mitochondria-targeted ICUEl with MitoTracker at mitochondria. FIG. 3C, Representative emission ratio time courses for untagged (ICUEl), plasma membrane-targeted (pm ICUEl), mitochondria-targeted (MitoCOX- and MitoDAKAPl -ICUEl) and nuclear-localized cAMP reporters (NLS- ICUEl) stimulated with ISO (10 μM). FIG. 3D, Representative emission ratio time courses for pm ICUEl 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-ICUEl in response to PGE₁ (10 μM) and ISO (10 μM) separated by a washing step.

[09] 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 ICUEl 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 ICUEl and NLS-ICUEl in the same cell stimulated with 10 μM ISO followed by 10 μM propranolol (n=4).

[10] FIG. 5. Graph showing emission ratio time courses for ICUE2 and targeted versions of ICUE2. Y axis, normalized emission ratio (cyan/yellow).

[11] FIG. 6. Graph showing emission ratio time courses for ICUE2 and ICUE3.

DETAILED DESCRIPTION OF THE INVENTION

[12] 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); 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.

[13] 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.

[14] 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. Sd. 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, CzV. Res. 94, 866-73, 2004).

Polypeptides

[15] Polypeptides used in cAMP reporters of the invention comprise a cAMP-binding domain of an Epac, e.g., Epacl or Epac2. Epacl 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 Epacl 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 Epacl and Epac2 proteins are provided in SEQ ID NOS: 1, 3, and 16-20. 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

[16] 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."

[17] 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. ScL 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.

[18] 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.

[19] 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

[20] 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.

[21] RFPs include Discosoma RFPs, such Discosoma DsRed (SEQ ID NO:9) or a mutant thereof which includes an Ilel25Arg 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).

[22] 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, Asnl45Ile, Metl53Thr, and Vall63Ala in CFP and Ser65Gly, Vall68Leu, 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. Patents 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079; Chalfie et al, Science 253:802-805, 1994. [23] 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.

[24] 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, andN212K.

Luminescent moieties

[25] 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. Patent 4,374,120; and U.S. Patent 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.

[26] 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 ah, Photochem. Photobiol. 27, 389-96, 1978; Ward et al, J. Biol. Chem. 254, 781-88, 1979; Ward et ah, 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, IN. Recombinantly produced firefly luciferase is available from Promega Corporation, Madison, WI. Jellyfish aequorin and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, GA.

[27] 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. Sd. USA 82, 7870-73, 1985.

[28] Luciferase substrates (luciferins) are well known and include coelenterazine (available from Molecular Probes, Eugene, OR) and ENDUREN™. 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. ScL USA 74, 4285-87, 1977).

Dark quenchers

[29] 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), QSY™-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, OR). Suitable quencher moieties are disclosed, for example, in US 2005/0118619; US 20050112673; and US 20040146959.

[30] 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), 27'-dimethoxy-4'5^I-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 l-dimethylaminonaphthyl-5-sulfonate, 1- anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).

[31] Other suitable fluorescent moieties include coumarins, such as 3-phenyl-7- isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridin- e and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-l-carboxy-pentyl)-3'- ethyl-5,5'-dimethy- loxacarbocyanine (CyA); lH,5H,lH,15H-Xantheno[2,3,4-ij:5,6,7- i'j']diquinol- izin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-l-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); BODIPY™ dyes; benzoxaazoles; stilbenes; pyrenes; and the like.

Subcellular targeting sequences

[32] 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.

[33] hi 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 acidsl57-316 of SEQ ID NO:1 (human Epacl).

Assembly ofcAMP reporters

[34] 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, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[35] 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.

[36] 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.

[37] 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, tetrafiuorylphenyl and pentafmoryl esters. Free carboxyl groups in the polypeptide can be reacted with carbodiimides such as l-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 moities 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.

[38] 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 ah, 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 43 IA 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 ofcAMP reporters to cells

[39] AU cAMP reporters of the invention can be introduced into cells in vitro using reversible permeabilization techniques. See U.S. Patent 6,127,177; U.S. Patent 6,902,931; Russo et ah, Nature Biotechnology 15, 278-82, March 1997; Santangelo et ah, Nucleic Acids Res. 32, 1-9, April 14, 2004.

[40] 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.

[41] Useful vectors and methods of delivering the vectors to cells in vivo are disclosed, for example, in U.S. Patent 6,825,012; U.S. Patent 6,878,549; U.S. Patent 6,645,942; U.S. Patent 6,692,737; U.S. Patent 6,689,758; U.S. Patent 6,669,935; and U.S. Patent 6,821,957.

Methods of detecting cAMP

[42] 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.

[43] 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. Patent 6,773,885; U.S. Patent 6,803,201; U.S. Patent 6,818,420; Ayoub et ah, 2002; Boute et ah, 2002; Domin et ah, Prog. Biomed. Optics and Imaging, Proc. SPIE, vol 5139, 2003, pp238-242; Evellin et ah, Methods MoI. biol. 284, 259-70, 2004; Honda et ah, Proc. Natl. Acad. ScL USA 98, 437-42, February 27, 2001; Honda et ah, Methods MoI. Biol. 3, 27-44, 1005; Mongillo et ah, Cir. Res. 95, 67-75, July 9, 2004; Mongillo et ah, Methods MoI. Biol. 307, 1-14, 2005; Nagai et ah, Proc. Natl. Acad. ScL USA 101, 10554-59, My 20, 2004; Nikolaev et ah, J. Biol. Chem. 279, 37215-18, 2004; Polit et ah, Eur. J. Biochem. 270, 1413-23, 2003; Ponsioen et ah, EMBO Rep. 5, 1176- 80, 2004; Santangelo et ah, Nucl. Acids Res. 32, 1-9, e-published April 14, 2004; and Warrier et ah, 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.

[44] 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. [45] 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, prostaglandin E₁), cAMP analogs (e.g., 8-(4-chloro-phenylthio)-2'-O-methyl adenosine 3 ',5 '-monophosphate; N⁶,2'-Odibutyryl 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-l- methylxanthine).

[46] 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.

[47] 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 l

Preparation and function ofcAMP reporters

[48] 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 Epacl (1-881, SEQ ID NO:1) and truncated forms of Eρac2, 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 Epacl (de Rooij et al., Nature 396, AlA-Il, 1998.) or Epac2 (SEQ ID NO:20; Ozaki et al, Nature Cell Biology 2, 805-11, 2000) as the templates, hi 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.

[49] 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 ICUEl, the sequence KKKKKSKTKCVM (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

[50] Cell Culture. HEK-293, HeLa and PC 12 cells were plated onto sterilized glass coverslips in 35mm dishes and grown to 50-90% confluency in DMEM (10% FBS at 37⁰C, 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.

[51] Imaging. Cells were washed twice with Hanks' balanced salt solution buffer after 12- to 24-h incubation at 37 ⁰C 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 '^'-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. ScL USA 98, 14997-15002, 2001.).

[52] 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.

[53] 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 (F_da) and after citrine photobleaching (Fa) and the equation E = I - (F_da/F_d) were then used to calculate the FRET efficiency.

EXAMPLE 3

Function ofcAMP reporters

[54] 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 ah, 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.

[55] To improve the dynamic range of the response and to develop a reporter for Epac activation, we sandwiched the full-length Epacl between ECFP and citrine (FIG. 1). When this reporter (designated as ICUEl) 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 Epacl fused to YFP (Qiao et al, J. Biol. Chem. 277, 26581-86, 2002). A similar expression pattern was also observed in HeLa and PC 12 cells.

[56] Stimulation of endogenous β-adrenergic receptor (β-AR) with isoproterenol generated a FRET decrease in HEK293 cells expressing ICUEl, resulting in an increase in the ratio of cyan to yellow emissions (FIG. 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.

[57] 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).

[58] 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 Epacl 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). [59] Different means of elevating intracellular cAMP revealed different kinetics for the FRET response (FIG. 2C). As shown in FIG. 2B and 2C₅ 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 El (PGE₁) to increase cAMP levels (Rich et al, Proc. Natl. Acad. ScL 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 mininutes to produce a half-maximal increase in emission ratios (ty₂) (FIG. 2C, 12.8% ± 0.9, n=6).

[60] 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 ICUEl 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 Epacl, or the FRET response of the reporter.

EXAMPLE 4

cAMP dynamics within subcellular compartments

[61] To directly monitor cAMP dynamics at different subcellular locations inside cells, we prepared several fusions of ICUEl 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 ICUEl. This targeting motif combined a farnesylated cysteine residue with a strongly polybasic sequence and effectively targeted the reporter to the plasma membrane (FIG. 3B).

[62] As shown in FIG. 3 C, plasma membrane targeted ICUEl generated a FRET response of 18.3% ± 1.2 (n=8) upon stimulation with isoproterenol. The response time (ti_# = 24.9 s ± 2.8, n= 8) was shortened by 40% compared to the time course for the cytoplasmically- distributed ICUEl (tv₂= 40.5 s ± 3.3, n=8), while both plasma membrane-targeted and cytoplasmically-distributed ICUEl generated rapid responses upon whole-cell cAMP uncaging. These results indicate that this delay in response of untargeted ICUEl 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 ah, J. Gen. Physiol. 116, 147-61, 2000).

EXAMPLE 5

cAMP dynamics andEpac activation in mitochondria

[63] 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 ICUEl (FIG. 3A). The first MitoCOX-ICUEl was generated by fusing the targeting sequence of subunit IV of cytochrome c oxidase (COX) to the N-terminus of ICUEl. This COX sequence delivers fused proteins to the mitochondrial matrix (Hurt et al, EMBO J. 4, 2061-68). As shown in FIG. 3B, MitoCOX-ICUEl 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. [64] 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 (ti_/2= 40.4 s ± 7.3, n=5), indicating that cAMP can enter mitochondria and accumulate in the matrix, hi a second mitochondria-targeted ICUEl (MitoD AKAPl -ICUEl), a mitochondria targeting motif taken from the N-terminal sequence of DAKAPIa (Ma & Taylor, J. Biol. Chem. 277, 27328-36, 2002) effectively targeted ICUEl to mitochondria (FIG. 3B), where the isoproterenol stimulated FRET response (14.5% ± 1.5, tm= 42.4 s ± 2.5, n=6) is similar to the cytosolic response (FIG. 3C).

[65] When fused to a nuclear localization signal (NLS), ICUEl 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 ICUEl (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 (t_1/2= 38.5 s ± 3.5, n=12) compared to that from untargeted ICUEl. 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.

[66] 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 ah, 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. [67] After removal of PGE₁, a second response of similar amplitude was induced by stimulation with isoproterenol. Both untargeted and mitochondria targeted ICUEl 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 ofcAMP dynamics and PKA phosphorylation

[68] Soluble AC and regulatory and catalytic subunits of protein kinase A (PKA) coexist in the nucleus of mammalian cells (Zippin et ah, 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 PE-A responses in the nucleus. Here, we took advantage of the targeted cAMP indicators and a PICA activity reporter, AKAR (Zhang et al., Proc. Natl. Acad. ScL USA 98, 14997-5002, 2001), to examine the temporal correlation of cAMP dynamics and PKA activation within single living cells.

[69] We co-expressed the plasma membrane-targeted ICUEl 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, MoI. Cell Biol. 4, 993-1002, 1993; Meinkoth et al, Proc. Natl. Acad. Sd. USA 87, 9595-99, 1990).

[70] As a control experiment, we also recorded cAMP responses from single cells co- expressing both plasma membrane-targeted and nuclear-localized ICUEl 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. hi 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 Epacl (ICUE2)

[71] In about 40% of transfected cells, untargeted ICUEl localizes to mitochondria and perinuclear region, which has been documented as the subcellular localization of endogenous Epacl. To create a more uniformly expressed reporter, we deleted the disheveled, EgI- 10, and pleckstrin homology (DEP) domain (amino acids 1-148) which is responsible for this localization. The truncation was generated by PCR amplification of ICUEl in pRSETB bacterial vector starting from glycinel49 of Epacl through to the end of citrine using the forward primer shown in SEQ ID NO: 15:

5'-CGCGGTACCCCCGTGGGAACTCATGAGATGG-S'

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.

[72] 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 ICUEl. Maximum FRET response was reached in 1.5-3 minutes upon stimulation with isoproterenol, which is on the same time scale as the ICUEl response. Targeted versions of ICUE2 exhibited the increased dynamic range in cyan/yellow emission ratio as well, therefore improving the signal-to-noise ratio.

[73] ICUE2 also responded to lower concentrations of isoproterenol. We observed FRET responses upon the addition of 0.1 μM, IuM, as well as lOμM isoproterenol, which was the lowest concentration of isoproterenol that generated a FRET response of ICUEl. 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)

[74] The dynamic range of ICUE2 was increased by replacing citrine with a circularly permuted YFP, cp Venus Ll 95 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).

[75] Upon stimulation with the adenylate cyclase activator forskolin, ICUE3 produced a maximum 93% increase in cyan/yellow emissions ratio (average 78% ± 0.05, N= 8) in HEK-293 cells, which is approximately double the average response measured by ICUE2. Like ICUE2, the ICUE3 response is also reversible when cells are stimulated by the β-adrenergic receptor agonist, isoproterenol.

Claims

1. A cAMP reporter comprising:

(a) a donor moiety;

(b) a polypeptide comprising 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 is excited.

2. The cAMP reporter of claim 1 wherein the polypeptide is Epacl or Epac2.

3. The cAMP reporter of claim 1 wherein the polypeptide:

(a) comprises SEQ ID NO:1;

(b) comprises SEQ ID NO:2;

(c) comprises SEQ ID NO:1 but for an R522E mutation;

(d) comprises SEQ ID NO:1 but for an R279E mutation;

(e) comprises of amino acids 149-318 of SEQ ID NO:1;

(d) consists of amino acids 149-881 of SEQ ID NO:1;

(e) comprises amino acids 1-160 of SEQ ID NO:20;

(f) consists of amino acids 1-160 of SEQ ID NO:20;

(g) comprises amino acids 280-463 of SEQ ID NO:20; (h) consists of amino acids 280-463 of SEQ ID NO:20; (i) comprises amino acids 315-501 of SEQ ID NO:20; (j) consists of amino acids 315-501 of SEQ ID NO:20; (k) comprises amino acids 350-501 of SEQ ID NO:20; (1) consists of amino acids 350-501 of SEQ ID NO:20; (m) comprises SEQ ID NO:16; (n) comprises SEQ ID NO: 17; (o) comprises SEQ ID NO: 18; or (p) comprises SEQ ID NO: 19.

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

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

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

7. The cAMP reporter of claim 6 wherein the luminescent moiety is selected from the group consisting of a luminescent protein and a lanthanide chelate.

8. The cAMP reporter of claim 1 wherein the acceptor moiety is selected from the group consisting of a coumarin, a xanthene, a fluorescein, a fluorescent protein, a circularly permuted fluorescent protein, a rhodol, a rhodamine, a resorufm, 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 zwitteriomc azopyridinium compound.

9. The cAMP reporter of claim 1 wherein the acceptor moiety is a dark quencher.

10. The cAMP reporter of claim 1 wherein the donor moiety is selected from the group consisting of 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 aminonaphthalήnide, an aminobenzofuran, an aminoquinoline, a dicyanohydroquinone, a semiconductor fluorescent nanocrystal, a fluorescent protein, a circularly permuted fluorescent protein, and fluorescent lanthanide chelate.

11. 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.

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

13. 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.

14. The cAMP reporter of claim 1 further comprising a subcellular targeting sequence.

15. The cAMP reporter of claim 14 wherein the subcellular targeting sequence targets the reporter to a subcellular location selected from the group consisting of 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, and a lumen of an endosome.

16. The cAMP reporter of claim 1 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.

17. A nucleic acid molecule which encodes the cAMP reporter of claim 1 wherein each of the donor and acceptor moieties is a protein.

18. The nucleic acid molecule of claim 17 which is an expression vector.

19. A host cell comprising the nucleic acid molecule of claim 17.

20. A method for cAMP, comprising: detecting a change in resonance energy transfer of a cAMP reporter subjected to a change in cAMP concentration, wherein the cAMP reporter comprises:

(a) a donor moiety;

(b) a polypeptide comprising 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 is excited. wherein a change in resonance energy transfer indicates a change in cAMP concentration.

21. The method of claim 20 wherein the cAMP concentration is changed by addition of a test compound.

22. The method of claim 20 wherein the cAMP reporter is in a cell-free system.

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

24. The method of claim 24 wherein the cell is in a tissue sample.

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

26. The method of claim 20 wherein the change in resonance energy is 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.