Lack of Evidence for AT1R/B2R Heterodimerization in COS-7, HEK293, and NIH3T3 Cells
2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês
10.1074/jbc.m804607200
ISSN1083-351X
AutoresJakob Lerche Hansen, Jonas Hansen, Tobias Speerschneider, Christina Lyngsø, Niels Erikstrup, Ethan S. Burstein, David M. Weiner, Thomas Walther, Noriko Makita, Taroh Iiri, Nicole Merten, Evi Kostenis, Søren P. Sheikh,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoIt has been suggested previously (AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, β-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling. It has been suggested previously (AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, β-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling. The angiotensin II type 1 (AT1) 5The abbreviations used are: AT1, angiotensin II type 1; AT2, angiotensin II type 2; Ang II, angiotensin II; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; B2R, bradykinin B2 receptor; BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; RSAT, receptor selection and amplification technology; 7TM, seven-transmembrane; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; P-ERK, phosphorylated ERK; Rluc, Renilla luciferase; GABAB, γ-aminobutyric acid, type B; wt, wild type; ER, endoplasmic reticulum; IP, inositol phosphate.5The abbreviations used are: AT1, angiotensin II type 1; AT2, angiotensin II type 2; Ang II, angiotensin II; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; B2R, bradykinin B2 receptor; BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; RSAT, receptor selection and amplification technology; 7TM, seven-transmembrane; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; P-ERK, phosphorylated ERK; Rluc, Renilla luciferase; GABAB, γ-aminobutyric acid, type B; wt, wild type; ER, endoplasmic reticulum; IP, inositol phosphate. receptor belongs to the superfamily of the seven-transmembrane (7TM) or G protein-coupled receptors. The AT1R is a key regulator of blood pressure and body fluid homeostasis, and its importance in renal and cardiovascular pathophysiology is underscored by the widespread use of receptor blockers and inhibitors of the angiotensin-converting enzyme in clinical practice (1Zaman M.A. Oparil S. Calhoun D.A. Nat. Rev. Drug Discov. 2002; 1: 621-636Crossref PubMed Scopus (338) Google Scholar, 2Healey J.S. Baranchuk A. Crystal E. Morillo C.A. Garfinkle M. Yusuf S. Connolly S.J. J. Am. Coll. Cardiol. 2005; 45: 1832-1839Crossref PubMed Scopus (696) Google Scholar). There is mounting evidence that the AT1R may form both homo- and heterodimers and that dimerization could be important for the receptor function (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 4Karip E. Turu G. Supeki K. Szidonya L. Hunyady L. Neurochem. Int. 2007; 51: 261-267Crossref PubMed Scopus (19) Google Scholar, 5AbdAlla S. 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The AT1R also co-immunoprecipitates with the Dopamine D1 and D3 receptor in tissue specimen obtained from Wistar-Kyoto and spontaneously hypertensive rats (13Zeng C. Luo Y. Asico L.D. Hopfer U. Eisner G.M. Felder R.A. Jose P.A. Hypertension. 2003; 42: 787-792Crossref PubMed Scopus (87) Google Scholar, 15Zeng C. Asico L.D. Wang X. Hopfer U. Eisner G.M. Felder R.A. Jose P.A. Hypertension. 2003; 41: 724-729Crossref PubMed Scopus (60) Google Scholar). Finally AbdAlla et al. (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar, 7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar, 16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar) have shown in a number of studies that the AT1R forms heterodimers with the B2R. From these studies, they derive three important pharmacological conclusions. First, the AT1R and the B2R form heterodimers in all the cellular systems examined including HEK293 cells (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), primary cells such as vascular smooth muscle cells (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), neurons (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar), and platelets (7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar). These data indicate that the system is "universal" and that AT1R/B2R heterodimerization occurs as a natural consequence of simultaneous AT1R and B2R expression within the same cell (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar, 7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar, 16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar). Second, when compared with the individually expressed receptors, the AT1R/B2R heterodimer signals with higher potency and efficacy upon Ang II stimulation, whereas bradykinin signaling remains largely unaffected (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar). Third, the B2R must be competent to engage its G protein signaling pathway to produce the functional AT1R potentiation (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar). From a medical perspective, the AT1R/B2R interaction would be of outstanding value because the authors show that it can be linked to experimental hypertension (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar), vascular smooth muscle cell contraction (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), and preeclampsia (7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar). The disease relevance of the dimer implies that development of AT1R/B2R heterodimer-specific antagonists could be beneficial for the treatment of cardiovascular diseases. The interaction is also of great biological interest because it could add an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems (17Shen B. Harrison-Bernard L.M. Fuller A.J. Vanderpool V. Saifudeen Z. El-Dahr S.S. J. Am. Soc. Nephrol. 2007; 18: 1140-1149Crossref PubMed Scopus (34) Google Scholar, 18Tan Y. Hutchison F.N. Jaffa A.A. Am. J. Physiol. 2004; 286: H926Crossref PubMed Scopus (30) Google Scholar). Both systems are intricately connected. (i) The AT1R mediates vasoconstriction, whereas the B2R is a vasodepressor (17Shen B. Harrison-Bernard L.M. Fuller A.J. Vanderpool V. Saifudeen Z. El-Dahr S.S. J. Am. Soc. Nephrol. 2007; 18: 1140-1149Crossref PubMed Scopus (34) Google Scholar, 18Tan Y. Hutchison F.N. Jaffa A.A. Am. J. Physiol. 2004; 286: H926Crossref PubMed Scopus (30) Google Scholar). (ii) Angiotensin-converting enzyme is responsible for both production of circulating Ang II and at the same time degradation of bradykinin (19Yang H.Y. Erdos E.G. Levin Y. Biochim. Biophys. Acta. 1970; 214: 374-376Crossref PubMed Scopus (477) Google Scholar). (iii) AT1R activation leads to up-regulation of the B2R (17Shen B. Harrison-Bernard L.M. Fuller A.J. Vanderpool V. Saifudeen Z. El-Dahr S.S. J. Am. Soc. Nephrol. 2007; 18: 1140-1149Crossref PubMed Scopus (34) Google Scholar, 18Tan Y. Hutchison F.N. Jaffa A.A. Am. J. Physiol. 2004; 286: H926Crossref PubMed Scopus (30) Google Scholar). It has also been shown that many biological effects of AT1R blockers such as losartan are in fact mediated by the release of bradykinin and, accordingly, can be blocked by the B2R inhibitor HOE130 (20Hu K. Gaudron P. Anders H.J. Weidemann F. Turschner O. Nahrendorf M. Ertl G. Cardiovasc. Res. 1998; 39: 401-412Crossref PubMed Scopus (67) Google Scholar, 21Zhu P. Zaugg C.E. Hornstein P.S. Allegrini P.R. Buser P.T. J. Cardiovasc. Pharmacol. 1999; 33: 785-790Crossref PubMed Scopus (30) Google Scholar, 22Sato M. Engelman R.M. Otani H. Maulik N. Rousou J.A. Flack III, J.E. Deaton D.W. Das D.K. Circulation. 2000; 102: III346-III351PubMed Google Scholar). Finally heterodimerization between the AT2 receptor and B2R has also been proposed to enhance NO release (23Abadir P.M. Periasamy A. Carey R.M. Siragy H.M. Hypertension. 2006; 48: 316-322Crossref PubMed Scopus (125) Google Scholar), and it has been implicated in left ventricular remodeling after myocardial infarction in mice (24Isbell D.C. Voros S. Yang Z. DiMaria J.M. Berr S.S. French B.A. Epstein F.H. Bishop S.P. Wang H. Roy R.J. Kemp B.A. Matsubara H. Carey R.M. Kramer C.M. Am. J. Physiol. 2007; 293: H3372Crossref PubMed Scopus (14) Google Scholar). The concept of 7TM receptor heterodimerization is of emerging importance, and it will be crucial to address general questions with respect to experimental pitfalls, specificity, generality, and biological significance of a given heterodimeric interaction. Scientists from four independent groups set out to study the putative AT1R/B2R heterodimerization in detail. Surprisingly although both the AT1Rs and the B2Rs were functional in our studies, we were not able to establish any functional potentiation or physical interaction between the two receptors. Despite careful experimental fine tuning, our data do not support the concept of an AT1R/B2R heterodimer occurring as a natural consequence of simultaneous AT1R and B2R expression in the same cell. The rAT1aR and the hAT1R have been described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Hansen J.L. Haunso S. Brann M.R. Sheikh S.P. Weiner D.M. Mol. Pharmacol. 2004; 65: 770-777Crossref PubMed Scopus (22) Google Scholar). Using PCR-based methods, the rB2R was cloned from rat heart cDNA, and the hB2R was cloned from human smooth muscle cDNA. Both receptors were fully sequence-verified and subcloned into the pCDNA3.1 (Invitrogen) and pSI (Promega) expression vectors, respectively. The wild type rat B2R sequence is identical to that of GenBank™ accession number M59967 (26McEachern A.E. Shelton E.R. Bhakta S. Obernolte R. Bach C. Zuppan P. Fujisaki J. Aldrich R.W. Jarnagin K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7724-7728Crossref PubMed Scopus (402) Google Scholar), and the human B2R is identical to that described in Hess et al. (27Hess J.F. Borkowski J.A. Young G.S. Strader C.D. Ransom R.W. Biochem. Biophys. Res. Commun. 1992; 184: 260-268Crossref PubMed Scopus (447) Google Scholar). The enhanced GFP-tagged bovine β-arrestin 2 and AT1R-Rluc were reported previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Hansen J.L. Haunso S. Brann M.R. Sheikh S.P. Weiner D.M. Mol. Pharmacol. 2004; 65: 770-777Crossref PubMed Scopus (22) Google Scholar). To perform this assay, we seeded 2.5 × 106 COS-7 cells in a p10 dish and transfected the cells with the amounts of receptor plasmid described under "Results" using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. After 1 day, the cells were seeded into 48-well plates coated with 0.25% poly-l-lysine (100,000 cells/well) and incubated in inositol-free Dulbecco's modified Eagle's medium (DMEM) supplemented with myo-[2-3H]inositol (1 μCi/ml) (Amersham Biosciences). Twenty hours after transfection, the cells were assayed as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Jensen A.A. Hansen J.L. Sheikh S.P. Brauner-Osborne H. Eur. J. Biochem. 2002; 269: 5076-5087Crossref PubMed Scopus (78) Google Scholar). This assay was performed as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Hansen J.L. Servant G. Baranski T.J. Fujita T. Iiri T. Sheikh S.P. Circ. Res. 2000; 87: 753-759Crossref PubMed Scopus (58) Google Scholar). Briefly 2.5 million COS-7 cells were seeded into a p10 dish and grown in 10% fetal calf serum, DMEM overnight. After 24 h, the cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol. The day after transfection they were seeded onto 6-well plates. Next 45 h after transfection, the cells were serum-starved for 3 h, incubated with agonist for 12 min at 37 °C, and then lysed. SDS-PAGE and immunoblotting were performed as described previously (30Aplin M. Christensen G.L. Schneider M. Heydorn A. Gammeltoft S. Kjolbye A.L. Sheikh S.P. Hansen J.L. Basic Clin. Pharmacol. Toxicol. 2007; 100: 296-301Crossref PubMed Scopus (67) Google Scholar, 31Aplin M. Christensen G.L. Schneider M. Heydorn A. Gammeltoft S. Kjolbye A.L. Sheikh S.P. Hansen J.L. Basic Clin. Pharmacol. Toxicol. 2007; 100: 289-295Crossref PubMed Scopus (58) Google Scholar), and the bands were visualized using the enhanced chemiluminescence system (BD Biosciences). To assess the relative efficacies of agonists, a densitometric gel quantification of the phosphorylated ERK (P-ERK) and total ERK band intensities was performed, and the ratios of P-ERK to total ERK were normalized with reference to the receptor maximum Ang II response (100%). This assay was performed as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 32Angers 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) except for minor changes. Briefly 2.5 million COS-7 cells were seeded into a p10 dish and grown in 10% fetal calf serum, DMEM overnight. After 24 h, the cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol. We used 20 μg of GFP2-β-arrestin 2 and 1 μg of rAT1R-Rluc or B2R-Luc DNA with either 3 μg of empty vector, B2R, or AT1R as described in the text. The cells were isolated and submitted to either basal or agonist treatment for 20 min at room temperature before measuring the BRET2 ratios as described in the text and previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). RSAT was performed as described previously (25Hansen J.L. Haunso S. Brann M.R. Sheikh S.P. Weiner D.M. Mol. Pharmacol. 2004; 65: 770-777Crossref PubMed Scopus (22) Google Scholar, 33Weiner D.M. Burstein E.S. Nash N. Croston G.E. Currier E.A. Vanover K.E. Harvey S.C. Donohue E. Hansen H.C. Andersson C.M. Spalding T.A. Gibson D.F. Krebs-Thomson K. Powell S.B. Geyer M.A. Hacksell U. Brann M.R. J. Pharmacol. Exp. Ther. 2001; 299: 268-276PubMed Google Scholar, 34Hansen J.L. Aplin M. Hansen J.T. Christensen G.L. Bonde M.M. Schneider M. Haunso S. Schiffer H.H. Burstein E.S. Weiner D.M. Sheikh S.P. Eur. J. Pharmacol. 2008; 590: 255-263Crossref PubMed Scopus (23) Google Scholar). Briefly NIH3T3 cells at 70-80% confluence were transfected with cDNA as follows (0-25 ng of receptor as described in the figures and 20 ng of β-galactosidase reporter/well of a 96-well plate) using the PolyFect Reagent (Qiagen, Valencia, CA) as described in the manufacturer's protocol. One day after transfection, ligands were added in DMEM supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 2% Cyto-SF3. After 6 days, the medium was removed by aspiration, cells were lysed, O-nitrophenyl-β-d-galactopyranoside was added, and the resulting absorbance was measured spectrophotometrically. All concentration-response curves were performed in duplicate. BRET2 Assay—Except for minor differences, the BRET2 assay was performed as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Jensen A.A. Hansen J.L. Sheikh S.P. Brauner-Osborne H. Eur. J. Biochem. 2002; 269: 5076-5087Crossref PubMed Scopus (78) Google Scholar). Briefly 0.5 million COS-7 cells/well were seeded into a 6-well plate and grown in 10% fetal calf serum, DMEM overnight. After 24 h the cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol (varying amounts of DNA were used to assure equal expression levels between the different combinations). After 2 days, the cells were washed twice with phosphate-buffered saline to remove the indicator dye before detachment in phosphate-buffered saline. The cells were then split into two portions. The first portion was used to examine the GFP2 levels by fluorescent measurements as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Jensen A.A. Hansen J.L. Sheikh S.P. Brauner-Osborne H. Eur. J. Biochem. 2002; 269: 5076-5087Crossref PubMed Scopus (78) Google Scholar), and next the Rluc expression was determined by measuring the Coelenterazine h-induced luminescence as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Jensen A.A. Hansen J.L. Sheikh S.P. Brauner-Osborne H. Eur. J. Biochem. 2002; 269: 5076-5087Crossref PubMed Scopus (78) Google Scholar). The second portion of the cells was submitted to DeepBlueC excitation, and the luminescence at the dual bands (515/30 and 410/80 nm) was measured on a Fusion reader (PerkinElmer Life Sciences). The BRET2 ratio was determined according to the principle described in (32Angers 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). The BRET2 ratio equals [(emission 515/30)-(emission 410/80) × Cf)]/(emission 410/80) where Cf denotes the cross-Rluc luminescence cross-talk ratio into the 515/30 filter defined as (emission 515/30)/(emission 410/80) when Rluc expressed alone is excited. Regulated Secretion/Aggregation Technology—This assay was developed by Rivera et al. (35Rivera V.M. Wang X. Wardwell S. Courage N.L. Volchuk A. Keenan T. Holt D.A. Gilman M. Orci L. Cerasoli Jr., F. Rothman J.E. Clackson T. Science. 2000; 287: 826-830Crossref PubMed Scopus (271) Google Scholar) and is available from ARIAD Pharmaceuticals. The assay was performed as described previously (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Briefly 2.5 × 106 COS-7 cells were seeded in a p10 dish and transfected with 10 μg of Fm4-AT1R (either wild type or mutant) cDNA alone or together with 2 μg of wild type B2R DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 24 h, the cells were split into 48-well plates coated with 0.25% poly-l-lysine (100,000 cells/well) and stimulated with or without AP21998 at 2 μm for 2 h prior to performing the PI hydrolysis assays as described above. The B2R Does Not Enhance AT1R-mediated G Protein Activation in Various Cellular Models—AbdAlla et al. (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar, 7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar, 16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar) have shown that simultaneous expression of functional B2Rs dramatically increases the ability of AT1Rs to activate Gαq signaling. These studies were performed very carefully in a variety of systems including HEK293 cells (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), primary cells such as vascular smooth muscle cells (16AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), neurons (5AbdAlla S. Abdel-Baset A. Lother H. J. Mol. Neurosci. 2005; 26: 185-192Crossref PubMed Scopus (76) Google Scholar), and platelets (7AbdAlla S. Lother H. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (400) Google Scholar). Given the natural interest in this interaction and its significant (patho)physiological relevance, we set out to examine this interaction in more detail. To be able to detect enhanced signaling, it is pivotal that the assay is within a dynamic range that allows for detection of further increases in signaling. We therefore transfected increasing amounts of AT1R cDNA into COS-7 cells and measured the Ang II-induced PI hydrolysis (Fig. 1A). We observed that an increasing amount of DNA would result in increased signaling up to the level of 10 μg of DNA/10-cm culture dish. Next we analyzed the effect of B2R expression on AT1R signaling. To do so, we compared the Ang II-induced signaling in cells transfected with either 2 μg of AT1R alone or with 0.5, 2, or 5 μg of B2R as indicated in Fig. 1B. Surprisingly we did not observe any changes in the AT1R efficacy or potency even though our system is indeed able to pick up changes as shown in Fig. 1A. AT1R expression did not change by co-transfection of the B2R and was consistently in the range of 20-22 fmol of receptor/100,000 cells as determined by radioligand binding as described previously (25Hansen J.L. Haunso S. Brann M.R. Sheikh S.P. Weiner D.M. Mol. Pharmacol. 2004; 65: 770-777Crossref PubMed Scopus (22) Google Scholar). The bradykinin receptor expression and function increased as a result of increasing amounts of B2R cDNA transfection. The expression levels of the B2R per 100,000 cells were 7 ± 3 fmol (for 0.5 μg of B2R), 23 ± 4 fmol (for 2 μg of B2R), and 50 ± 6 fmol (for 5 μg of B2R). Functional activity of the B2R was verified in the PI hydrolysis assay (Fig. 1C). In addition, we have tried several combinations of cDNAs in COS-7 cells, and we also have performed analysis in HEK-293 cells, but we have not observed any gain of function of the AT1R as a result of B2R co-transfection (data not shown). The B2R Does Not Enhance AT1R-mediated β-Arrestin 2 Recruitment or ERK Activation in Different Cellular Models—To test whether potentiation by the B2R of AT1R signaling could be detected with other functional assays, Ang II-mediated activation of the ERK cascade and β-arrestin 2 recruitment has been investigated. To analyze the Ang II-induced ERK activation, we first confirmed that the system was dynamic as described for the inositol 1,4,5-trisphosphate assay (Fig. 2A) and then examined the effects on Ang II-mediated AT1R stimulation as a consequence of B2R co-transfection, but we did not see any changes in this assay either (Fig. 2B). To analyze the β-arrestin 2 recruitment, COS-7 cells were transfected with AT1R-Rluc and β-arrestin 2-GFP2 either alone or in combination with the B2R, and the Ang II-induced BRET2 signal was measured. As depicted in Fig. 2C, we failed to detect any changes in the AT1R-mediated β-arrestin 2 recruitment. Failure to detect enhancement by B2R of AT1R-mediated β-arrestin 2 recruitment is not due to lack of activity of the B2R in this functional assay because the B2R-Rluc is capable of inducing a strong β-arrestin 2 translocation response (Fig. 2D). We have shown previously that it is possible to "transactivate" β-arrestin 2 recruitment to an AT1R mutant (K199A) that does not bind Ang II by co-expression of the AT1R-K199A-Rluc, β-arrestin 2-GFP2, and an untagged version of the wild type AT1R (3Hansen J.L. Theilade J. Haunsø S. Sheikh S.P. J. Biol. Chem. 2004; 279: 24108-24115Abstract Full Text Full
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