Ligand-dependent Differences in the Internalization of Endothelin A and Endothelin B Receptor Heterodimers
2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês
10.1074/jbc.m403601200
ISSN1083-351X
AutoresBernd Gregan, Jana Jürgensen, G Papsdorf, Jens Furkert, Michael Schaefer, Michael Beyermann, Walter Rosenthal, A. Oksche,
Tópico(s)Renin-Angiotensin System Studies
ResumoEndothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETA) and endothelin B (ETB) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ETB-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ETA receptor and CFP (ETAmyc·CFP) and a fusion protein consisting of an ETB receptor and YFP (ETB·YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ETB and myc-tagged ETA receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ETAmyc·CFP and ETB·YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ETB receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A·dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ETAmyc·CFP/ETBflag·YFP receptors revealed a slower sequestration of the ETBflag·YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ETBflag·YFP receptors alone. The data suggest that ETA and ETB receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ETB-selective stimulation. Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETA) and endothelin B (ETB) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ETB-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ETA receptor and CFP (ETAmyc·CFP) and a fusion protein consisting of an ETB receptor and YFP (ETB·YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ETB and myc-tagged ETA receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ETAmyc·CFP and ETB·YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ETB receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A·dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ETAmyc·CFP/ETBflag·YFP receptors revealed a slower sequestration of the ETBflag·YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ETBflag·YFP receptors alone. The data suggest that ETA and ETB receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ETB-selective stimulation. Endothelins (ET-1, ET-2, and ET-3) 1The abbreviations used are: ET-1, endothelin 1; ET-2, endothelial 2; ET-3, endothelin 3; CFP, cyan fluorescent protein; CXCR2, chemokine receptor; Cy3, cyanin 3; ELISA, enzyme-linked immunosorbent assay; ETA receptor, endothelin A receptor; ETB receptor, endothelin B receptor; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HEK293 cells, human embryonal kidney 293 cells; PBS, phosphate-buffered saline; YFP, yellow fluorescent protein; endoH, endoglycosidase H. are important regulators in the vascular system. They act via two receptors: the endothelin A (ETA) and endothelin B (ETB) receptors (1Arai H. Hori S. Aramori I. Ohkubo H. Nakanishi S. Nature. 1990; 348: 730-732Crossref PubMed Scopus (2513) Google Scholar, 2Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Yasuda D. Kimura K. Koyanagi Y. Aoki T. Kakuta T. Sakurai H. Tsuchida A. Yoshimatsu A. Ozawa H. et al.Nature. 1990; 348: 732-735Crossref PubMed Scopus (2366) Google Scholar). Although human ETA and ETB receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ETA receptor stimulates G proteins of the Gq/11 and G12/13 families, the ETB receptor activates mainly G proteins of the Gi and Gq/11 families (3Cramer H. Schmenger K. Heinrich K. Horstmeyer A. Boning H. Breit A. Piiper A. Lundstrom K. Müller-Esterl W. Schroeder C. Eur. J. Biochem. 2001; 268: 5449-5459Crossref PubMed Scopus (56) Google Scholar, 4Eguchi S. Hirata Y. Marumo F. J. Cardiovasc. 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Chem. 2000; 275: 6439-6446Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 9Bremnes T. Paasche J.D. Mehlum A. Sandberg C. Bremnes B. Attramadal H. J. Biol. Chem. 2000; 275: 17596-17604Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In contrast, the ETB receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9Bremnes T. Paasche J.D. Mehlum A. Sandberg C. Bremnes B. Attramadal H. J. Biol. Chem. 2000; 275: 17596-17604Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 10Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar). The ETA receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca2+ concentrations and activation of Rho proteins (11Seo B. Oemar B.S. Siebenmann R. von Segesser L. Luscher T.F. Circulation. 1994; 89: 1203-1208Crossref PubMed Scopus (482) Google Scholar, 12Seko T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (280) Google Scholar). The ETB receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13de Nucci G. Gryglewski R.J. Warner T.D. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2334-2338Crossref PubMed Scopus (239) Google Scholar). In addition, ETA and ETB receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14Angelova K. Puett D. Narayan P. Endocr. J. 1997; 7: 287-293Crossref Google Scholar, 15Kitsukawa Y. Gu Z.F. Hildebrand P. Jensen R.T. Am. J. Physiol. 1994; 266: G713-G721PubMed Google Scholar, 16Harada N. Himeno A. Shigematsu K. Sumikawa K. Niwa M. Cell. Mol. Neurobiol. 2002; 22: 207-226Crossref PubMed Scopus (66) Google Scholar). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ETA and ETB receptors (17Iwasa S. Fan J. Shimokama T. Nagata M. Watanabe T. Atherosclerosis. 1999; 146: 93-100Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Because atypical ligand binding was observed for cells co-expressing ETA and ETB receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15Kitsukawa Y. Gu Z.F. Hildebrand P. Jensen R.T. Am. J. Physiol. 1994; 266: G713-G721PubMed Google Scholar, 16Harada N. Himeno A. Shigematsu K. Sumikawa K. Niwa M. Cell. Mol. Neurobiol. 2002; 22: 207-226Crossref PubMed Scopus (66) Google Scholar, 18Ehrenreich H. Am. J. Physiol. 1999; 277: C614-C615Crossref PubMed Google Scholar). For example, in epithelial cells of the anterior pituitary, ETB receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125IET-1 binding only in the presence of the ETA receptor-selective antagonist BQ123 (16Harada N. Himeno A. Shigematsu K. Sumikawa K. Niwa M. Cell. Mol. Neurobiol. 2002; 22: 207-226Crossref PubMed Scopus (66) Google Scholar). In astrocytes, ETA and ETB receptors cooperatively control ET-1 clearance, because only the combination of ETA and ETB receptor-selective antagonists, but not their individual application increased ET-1 in the extracellular fluid (19Hasselblatt M. Kamrowski-Kruck H. Jensen N. Schilling L. Kratzin H. Siren A.L. Ehrenreich H. Brain Res. 1998; 785: 253-261Crossref PubMed Scopus (33) Google Scholar). Similarly, the gap junction permeability of astrocytes is cooperatively inhibited via ETA and ETB receptors: only the combined application of ETA and ETB receptor-selective antagonists block ET-1 action (20Blomstrand F. 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EMBO Rep. 2004; 5: 30-34Crossref PubMed Scopus (550) Google Scholar). The cooperativity of ET-1 action on both ETA and ETB receptors may be explained by the fact that ET-1 is a bivalent ligand, which binds to the ETA receptor via its cyclic N terminus and to the ETB receptor via its extended C terminus. It might even be possible that the bivalent ligand ET-1 could mediate the formation of ETA/ETB heterodimers by bridging ETA and ETB receptors via its N- and C-terminal parts, respectively (16Harada N. Himeno A. Shigematsu K. Sumikawa K. Niwa M. Cell. Mol. Neurobiol. 2002; 22: 207-226Crossref PubMed Scopus (66) Google Scholar, 26Sakamoto A. Yanagisawa M. Sawamura T. Enoki T. Ohtani T. Sakurai T. Nakao K. Toyo-oka T. Masaki T. J. Biol. Chem. 1993; 268: 8547-8553Abstract Full Text PDF PubMed Google Scholar). To address the questions of whether endothelin receptor subtypes form heterodimers and what functional consequences this might have, we performed co-immunoprecipitation experiments with HEK293 cells stably co-expressing ETAmyc·CFP and ETB·YFP receptors or transiently co-expressing ETAmyc and ETBflag receptors. In addition, heterodimerization and its regulation by mixed and selective agonists and antagonists was investigated in fluorescence resonance energy transfer (FRET) experiments with living HEK293 cells transiently co-expressing ETAmyc·CFP and ETB·YFP receptors. Moreover, we studied ligand-induced receptor sequestration by ELISA and laser scanning microscopy. Materials—Bacitracin and aprotinin were from Merck (Darmstadt, Germany); G418, monensin, and nigericin from Calbiochem-Novabiochem (Bad Soden, Germany); Zeocin and LipofectAMINE from Invitrogen; FuGENE 6 from Roche Diagnostics; and fetal calf serum from Biochrom (Berlin, Germany). 125I-ET-1, 125I-PD151242, and 125I-ET-3 (both 2000 Ci/mmol) were from Amersham Biosciences. BQ123 and BQ788 were from Alexis (Läufelfingen, Schweiz); ET-3 and BQ788 were from Calbiochem-Novabiochem. All other reagents were from Sigma. Monoclonal c-myc (9E10) antibody was from Roche Diagnostics; monoclonal FLAG (M2) antibody from Sigma; and monoclonal GFP antibody (JL-8) from BD Biosciences. Epidermal growth factor receptor cDNA (HER1) was kindly provided by Dr. A. Sorkin (University of Colorado Health Science Center, Denver, CO). K44A·dynamin I was kindly provided by Dr. S. L. Schmid (The Scripps Research Institute, La Jolla, CA). Peptide Synthesis and Fluorescence Labeling—ET-1 and BQ3020 (N-acetyl-[Ala11,15]6-21-endothelin-1) were synthesized and purified essentially as described (10Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar, 27Oksche A. Boese G. Horstmeyer A. Papsdorf G. Furkert J. Beyermann M. Bienert M. Rosenthal W. J. Cardiovasc. Pharmacol. 2000; 36: S44-S47Crossref PubMed Google Scholar). The masses of purified ET-1 and BQ3020 were verified by electrospray mass spectometry. Fluorescence labeling of ET-1 and BQ3020 was carried out by selective modification of the ϵ-amino groups of Lys-9 of ET-1 and Lys-4 of BQ3020 using the Cy3 monoreactive succinimidyl ester (Amersham Biosciences) in 0.1 m NaHCO3 at pH 9.3 followed by preparative high performance liquid chromatography purification. Cell Culture—HEK293 cells (obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in a humidified atmosphere of 95% air, 5% CO2. For laser scanning microscopy, cells were grown on glass coverslips for 48 h. For biochemical analyses, cells were grown for 48-72 h until 80% confluence. Transient and Stable Transfection of HEK293 Cells—The procedures for the stable transfection of HEK293 cells with LipofectAMINE and isolation of cell clones expressing ETB·GFP, ETBflag·GFP, ETAmyc·CFP/ETB·YFP, and ETAmyc·CFP/ETBflag·YFP were essentially performed as described (10Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar), with the exception that for the selection and maintenance of ETAmyc·CFP and ETAmyc·CFP/ETBflag·YFP cell clones, Zeocin and Zeocin/G418 were used, respectively. For transient transfection, FuGENE 6 was used according to the instructions of the manufacturer (4 μl of FuGENE 6/μg of DNA). Generation and Affinity Purification of Polyclonal Antibodies—A polyclonal NT-ETB serum was raised against a conjugate consisting of a synthetic peptide corresponding to amino acids 19-37 in the N terminus of the ETB receptor (Swiss-Prot accession number P24530; CGLSRIWGEERGFPPDRATP) and the carrier protein keyhole limpet hemocyanin (Calbiochem-Novabiochem). The NT-ETB antibody was affinity purified with the peptide coupled to protein-Sepharose 6B (Amersham Biosciences) according to the manufacturers protocol. The antibody fraction was dialyzed against sodium phosphate buffer (20 mm sodium phosphate buffer, 150 mm NaCl, pH 7.5) and stored in aliquots at -20 °C. 125I-ET-1 Displacement Binding Experiments—Radioligand experiments were performed as described (10Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar). In brief, membranes (0.1-0.5 μg) were incubated in 200 μl of Tris/bacitracin/aprotinin/MgCl2/EGTA buffer containing 50 pm125I-ET-1 without or with increasing concentrations of unlabeled ligand (1 × 10-13 to 1 × 10-4m) for 3 h at 25 °C in a shaking water bath. The samples were then transferred to GF/C filters (Whatman) pretreated with 0.1% (w/v) polyethylenimine. The filters were washed twice with PBS using a Brandel cell harvester and transferred into 5-ml vials. Radioactivity was determined in a γ-counter. Data were analyzed with RadLig Software 4.0 (Cambridge, UK), and graphs were generated with Prism Software 2.01 (GraphPad, San Diego, CA). Immunoblots—HEK293 cell clones stably expressing the fusion proteins were washed twice with PBS (137 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, 8.0 mm Na2HPO4, pH 7.4) and harvested with lysis buffer (20 mm Tris-HCl, 1% (w/v) Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1.5 mm MgCl2, 150 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 2 mg/ml soybean trypsin inhibitor, 1.43 mg/ml aprotinin, 0.5 mm benzamidine, pH 7.5). Lysates (30 μg of protein per lane) were separated by SDS-PAGE (10% gels) and transferred to nitrocellulose filters (Schleicher and Schuell). Filters were probed with polyclonal NT-ETB antibody (diluted 1:5000), monoclonal c-myc, or monoclonal GFP antibodies (diluted 1:2000). Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG or with horseradish peroxidase-conjugated donkey anti-mouse IgG (both diluted 1:2000, Jackson ImmunoResearch Laboratories) using Lumi-Light Western blotting substrate (Roche Diagnostics). Immunoprecipitation—HEK293 cell clones were grown in 75-cm2 cell culture flasks for 48 h to near confluence. Cells were washed twice with PBS, and finally lysed with lysis buffer (1.2 ml). The cells were then harvested with a rubber policeman and homogenized five times by passage through a 27-gauge needle. The homogenate was centrifuged (800 × g) for 10 min at 4 °C, and the supernatant was transferred to a new reaction tube and re-centrifuged (26,000 × g) for 30 min. The new supernatant was then mixed with the NT-ETB antibody (diluted 1:2000) and protein A-Sepharose (3.5 mg) in a final volume of 1.0 ml and incubated for 12 h in a shaker at 4 °C. After 3 washes with lysis buffer, the pellet was resuspended in Laemmli buffer (10% β-mercaptoethanol (w/v), 4% SDS (w/v), 2% bromphenol blue (w/v), 20% glycerol (w/v), 250 mm Tris, pH 6.8) and analyzed in immunoblot experiments. FRET—Cells were grown on glass coverslips for 48 h. Coverslips were then mounted in a custom-made chamber and covered with incubation buffer (138 mm NaCl, 6 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 5.5 mm glucose, 2 mg/ml bovine serum albumin, and 10 mm Hepes, pH 7.5). FRET analysis was performed as described (28Brock C. Schaefer M. Reusch H.P. Czupalla C. Michalke M. Spicher K. Schultz G. Nurnberg B. J. Cell Biol. 2003; 160: 89-99Crossref PubMed Scopus (213) Google Scholar), using an inverted microscope Axiovert 100 equipped with a Plan-Apochromat ×63/1.4 objective (Carl Zeiss, Göttingen, Germany). In brief, CFP and YFP were alternately excited at 410 and 515 nm with a monochromator (Polychrome II; TILL Photonics, Gräfelfing, Germany) in combination with a dual reflectivity dichroic mirror ( 570 nm for Cy3. Generation of HEK293 Cell Clones Stably Expressing ETAand ETBReceptors—HEK293 cells were stably transfected with plasmids encoding fusion proteins comprising an N-terminal c-myc epitope-tagged ETA receptor, C-terminal fused to CFP (ETAmyc·CFP) or an ETB receptor, C-terminal fused to GFP or YFP (ETB·GFP; ETB·YFP), or an ETB receptor, N-terminal FLAG epitope-tagged and C-terminal fused to YFP (ETBflag·YFP). Several laboratories including ours have previously shown that the GFP fused to the C terminus of ETA and ETB receptors does not alter their functional properties (9Bremnes T. Paasche J.D. Mehlum A. Sandberg C. Bremnes B. Attramadal H. J. Biol. Chem. 2000; 275: 17596-17604Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 10Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar, 29Grantcharova E. Furkert J. Reusch H.P. Krell H.W. Papsdorf G. Beyermann M. Schülein R. Rosenthal W. Oksche A. J. Biol. Chem. 2002; 277: 43933-43941Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Similarly, the myc and FLAG epitopes, inserted after the signal peptide of the ETA and ETB receptors, respectively, did not influence ligand binding. 2J. Jürgensen, M. Jandke, G. Papsdorf, W. Rosenthal, and A. Oksche, manuscript in preparation. In saturation binding experiments with 125I-ET-1 as radioligand at least two independently obtained cell clones were analyzed, which express ETAmyc·CFP, ETB·GFP, ETB·YFP, and ETBflag·YFP receptors individually or in combination (ETAmyc·CFP/ETB·YFP and ETAmyc·CFP/ETBflag·YFP). We obtained very similar KD values for the receptor fusion proteins in the different cell clones (Table I). 125I-PD151242 (ETA receptor-selective) and 125I-ET-3 (ETB receptor-selective) were used as radioligands in saturation binding experiments to determine the contribution of the ETAmyc·CFP and ETBflag·YFP receptors to 125I-ET1 binding in ETAmyc·CFP/ETB·YFP cell clones. We found that the ratio of ETAmyc·CFP and ETBflag·YFP receptors in these clones varies between 70:30 and 60:40 (Table I). Laser scanning microscopy of the cell clones co-expressing ETAmyc·CFP and ETB·YFP receptors revealed a uniform and predominant expression of both receptor subtypes in the plasma membrane (Fig. 1).Table ISynopsis of Kd and Bmax values of HEK293 cell clones stably expressing ETAmyc·CFP, ETB·YFP, or ETAmyc·CFP/ETB·YFP receptorsKdBmax125I-ET-1125I-ET-1125I-ET-3125I-PD-151242pMpmol/mg proteinETAmyc·CFP26.7 ± 45.6 ± 0.4NDaND, not determined.ND31.0 ± 3.99.0 ± 1.0NDNDETB·YFP10.5 ± 0.96.2 ± 1.6NDND12.3 ± 2.07.2 ± 1.1NDNDETAmyc·CFP/ETB·YFP28.0 ± 19.513.7 ± 0.74.2 ± 0.310 ± 2.429.7 ± 7.815.2 ± 6.46.5 ± 0.611 ± 2.2a ND, not determined. Open table in a new tab HEK293 Cell Clones Express Mature, Complex-glycosylated Endothelin Receptor Fusion Proteins—Prior to immunoprecipitation experiments, we verified the integrity of the receptor fusion proteins expressed in HEK293 cell clones by analyzing cell lysates in immunoblot experiments. The ETB·GFP receptor was detected with a polyclonal antibody directed against the N terminus of the ETB receptor (NT-ETB, see "Experimental Procedures"). In lysates from ETB·GFP or ETAmyc·CFP/ETB·YFP cell clones, we detected a prominent band at 75 kDa corresponding to the ETB·GFP or ETB·YFP receptors (Fig. 2, A and B, arrow). When we preincubated the NT-ETB antibody with the peptide used for immunization, the band was not observed (Fig. 2A). Thus, the NT-ETB antibody specifically detects the ETB·GFP receptor. We further investigated the glycosylation pattern of the ETB·GFP receptor by treatment of the lysates with endoH and PNGaseF. EndoH, which removes high man-nose glycans from immature, core-glycosylated proteins did not increase the mobility of the 75-kDa band (Fig. 2B). Treatment with PNGaseF, which removes N-linked glycans from both mature, complex-glycosylated and immature, core-glycosylated proteins, increased the mobility of the immunoreactive band, which now migrated at about 67 kDa (Fig. 2B, asterisk). Thus, the ETB·GFP receptor is expressed as a mature protein in HEK293 cell clones. For the detection of the ETAmyc·CFP receptor in immunoblots with lysates from ETAmyc·CFP/ETB·YFP cell clones, we used a monoclonal c-myc antibody. Here, we observed a prominent band at about 87 kDa (Fig. 2C, double arrow). Incubation with endoH had no effect on the protein mobility of the ETAmyc·CFP receptor, whereas treatment with PNGaseF increased the mobility, resulting in a protein migrating at about 75 kDa (Fig. 2C, arrowhead). The results demonstrate that ET receptor fusion proteins are mainly expressed in HEK293 cell clones as complex-glycosylated, mature proteins. This is in good agreement with the microscopic analysis (Fig. 1), according to which both ET receptor fusion proteins are detected at the plasma membrane. Co-immunoprecipitation Reveals ETA/ETBHeterodimers—First we tested whether NT-ETB and c-myc antibodies were suitable for immunoprecipitation experiments. Whereas immunoprecipitation of the ETB·GFP receptor was possible with the polyclonal NT-ETB antibody (see below), the monoclonal c-myc antibody was unable to precipitate the ETAmyc·CFP receptor (data not shown). When the ETB receptor was precipitated with the NT-ETB antibody from detergent extracts of cell clones expressing only the ETB·GFP receptor, in immunoblots with a GFP antibody a 75-kDa band was detected, corresponding to the ETB·GFP receptor (Fig. 3A, lane 3). Immunoprecipitation of detergent extracts from ETAmyc·CFP/ETB·YFP cell clones with the NT-ETB antibody and subsequent immunoblots with a monoclonal anti-GFP antibody revealed two bands: 75- and 87-kDa bands, corresponding to ETB·YFP and ETAmyc·CFP receptors, respectively (Fig. 3A, lane 4). The presence of the ETAmyc·CFP receptor in the immunoprecipitate was verified by using a c-myc antibody, which detected a single band at 87 kDa, representing the ETAmyc·CFP receptor (Fig. 3A, lane 5). The 75- and 87-kDa bands were also observed using NT-ETB (Fig. 3A, lane 1) and c-myc (Fig. 3A, lane 2) antibodies in immunoblots from lysates of ETAmyc·CFP/ETB·YFP cell clones. Thus, upon immunoprecipitation of the ETB·YFP receptor from detergent extracts of cell clones expressing ETAmyc·CFP and ETB·YFP receptors, we observed co-precipitation of the ETAmyc·CFP receptor. To confirm that heterodimerization is not because of secondary aggregation of fusion proteins after cell disruption or the lack of specificity of the affinity-purified antibody, we performed immunoprecipitation experiments with a mixture of detergent extracts derived from cell clones individually expressin
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