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• W1966985666 abstract "We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the α1a- and α1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the α1a- and α1b-AR can form homo-oligomers with similar transfer efficiency of ∼0.10. Hetero-oligomers could also be observed between the α1b- and the α1a-AR subtypes but not between the α1b-AR and the β2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the α1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the α1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type α1b-AR. Confocal imaging revealed that oligomerization of the α1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The α1a-selective agonist oxymetazoline induced the co-internalization of the α1a- and α1b-AR, whereas the α1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the α1a- and α1b-AR subtypes. We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the α1a- and α1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the α1a- and α1b-AR can form homo-oligomers with similar transfer efficiency of ∼0.10. Hetero-oligomers could also be observed between the α1b- and the α1a-AR subtypes but not between the α1b-AR and the β2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the α1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the α1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type α1b-AR. Confocal imaging revealed that oligomerization of the α1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The α1a-selective agonist oxymetazoline induced the co-internalization of the α1a- and α1b-AR, whereas the α1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the α1a- and α1b-AR subtypes. G protein-coupled receptors (GPCR), 1The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor(s); G protein, guanylyl nucleotide binding regulatory protein; IP, inositol phosphate; DMEM, Dulbecco's modified Eagle's medium; FRET, fluorescence resonance energy transfer; [125I]HEAT, [125I]iodo-2-[β-(4-hydroxyphenyl)-ethylaminomethyl]tetralone; CFP, cyan fluorescent protein; GFP, green fluorescent proteins; HA, hemagglutinin; GABAB, γ-aminobutyric acid, type B; RANTES, regulated on activation normal T cell expressed and secreted; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; YFP, yellow fluorescent protein. known also as heptahelical receptors, form the largest family of transmembrane signaling proteins transducing signals arising from ions, hormones, neurotransmitters, odorants, chemoattractants, and photons. GPCRs were for a long time presumed to function as monomers according to the prevailing model: one ligand molecule-one receptor-one G protein. Recently, increasing complexity of GPCR function and regulation has progressively emerged. For example, one GPCR can adopt multiple conformational states able to interact differentially with signaling and regulatory proteins (1Ghanouni P. Gryczynski Z. Steenhuis J.J. Lee T.W. Farrens D.L. Lakowicz J.R. Kobilka B.K. J. Biol. Chem. 2001; 276: 24433-24436Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Watson C. Chen G. Irving P. Way J. Chen W.J. Kenakin T. Mol. Pharmacol. 2000; 58: 1230-1238Crossref PubMed Scopus (103) Google Scholar). In addition, receptor cross-talks both at the G protein and at downstream signaling levels have often been described (3Hur E.M. Kim K.T. Cell. Signal. 2002; 14: 397-405Crossref PubMed Scopus (159) Google Scholar, 4Selbie L.A. Hill S.J. Trends Pharmacol. Sci. 1998; 19: 87-93Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Recently, it was shown that cross-talk among GPCRs can also occur at the receptor level by means of receptor oligomerization (reviewed in Refs. 5Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar, 6Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Gomes I. Jordan B.A. Gupta A. Rios C. Trapaidze N. Devi L.A J. Mol. Med. 2001; 79: 226-242Crossref PubMed Scopus (151) Google Scholar). One of the early indications suggesting the capacity of GPCRs to oligomerize came from pharmacological studies showing positive or negative cooperativity of ligand binding curves. More recent support was provided by some “complementation” studies in which co-expression of two GPCR mutants could rescue their functional defects (8Maggio R. Barbier P. Colelli A. Salvadori F. Demontis G. Corsini G.U. J. Pharmacol. Exp. Ther. 1999; 291: 251-257PubMed Google Scholar). These results were interpreted as evidence of inter-molecular interactions between two receptor mutants according to a “domain swapping” model (9Gouldson P.R. Higgs C. Smith R.E. Dean M.K. Gkoutos G.V. Reynolds C.A. Neuropsychopharmacology. 2000; 23: S60-S77Crossref PubMed Scopus (127) Google Scholar). Further indication of GPCR oligomerization came from a large number of studies using co-immunoprecipitation of epitope-tagged receptors co-expressed in the same cells or fluorescence spectroscopy to monitor such oligomers in live cells (reviewed in Refs. 5Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar, 6Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Gomes I. Jordan B.A. Gupta A. Rios C. Trapaidze N. Devi L.A J. Mol. Med. 2001; 79: 226-242Crossref PubMed Scopus (151) Google Scholar). The molecular basis of GPCR oligomerization is not yet fully understood. Co-immunoprecipitation relies on receptor solubilization, which can result in artifactual aggregation of the proteins. To overcome these limits fluorescence spectroscopy techniques, like fluorescence or bioluminescence resonance energy transfer, have been increasingly used. It is widely accepted that energy transfer measurements are well suited to monitor protein-protein interactions or proximity in live cells, as FRET only occurs when the distance between the two fluorophores falls below ∼100 Å (10Pollok B.A. Heim R. Trends Cell Biol. 1999; 9: 57-60Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). So far, in virtually all studies an increased energy transfer signal between GPCRs has been interpreted as the evidence for receptor oligomerization. However, whether receptor oligomerization involves intramolecular interactions among receptors versus their increased proximity within the cell membrane without direct contact cannot be unequivocally demonstrated either by the energy transfer measurements or by the results of co-immunoprecipitation experiments. Therefore, in this study the term receptor “oligomerization” will be used bearing in mind that the precise molecular events underlying this phenomenon are not fully understood. The functional implication of the existence of GPCR homo- and hetero-oligomers has been addressed by several studies. The strongest evidence supporting the functional importance of hetero-oligomerization came from studies on the metabotropic GABAB receptor. The full reconstitution of the functional activity of the GABAB receptor requires the oligomerization between the GABAB-R1 and GABAB-R2 receptor monomers (11Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). For other GPCRs, a role of oligomerization in receptor signaling as well as internalization has been suggested (reviewed in Refs. 5Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar, 6Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Gomes I. Jordan B.A. Gupta A. Rios C. Trapaidze N. Devi L.A J. Mol. Med. 2001; 79: 226-242Crossref PubMed Scopus (151) Google Scholar). However, much work is still required to elucidate how oligomerization is involved in these distinct processes. In addition, whereas the majority of studies was performed in recombinant systems, only in a few cases was evidence provided that GPCR oligomerization occurs in physiological systems. For example, in a recent study hetero-oligomers formed by the adenosine A1 and glutamate mGluR1 receptors were isolated from cerebellar neuronal cultures (12Ciruela F. Escriche M. Burgueno J. Angulo E. Casado V. Soloviev M.M. Canela E.I. Mallol J. Chan W.Y. Lluis C. McIlhinney R.A. Franco R. J. Biol. Chem. 2001; 276: 18345-18351Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In addition, hetero-oligomers formed by the angiotensin II AT1 and bradykinin B2 receptors were isolated in platelets of pre-eclamptic pregnant women (13AbdAlla S. Lother H. el Massiery A. Quitterer U. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (417) Google Scholar). Within the adrenergic receptor (AR) family, oligomerization has been extensively studied for the β2-AR (14Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 15Hebert T.E. Loisel T.P. Adam L. Ethier N. Onge S.S. Bouvier M. Biochem. J. 1998; 330: 287-293Crossref PubMed Scopus (85) Google Scholar, 16Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar), and these studies have often represented an important reference for investigating the oligomerization of other GPCRs. The β2-AR can form both homo- and hetero-oligomers with the δ- and κ-opioid receptors (17Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar) as well as with the β1-AR (18Lavoie C. Mercier J.F. Salahpour A. Umapathy D. Breit A. Villeneuve L.R. Zhu W.Z. Xiao R.P. Lakatta E.G. Bouvier M. Hebert T.E. J. Biol. Chem. 2002; 277: 35402-35410Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Homo-oligomerization of the β2-AR is constitutive, but it can also be enhanced by exposure to the agonist (16Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar). In contrast to the amount of information available for the β2-AR and several other GPCRs, nothing is known so far on the putative oligomeric state of the α1-AR subtypes. In this study we extensively investigated the ability of the α1a- and α1b-AR subtypes to oligomerize using biophysical, biochemical, as well as pharmacological approaches. FRET measurements provided solid evidence that both recombinant α1a- and α1b-AR subtypes can selectively form homo- and hetero-oligomers. Our results suggest that the homo-oligomerization of the α1b-AR involves the participation of helix I and, to a lesser extent, of helix VII. The results of confocal imaging strongly suggest that receptor oligomerization plays a role in receptor endocytosis, which might have implications for the physiological responses mediated by the α1a- and α1b-AR subtypes. Materials—The plasmid encoding the chemokine CCR5 receptor and RANTES were kind gifts of Dr. Jean-Luc Galzi, UPR 9050 CNRS, Illkirch, France. The NK1-pEGFP-N1 plasmid was a kind gift of Dr. Bruno Meyer, EPFL, Lausanne, Switzerland. The pEGFP-N1, pECFP-N1, and pEYFP-N1 vectors were from Clontech. Immobilon membranes were from Millipore. Prolong mounting medium was from Molecular Probes. Monoclonal anti-c-Myc antibodies, protein A-Sepharose, epinephrine, and prazosin from Sigma. Pwo DNA polymerase, complete protease inhibitor mixture, and restriction enzymes were from Roche Applied Science. Monoclonal and polyclonal anti-HA antibodies were from Santa Cruz Biotechnology. Fluorescein- and rhodamine-coupled anti-rabbit and rhodamine-coupled anti-mouse antibodies were from The Jackson Laboratories. Anti-rabbit and anti-mouse horseradish peroxidase-coupled antibodies, [125I]iodocyanopindolol, and ECL Western blotting detection reagent were from Amersham Biosciences. Protein assay (Bradford) was from Bio-Rad. Effectene transfection reagent was from Qiagen. DMEM, fetal calf serum, fungizone, and gentamycin were from Invitrogen. [125I]HEAT and [3H]inositol were from PerkinElmer Life Sciences. Construction of Receptor-GFP and CFP Fusion Proteins and Site-directed Mutagenesis—The full-length cDNA encoding the hamster α1b-AR (19Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar) was PCR-amplified and inserted into the pEGFP-N1 and pECFP-N1 vectors using EcoRI/AgeI to give the α1b-pEGFP-N1 and α1b-pECFP-N1 vectors, respectively. The cDNA encoding the α1b-AR fused to GFP (α1b-GFP) was also subcloned in the pRK5 using EcoRI/XbaI to give the α1b-GFP-pRK5 vector. The Thr369-GFP, Thr369-CFP, and Thr369-YFP constructs were obtained by PCR-amplifying a DNA fragment of the α1b encompassing amino acids 1-369 and subcloning it at the EcoRI-AgeI sites into the α1b-GFP-pRK5, α1b-pECFP-N1, and pEYFP-N1 vectors, respectively. The R143E-GFP and A293E-GFP constructs were obtained by replacing the EcoRI-BssHII fragment of the previously described R143E and A293E mutants (20Scheer A. Costa T. Fanelli F. De Benedetti P.G. Mhaouty-Kodja S. Abuin L. Nenniger-Tosato M. Cotecchia S. Mol. Pharmacol. 2000; 57: 219-231PubMed Google Scholar, 21Mhaouty-Kodja S. Barak L.S. Scheer A. Abuin L. Diviani D. Caron M.G. Cotecchia S. Mol. Pharmacol. 1999; 55: 339-347Crossref PubMed Scopus (78) Google Scholar) into the α1b-GFP-pRK5 vector. The G53L and G301L mutants were constructed by PCR mutagenesis and subcloned into the α1b-GFP-pRK5 vector to give the G53L-GFP and G301L-GFP constructs, respectively. The construction of the N-glycosylation-deficient mutant N4 fused to GFP (N4-GFP) was described previously (22Bjorklof K. Lundstrom K. Abuin L. Greasley P.J. Cotecchia S. Biochemistry. 2002; 41: 4281-4291Crossref PubMed Scopus (15) Google Scholar). Fragments of the α1b-AR lacking different transmembrane helices were constructed by PCR introducing EcoRI and BssHII sites at their 5′ and 3′ ends, respectively, and subcloned into the Thr369-YFP at the EcoRI site (which is in the 5′-untranslated) and BssHII site (corresponding to amino acids 356 and 357). The receptor fragments engineered were the following: α1b(I-III), amino acids 1-148; α1b(I-V), amino acids 1-231; α1b(III-VII), amino acids 112-369; and α1b(V-VII), amino acids 195-369. All the fragments included 4-5 residues beyond the helical portion as well as also amino acids 356-369 of the C-tail before the fluorophore. The α1b/β2 adrenergic chimeric receptors were constructed using PCR by replacing each helix of the hamster α1b-AR with the corresponding segment of the human β2-AR. The helices were defined based on sequence alignment and on the x-ray structure of rhodopsin. The residues of the α1b-AR replaced with those of the β2-AR for each helix (h) were the following: hI (Ala45-Ala71 α1b/Val33-Ala59 β2); hII (Thr80—Val107 α1b/Thr68-Leu95 β2); hIII (Phe117-Tyr144 α1b/Trp105-Tyr133 β2); hIV (Ala162-Leu181 α1b/Ala150-Ile169 β2); hV (Tyr203-Val226α1b/Tyr199-Val222 β2); hVI (Arg288-Leu316 α1b/Lys267-Val295 β2); hVII (Val329-Ile362 α1b/Val307-Leu340 β2). The percentage of sequence identity between the portions replaced in each helix was 38% for hI, 50% for hII, 57% for hIII, 30% for hIV, 50% for hV, 65% for hVI, and 41% for hVII. The full-length cDNA encoding the human α1a-AR (23Schwinn D.A. Johnston G.I. Page S.O. Mosley M.J. Wilson K.H. Worman N.P. Campbell S. Fidock M.D. Furness L.M. Parry-Smith D.J. J. Pharmacol. Exp. Ther. 1995; 272: 134-142PubMed Google Scholar) was PCR-amplified and inserted into the pEGFP-N1 and pECFP-N1 vectors using EcoRI/AgeI (blunted) to give the α1a-GFP and α1a-CFP constructs, respectively. The full-length cDNA encoding the human β2-AR was PCR-amplified and subcloned into the pEGFP-N1 using HindIII/AgeI to give the β2-GFP construct. The full-length cDNA encoding the chemokine CCR5 receptor was PCR-amplified and subcloned into the pECFP-N1 using HindIII/AgeI to give the CCR5-CFP construct. Construction of Epitope-tagged Receptors—The C-terminal fragments of the α1a-AR and the α1b-AR were amplified using primers encoding the HA (YPYDVPDYA) or Myc (EQKLISEEDL) epitopes at the 3′ end and inserted into the α1b-AR-pRK5 (using BamHI/HindIII) or α1a-AR-pRK5 (using EcoRV/BamHI) vectors, respectively. The full-length cDNA encoding the chemokine CCR5 receptor was PCR-amplified using a primer encoding the Myc epitope at the 5′ end and subcloned into the pRK5 vector using BamHI/HindIII. The full-length cDNA encoding the tachykinin NK1 receptor was amplified using a primer encoding the Myc epitope at the 3′ end and subcloned into the pRK5 vector using EcoRI/HindIII. Cell Culture and Transfection—HEK-293 cells were grown in DMEM supplemented with 10% fetal calf serum and gentamycin (100 μg/ml) (37 °C and 5% CO2) and transfected using the calcium-phosphate method or the transfection reagent Effectene following the manufacturer's protocol. For inositol phosphate determination, cells (0.15 × 106) were seeded in 12-well plates and transfected with 0.2-0.5 μg/well using Effectene. For ligand binding, FRET, and immunoprecipitation experiments, cells were grown in 100-mm dishes and transfected with a maximum of 20 μg of DNA/dish using the calcium-phosphate method. For confocal imaging cells were grown on glass coverslips in 6-well dishes and transfected with 1-2 μg of DNA/well using the calcium-phosphate method. For cells transfected with different combinations of plasmids, the total amount of transfected DNA was kept constant in the samples using pRK5. Membrane Preparation and Ligand Binding—48 h after transfection, cells were washed in PBS, scraped off the culture plates, and collected in ice-cold membrane buffer (5 mm Tris, 0.5 mm EDTA, pH 7.4). After centrifugation at 40,000 × g, the pellet was resuspended in ice-cold membrane buffer and Polytron-homogenized. Protein concentration was determined using the Bradford protein assay. For ligand binding of [125I]HEAT, the membranes were resuspended in binding buffer (50 mm Tris, 0.5 mm EDTA, 150 mm NaCl, pH 7.4) and incubated with the radioligand for 1 h at 25 °C. [125I]HEAT was used at a concentration of 250 pm for measuring receptor expression at a single concentration and of 80 pm for competition binding analysis. For saturation binding experiments, the radioligand concentration ranged between 10 and 400 pm. Prazosin at 10-6m was used to determine nonspecific binding. Saturation analysis and competition curves were analyzed using Prism 3.02 (GraphPad Software Inc., San Diego). Inositol Phosphate Accumulation—24-36 h after transfection, cells were labeled for 12 h with myo-[3H]inositol at 4 μCi/ml in inositol-free DMEM supplemented with 1% fetal bovine serum. Cells were pre-incubated for 10 min in PBS containing 20 mm LiCl and then stimulated for 45 min with different concentrations of epinephrine. Total inositol phosphates were extracted and separated as described previously (19Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE and Western Blotting—Samples were denatured in SDS-PAGE loading buffer (65 mm Tris, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, pH 6.8) for 1 h at room temperature, separated on 10% acrylamide gels, and electroblotted onto Immobilon membranes. Blots were incubated in TBS buffer (50 mm Tris, 150 mm NaCl, pH 8) containing 0.05% Tween 20, 5% (w/v) powder milk, and the primary antibody (mouse monoclonal anti-HA or anti-Myc) diluted 1:200. After the primary antibody was washed, the secondary anti-mouse antibody linked to horse-radish peroxidase was added, and the blots were developed using the enhanced chemiluminescence (ECL) detection system. Immunoprecipitation of Receptors—36 h after transfection, cells were washed twice with PBS and scraped off the culture plates in ice-cold buffer containing 5 mm Tris and 5 mm EDTA, pH 7.4. After centrifugation at 40,000 × g, the pellet was resuspended in 1 ml of ice-cold lysis buffer (20 mm Tris, 0.5 mm EDTA, 1% digitonin, 100 mm NaCl, pH 7.4) containing a complete protease inhibitor mixture. Solubilization was carried out for 3 h at 4 °C on a spinning wheel. Unsolubilized material was pelleted by centrifugation for 15 min at 20,000 × g on a bench-top centrifuge. The supernatant was incubated with 5 μg of anti-HA polyclonal antibody overnight on the spinning wheel at 4 °C. After addition of 2.5% (w/v) protein A-Sepharose, the incubation was continued for 2 h at 4 °C, followed by a brief centrifugation on a bench-top centrifuge. The pellet was washed three times in the lysis buffer, once in PBS, and then dissolved in SDS-PAGE loading buffer for 1 h at 37 °C. Fluorescence Spectroscopy—48 h after transfection, cells were washed in PBS and detached from the plates using PBS containing 0.5 mm EDTA. The cells from one 10-cm culture dish were resuspended in 1 ml of isotonic buffer (137.5 mm NaCl, 1.25 mm MgCl2, 1.25 mm CaCl2, 6 mm KCl, 5.6 mm glucose, 10 mm HEPES, 0.4 mm NaH2PO4, pH 7.4) and used in spectrofluorimetric measurements. Cells were routinely co-transfected with equal amounts of plasmids encoding the receptors fused to GFP or to CFP (5-10 μg of each plasmid/100-mm diameter dish) in order to approach a CFP:GFP ratio of 1. Fluorescence spectra of cell suspensions were recorded on a SPEX Fluorolog II (Instruments S.A., Stanmore, UK). Samples were placed in a 10 × 4-mm2 quartz cuvette (Hellma, Germany) and continuously stirred by a magnetic stirrer. Excitation and emission slits were set at 2-nm band path. CFP and GFP were excited at 430 and 488 nm, respectively, and their fluorescence measured at 450-540 and 500-540 nm, respectively. The spectral contributions arising from light scattering and nonspecific fluorescence of the cells were eliminated by subtracting the emission spectra of mock-transfected cells from the fluorescence spectra of cells expressing the receptor-CFP and GFP constructs. To do so, we measured the emission intensity at 450 nm upon excitation at 430 nm, considering that CFP or GFP fluorescence is negligible and that only light scattering contributes to emission at this wavelength. We then calculated the ratio between the intensities at 450 nm in mock-transfected versus cells expressing the fluorescent receptors and used it as a correction factor F(background). The spectrum of the mock-transfected cells divided by the F(background) was then subtracted from the spectrum of the cells expressing receptor-CFP and GFP constructs. To take into account the differences in expression level of the receptor-CFP and -GFP constructs in co-transfected cells, a second normalization step was adopted. The fluorescence spectra of cells individually expressing the receptor-CFP and -GFP constructs were measured and divided by a correction factor F. For the receptor-CFP excited at 430 nm: F(CFP) = CFP(430/465)/CFP and GFP(430/465). For the receptor-GFP excited at 430 nm: F(GFP) = GFP(490/510)/CFP and GFP(490/510). After normalizing for cell density, FRET was measured by subtracting from the emission spectrum of cells expressing both fluorescent receptors the emission spectra obtained by excitation at 430 nm of cells individually expressing the receptor-CFP and GFP constructs, each divided by its respective correction factor F. The resulting spectrum in cells expressing both fluorescent receptors corresponds to GFP emission arising exclusively from FRET. The transfer efficiency E was determined as shown in Equations 1 and 2, E=AA(λD)AD(λD)IAD(λD,λA(emi)IA(λD,λA(emi)-1(Eq. 1) where AA(λD)AD(λD)=ϵA(λD)ϵD(λD)·IA(λA,λA(emi))·ϵD(λD)ϕD(λD(emi))IDA(λD,λD(emi))1-E·ϵA(λA)ϕA(λA(emi))(Eq. 2) and λD, 430 nm; λA, 490 nm; λD(emi), 475 nm; λA(emi), 510 nm; absorption coefficient of the donor at λD, ϵA(λD) = 13,750 cm-1m-1; absorption coefficient of the acceptor at λA, ϵA(λA) = 55,000 cm-1m-1; fluorescence quantum yield of the donor, [phis]D = 0.4; fluorescence quantum yield of the acceptor, [phis]A = 0.6; A A(λD), acceptor absorbance at the wavelength of donor excitation; A D(λD), donor absorbance at the wavelength of donor excitation; I A(λD, λA(emi)), fluorescence intensity of the acceptor at λA(emi) when excited at λD; I AD(λD, λA(emi)), fluorescence intensity of the acceptor at λA(emi) when excited at λD in the presence of the donor; I DA(λD, λDem), fluorescence intensity of the donor at λD(emi) when excited at λD in the presence of the acceptor. The GFP:CFP ratio was given by Equation 3, CACD=IA(λA,λA(emi))·ϵD(λD)ϕD(λD(emi))IDA(λD,λD(emi))1-E·ϵA(λA)ϕA(λA(emi))(Eq. 3) where CD is the concentration of the donor (CFP); CA is the concentration of the acceptor (GFP). For FRET measurements on receptor fragments fused to YFP, the elaboration of the results described above was adapted to fit the YFP properties. CFP and YFP were excited at 430 and 500 nm, respectively, and their fluorescence was measured at 450-570 and 520-570 nm, respectively. In this case, the acceptor parameters used are as follows: λA, 500 nm; λA(emi), 530 nm; quantum yield of acceptor [phis]A, 0.6; ϵA(λD), 3530 cm-1m-1; ϵA(λA) = 43,700 cm-1m-1. To determine the effect of ligands on FRET efficiency, cell suspensions were incubated at saturating concentrations of epinephrine (10-4m) or prazosin (10-6m) for 15 min at 37 °C under gentle shaking. Spectra of treated and untreated cells were recorded at 37 °C. Internalization Experiments—36 h after transfection, cells grown on glass coverslips were treated for 1 h with vehicle or with a saturating concentration of different agonists (10-4m epinephrine, 10-5m oxymetazoline, 10-4m substance P, or 10-6m RANTES). After treatment, cells were washed with PBS, fixed with formaldehyde, and permeabilized for 5 min in 0.2% Triton. For immunostaining of cells expressing the HA-tagged receptors, the cells were incubated for 1 h in PBS containing 1% BSA and for another hour in PBS containing 0.1% BSA, and the polyclonal anti-HA antibody was diluted at 1:100. After a second incubation with the anti-rabbit rhodamine-coupled antibody diluted at 1:100 in PBS containing 0.1% BSA, the coverslips were washed three times in PBS and mounted in Prolong mounting medium. For immunostaining of cells co-expressing the α1b-HA and myc-CCR5, the polyclonal anti-HA and the monoclonal anti-Myc antibodies were used, followed by the anti-rabbit FITC-coupled and the anti-mouse rhodamine-coupled secondary antibodies. Cells immunostained with fluorescent antibodies were analyzed by confocal imaging. Confocal Imaging—Cells expressing different fluorescent receptors were imaged using a Zeiss LSM510 confocal microscope equipped with a C-apochromat ×63/1.2-watt water immersion objective (Zeiss, Germany). The following laser lines were used for excitation: 458 nm for CFP, 488 nm for GFP and FITC, and 543 nm for rhodamine. The following Zeiss filter sets were used to detect the fluorescence of a particular fluorophore: LP475 for CFP, BP505-550 for GFP and FITC, and LP543 for rhodamine. The pinhole was kept at ∼1 airy unit for all recordings. Functional Characterization of the Tagged Receptors—To investigate the pharmacological properties of the α1-AR subtypes tagged with different epitopes (HA and Myc) or fluorescent proteins (GFP and CFP), the receptors were expressed in HEK-293 cells and tested for their ability to bind the radioligand [125I]HEAT and epinephrine. Saturation binding experiments indicated that the K D values of [125I]HEAT were similar at the various tagged or non-tagged α1-AR constructs (results not shown). As shown in Table I, the expression levels of the various receptors ranged between 300 and 900 fmol/well. The IC50 values of epinephrine were similar at the tagged or non-tagged wild typ" @default.
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• W1966985666 date "2003-10-01" @default.
• W1966985666 modified "2023-10-16" @default.
• W1966985666 title "Oligomerization of the α1a- and α1b-Adrenergic Receptor Subtypes" @default.
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• W1966985666 doi "https://doi.org/10.1074/jbc.m306085200" @default.
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