All cyclic nucleotides, as well as the free Pharos chromophore were derived from Biolog Life Science Institute (Bremen, Germany). The solubility of the novel Pharos compound is approximately 50 mM in water. LogKw data were determined by a retention-based lipophilicity ranking using a LiChrograph HPLC (Merck-Hitachi, Darmstadt, Germany) equipped with a reversed phase YMC RP-18 phase (250 × 4 mm) running at a flow rate of 1.0 ml/min. Nucleotides were detected at λ = 280 nm . A. The purity of cyclic nucleotides was analyzed with an elution system consisting of 25% acetonitrile and 10 mM triethyl ammonium formiate at a flow rate of 1.5 ml/min and was found to be > 99%. No trace of free fluorophore was detected in 8-[ϕ-575]-cAMP.
The Pharos dye was diluted in 20 mM 2-(4-morpholino)-ethane sulfonic acid (MES) buffer adjusted to pH 6.0, pH 7.0 and pH 7.4, and all samples were degassed by bubbling nitrogen through the solution before measuring the absorption or fluorescence spectra.
For Stokes shift and quantum yield determination, absorption and fluorescence spectra were recorded in cells with 1 cm path length using a PerkinElmer Lambda 900 UV-Vis spectrophotometer and a Hitachi F-4500 fluorescence spectrophotometer, respectively.
To calculate the relative quantum yield ϕ of the free Pharos dye, the absorption (500 nm to 640 nm) and fluorescence (λex = 553 nm, λem ranging from 558 nm to 800 nm) spectra of several Pharos dye concentrations (640 nM to 64.2 μM) were recorded in A. bidest. The integrated fluorescence intensity (I) was plotted versus the absorbance (A) at the excitation wavelength. For low concentrations the dye molecules are not influenced by each other and thus exhibit a linear behavior concerning absorption and emission of light. Linear regression yields the slope ΔI/ΔA that is compared to the corresponding data of a quantum yield standard by using the equation ϕsa = (ΔIsa/ΔAsa)·(ΔAst/ΔIst)·ϕst. As a standard, a solution of quinine sulfate in 1.0 N sulfuric acid (ϕst = 0.546 with λex = 365 nm) was used [21, 22].
For emission and excitation spectra (Kontron SFM25) with and without purified regulatory subunit of PKA (see below), 100 nM 8-[ϕ-575]-cAMP was measured with and without fourfold molar excess of regulatory subunit in 20 mM MOPS, 150 mM NaCl, 1 mM β-mercaptoethanol, pH (buffer A) at room temperature. The excitation spectra were detected at λem = 617 nm with λex ranging from 610 nm to 430 nm; emission spectra were measured with λex = 575 nm with λem ranging from 800 nm to 580 nm.
Expression and purification of PKA subunits
For expression of PKA regulatory subunits, one liter of Luria Broth medium containing 100 μg/ml of ampicillin was inoculated with E. coli BL21 (DE3) Codon Plus RIL cells (Stratagene) transformed with human RIα, RIβ, RIIα (in pRSETB) or rat RIIβ (in pETIIc) and grown at 37°C to an OD600 nm of 0.8. Recombinant protein expression was induced by addition of 0.2 M isopropyl-β-D-thiogalactopyranoside (IPTG) and the culture was incubated at 25°C for additional 17–18 h. The pellets were stored at -20°C.
For purification of RI isoforms (Moll et al., submitted), cell lysis of protein expressing E. coli cells was performed two times with a French Pressure Cell (Thermo Electron Corp., Needham Heights, USA) in lysis buffer (20 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 100 mM NaCl, 1 mM β-mercaptoethanol, 2 mM EDTA, 2 mM EGTA, pH7.0). The lysate was centrifuged at 27 000 ×g for 30 min and 4°C. 1.2 μmol Sp-8-AEA-cAMPS-agarose (Biolog Life Science Inst.) was used per purification, corresponding to 300–450 μl agarose-slurry. The protein content in 12 ml clarified supernatant was batch bound by gentle rotation over night by 4°C. The agarose was washed seven times with 1.25 ml lysis buffer. The protein elution step was performed with 1.25 ml of 10 mM cGMP in buffer A by gentle rotation at 4°C for 1 h. The agarose was rinsed with two additional wash steps (each 825 μl) with buffer A. Subsequently, the R subunits were subjected to gel filtration (PD10, Amersham Pharmacia Biotech, Freiburg, Germany) into buffer A. To remove all cGMP, the R subunits were dialyzed excessively against buffer A.
For purification of RII isoforms (Moll et al., submitted), cell lysis was performed in buffer consisting 20 mM MES, pH6.5, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA and 5 mM β-mercaptoethanol with added protease inhibitors (PI): Leupeptin (0.025 mg/100 ml), TPCK and TLCK (each 1 mg/100 ml) (buffer B). after centrifugation at 27 000 ×g for 30 min and 4°C, the supernatant was precipitated at 4°C with 50% saturated ammonium sulfate (AS) for RIIα and 45% AS for RIIβ and centrifuged by 10 000 ×g, 15 min 4°C. The AS pellets were re-suspended in buffer B and protein was batch bound to 1.4 μmol settled Sp-8-AEA-cAMPS-agarose. The agarose was rinsed twice with 20 mM MOPS, pH7.0, 1 M NaCl, 5 mM β-mercaptoethanol and then two times with 10 ml buffer B. Subsequently, two elution steps were carried out with 25 mM cGMP in buffer B. RII subunits were subjected to gel filtration into 20 mM MES, pH 6.5,150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM β-mercaptoethanol.
Murine Cα subunit (in pRSETB) was expressed in E. coli BL21 (DE3) (Stratagene) and purified as published previously [45, 46]. Protein expression and purification was followed by SDS-polyacrylamid gel electrophoresis . Typically, the recombinant proteins were purified to 95% homogeneity or higher.
Fluorescence Polarization (FP)
The fluorescence polarization displacement assay was performed as described before. Increasing concentrations of cAMP or 8-[ϕ-575]-cAMP were mixed with 1 nM 8-Fluo-cAMP before adding 2.5 nM regulatory subunit RIα, RIIα, RIIβ. Fluorescence polarization was measured after 5 minutes of incubation at room temperature.
Determination of activation constants
PKA activity was assayed by the coupled spectrophotometric assay first described by Cook et al.  using 260 μM Kemptide (LRRASLG) as the substrate. Holoenzyme formation was carried out for 3 minutes at room temperature with 20 nM murine PKA-Cα subunit and an about 1.2 fold molar excess cAMP-free RIα, RIIα, or RIIβ subunit in assay mixture (10 mM MgCl2, 100 μM ATP, 100 mM MOPS, 1 mM PEP, LDH, pyruvate kinase, NADH, 5 mM β-mercaptoethanol, pH 7.0). Apparent activation constants (Kact, EC50) were determined by adding increasing amounts of cAMP or 8-[ϕ-575]-cAMP (0.3 nM to 10 μM).
COS-7 cells were used for BRET experiments. They were routinely passaged and seeded in opaque 96-well microplates (CulturPlate™-96, PerkinElmer) 24 hours prior to co-transfection with the previously described PKA-IIα sensor, comprised of RIIα-RLuc (donor) and GFP2-Cα (acceptor) . Two days following transfection with 0.5 μg donor and acceptor DNA, respectively, cells were rinsed with glucose-supplemented Dulbecco's PBS (D-PBS, Invitrogen), and subsequently incubated with 8-[ϕ-575]-cAMP (0.01–6 mM final concentration in D-PBS, prepared from a 20 mM stock solution), or mock treated for 30 minutes at room temperature. For the BRET read-out, the luciferase substrate DeepBlueC™ (PerkinElmer) was added at a final concentration of 5 μM in a total volume of 50 μl D-PBS. Light output was detected consecutively using a Fusion™ α-FP microplate reader (PerkinElmer, read time 1s, gain 25) equipped with appropriate filters for the donor (RLuc; λ = 410 nm ± 80 nm) and for the acceptor fluorophore (GFP2; λ = 515 nm ± 30 nm) emission. Emission values obtained with untransfected (n.t.) cells were routinely subtracted, and BRET signals were calculated as follows: (emission(515nm) – n.t. cells(515nm))/(emission(410nm) – n.t. cells(410nm)). Control measurements with cells expressing RLuc and GFP proteins without a fusion partner yield the background BRET signal. A BRET titration (bystander BRET test) was performed by co-transfection of COS-7 cells with a constant donor-expression plasmid (0.5 μg) with an increasing amount of acceptor-expression plasmid (0–2 μg). The cells were treated as described above. Prior to the BRET read-out, cells were incubated with or without 0.6 mM 8-[ϕ-575]-cAMP for 30 minutes.
Uptake of 8-[ϕ-575]-cAMP in living cells
HEK293 and CHO cells were grown in the Dulbecco's modified Eagle medium (DMEM, Invitrogen) and F12 nutrient mixture (HAM, Invitrogen) mediums, respectively, containing 10% FBS and supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all: Sigma-Aldrich), in a 37°C humidified atmosphere containing 5% CO2. These cell lines are routinely used in our laboratory for FRET experiments using various sensors and they have been thoroughly characterized for fluorescent sensor expression levels.
For transient expression of the H30 sensor, cells were seeded onto 24-mm diameter round glass coverslips, and transfections were performed at 50–70% confluence with FuGENE-6 transfection reagent (Roche). Imaging experiments were performed after about 24 h. Cells were maintained in Hepes-buffered Ringer-modified solution, containing 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgS04, 5.5 mM glucose, 1 mM CaCl2, and 20 mM Hepes, pH 7.4, at room temperature and treated with 100 μM 3-Isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich) 10 minutes before the experiments. Cells were imaged on an inverted microscope (IX50; Olympus) with a 60× oil immersion objective (Olympus). The microscope was equipped with a monochromator (Polychrome IV; TILL Photonics) and a beam-splitter optical device (Multispec Microimager; Optical Insights). FRET variations were measured as changes of the ratio between the background-subtracted fluorescence emission intensities at 480 nm and 545 nm, on excitation at 430 nm. Forskolin (25 μM, Sigma-Aldrich) was added to saturate the FRET probe and to determine the maximal FRET response.
For the imaging of the intracellular distribution of 8-[ϕ-575]-cAMP and the Pharos dye, cells were grown and seeded as above. Before the image acquisitions, cells were treated with 100 μM IBMX and either 500 μM 8-[ϕ-575]-cAMP or the Pharos dye for 1 hour. Cells were then washed with the Hepes-buffered Ringer-modified solution described above and imaged on a confocal microscope (Leica) with a 20× oil immersion objective (Leica). Images were obtained by collecting the emission light from 600 nm to 640 nm, on excitation at 514 nm.
Intracellular co-localization of 8-[ϕ-575]-cAMP and GFP-hRIα or GFP-hRIIα was examined in COS-7 cells after two days transient expression of the GFP-tagged R subunits . Cloning of the R subunits into the expression vector hpGFP2-N2 (PerkinElmer) was performed as described previously . The cells were incubated for 30 minutes at 37°C with 500 μM 8-[ϕ-575]-cAMP in D-PBS, rinsed tree times with D-PBS, followed by a standard fixation procedure . Cellular imaging was performed on a confocal microscope (Leica) with a Plan apo 100× oil immersion objective (Leica).
Measurement and statistical evaluation of FP, kinase activity and BRET assays was carried out using GraphPad Prism software version 4 (GraphPad Software).