Materials came from the following sources: cell culture media, fetal bovine serum, G418 and Lipofectamine were from Invitrogen. Isoproterenol, propranolol, anti-FLAG monoclonal antibody (M1 clone), anti-HA polyclonal antibody and Hoechst 33258 were from Sigma. Phospho-p44/42 MAPK rabbit monoclonal antibody was from Cell Signalling Technology. Alexa-Fluor 488-conjugated transferrin, and antibodies Alexa-Fluor 488 and Alexa-Fluor 594 goat anti-mouse IgG, were from Molecular Probes. Nitrocellulose transfer membrane was from Schleicher&Schuell. Enhanced chemiluminescent substrate (ECL) for detection of HRP was from PIERCE.
The construction of full-length cDNA encoding the Flag-tagged β2AR-Gαs fusion protein was described previously .
The amino terminal Flag tagged wild receptor was obtained in a similar way. Briefly, the cleavable prolactin signal peptide tethered to the Flag epitope (DYKDDDDK) was added immediately before the Gly2 in β2 AR by PCR-based strategies and cloned in a pcDNA3 vector (Invitrogen).
The cDNA encoding for 3HA-β2AR was obtained by the annealing of two synthetic oligonucleotides containing the sequences of three sequential modules of the influenza hemagglutinin HA epitope (AYPYDVPDYA), and was cloned into pcDNA3 vector. Then, by PCR, we obtained the cDNA encoding for the β2AR deprived of the Met initiator codon, which was subcloned into the vector, in frame with the 3 × HA epitope.
Cell Culture and Transfection
Human embryonic kidney (HEK293) cells stably expressing the wild type β2AR or chimeric protein β2AR-Gαs were generated as described previously . The total number of expressed receptors was measured in membranes prepared from the transfected cells using radioligand binding assays, as described previously . The levels of receptor expression were 15 and 10.8 pmol/mg in cells expressing wild type β2AR and β2AR-Gαs, respectively.
Cells were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS; GIBCO), 100 units/ml penicillin, 100 μg/ml streptomycin sulphate, and 200 μg/ml G418 (GIBCO) in a humidified atmosphere of 5% CO2 at 37°C.
For transient transfections, cells cultured in 35-mm tissue culture dishes were transfected with 0.3 μg of pcDNA3/HA-β2AR and 0.7 μg of empty vector using Lipofectamine (Invitrogen), according to the manufacturer's instructions. The cells were allowed to express the transfected gene for 48 hrs before harvesting.
Cells were grown on 35-mm tissue culture dishes and following the various treatments were fixed with 4% buffered formaldehyde for 20 minutes. If necessary, fixed cells were permeabilized with 0.2% Nonidet P-40 (NP-40) in phosphate-buffered saline (PBS) for 10 min to assure the accessibility of intracellular and intravesicular antigens. For the detection of epitope-tagged receptors, anti-Flag mouse monoclonal antibody was added in blocking buffer (1% BSA) for 50 min at room temperature (r.t.) followed by Alexa-Fluor 594 conjugated anti-mouse IgG (Molecular Probes). In dual staining experiments, co-localization of Flag-tagged receptors and HA-tagged receptors in a single cell line, was performed by incubating cells with anti-Flag and anti-HA rabbit polyclonal antibody (Sigma).
Stained specimens were examined by conventional epifluorescence microscope (Olympus BX51; Tokyo, Japan) or confocal microscope.
Confocal Laser Scanning Microscopy
Fluorescently labelled preparations were also observed by a confocal fluorescent imaging system using the confocal laser scanning microscope LEICA TCS 4D (Leach Instruments, Heidelberg, Germany) supplemented with an Argon/Kripton laser and equipped with 40 × 1.00–0.5 and 100 × 1.3–0.6 oil immersion lenses. The excitation/emission wavelengths employed were 488 nm/510 nm, and 568 nm/590 nm for specific Alexa-Fluor labelling. Confocal sections were acquired at intervals of 0.5 μm from the middle to the bottom toward the cells, and a 3D reconstruction image of the fluorescent signal was obtained. In double staining experiments, confocal sections for both fluorescent signals were taken simultaneously, the 3D reconstruction images were recorded, and merged images of the two signals were obtained using confocal microscope software.
Receptor Internalization Assays
Cells, cultured on 96 multiwell plates (Nunc) until they reached confluence, were stimulated with 1 μM isoproterenol (Sigma) at 37°C for different times to drive agonist-induced internalization. At the end of the time-course, cells were immediately fixed with 4% buffered formaldehyde for 20 min, incubated in blocking buffer for 30 min and immuno-stained for 50 min with anti-FLAG mouse monoclonal antibody (Sigma), followed by Alexa-Fluor 488 conjugated anti-mouse IgG (Molecular Probes). Nuclei were stained with Hoechst 33258 (1 μg/ml). Fluorescence was measured by a multi-label counter (Victor 2; Wallac), setting excitation and emission filters as follows: λex = 485/λem = 535, and λex = 355/λem = 460 for Alexa-Fluor 488 and Hoechst, respectively. Each point was performed in triplicate and the receptor internalization was monitored in relationship to the decrease of fluorescence from the plasma membrane.
Receptor Recycling Assays
For recycling studies, cells cultured on 96 multiwell plates were incubated with 1 μM isoproterenol for 5 hrs to induce maximal receptor internalization. Reversal of internalization was initiated by adding the antagonist, 1 μM Propranolol (Sigma), for different times. In other experiments, 50 μg/ml cycloheximide (CHX; Sigma) was added during the last 2 hrs of agonist treatment. At the end of the time-course, the cells were fixed and subjected to immunofluorence followed by fluorimetry analysis as above.
Receptor recycling from the endocytic pathway was estimated by assaying the recovery of immunoreactive epitopes at the cell surface that were accessible by monoclonal antibody.
Localization of Receptor with Endocytosed Transferrin
In order to estimate the degree of co-localization of the receptor with endocytosed transferrin, cells were incubated for 30 min at 37°C in culture medium containing 100 μM Alexa-Fluor 488-conjugated transferrin (Molecular Probes), and then 1 μM isoproterenol was added for an additional 30 min.
Next, cells were washed twice in pre-warmed PBS to remove non-internalized transferrin and were subjected to immunofluorescence as above. Briefly, fixed cells were permeabilized and immuno-stained for detection of Flag-tagged receptors. Dual-label fluorescence microscopy was performed as described above.
Immunoblotting for Phospho p42/44 MAP Kinases
Cells were first sensitized by incubating with the agonist isoproterenol (1 μM) for different times and were then dissolved in Laemmli's sample buffer. Samples were electrophoresed through a 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes (Schleicher&Schuell). Blots were incubated in blocking buffer (5% w/v non fat dry-milk, 0,1% Tween-20 in Tris buffered saline) for 1 hrs at r.t., followed by incubation overnight at 4°C with Phospho-p44/42 MAP kinases rabbit monoclonal antibody (Cell Signalling Technology), which detects endogenous levels of p42 and p44 MAP kinases (ERK1/2) when phosphorylated at Thr202 and Tyr204, respectively. Immunoreactive bands probed with horseradish peroxidase-conjugated anti rabbit IgG antibody were visualized by chemiluminescence (ECL; PIERCE), according to the manufacturer's instructions.
Samples were normalized for protein content by reprobing blots with an alpha-tubulin monoclonal antibody (Sigma).