The Mode of Bone Morphogenetic Protein (BMP) Receptor Oligomerization Determines Different BMP-2 Signaling Pathways
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m102750200
ISSN1083-351X
AutoresAnja Nohe, Sylke Haßel, Marcelo Ehrlich, Florian Neubauer, Walter Sebald, Yoav I. Henis, Petra Knaus,
Tópico(s)Connective Tissue Growth Factor Research
ResumoBone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-inducedversus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK. Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-inducedversus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK. Bone morphogenetic proteins (BMPs) 1The abbreviations used are:BMPbone morphogenetic proteinBRIBMP-receptor type IBRIIBMP-receptor type IIBRII-SFBMP-receptor type II-ShortFormFITCfluorescein isothiocyanateALPalkaline phosphataseTGF-βtransforming growth factor-βJNKc-Jun N-terminal kinaseMAPKmitogen-activated protein kinaseHAhemagglutininTfRtransferrin receptorwtwild typeDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideBSAbovine serum albuminTBSTris-buffered salineERKextracellular signal-regulated kinase are members of the transforming growth factor-β (TGF-β) superfamily that play important roles in most morphogenetic processes during development (1Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1739) Google Scholar). BMPs are able to induce the formation of bone at nonbony sites in the adult animal by influencing the differentiation of mesenchymal progenitor cells along the cartilage lineage pathway. BMPs act on osteoblasts as well as chondrocytes but also on many other cell types such as neuronal cells (2Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (462) Google Scholar, 3Reddi A.H. Nat. Biotechnol. 1998; 16: 247-252Crossref PubMed Scopus (715) Google Scholar). bone morphogenetic protein BMP-receptor type I BMP-receptor type II BMP-receptor type II-ShortForm fluorescein isothiocyanate alkaline phosphatase transforming growth factor-β c-Jun N-terminal kinase mitogen-activated protein kinase hemagglutinin transferrin receptor wild type Dulbecco's modified Eagle's medium fetal calf serum phosphate-buffered saline phenylmethylsulfonyl fluoride bovine serum albumin Tris-buffered saline extracellular signal-regulated kinase Signaling by BMP-2 involves two types of transmembrane serine/threonine kinases, termed type I (BRI) and type II (BRII) receptors (4Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 5Kawabata M. Chytil A. Moses H.L. J. Biol. Chem. 1995; 270: 5625-5630Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 6Liu F. Ventura F. Doody J. Massague J. Mol. Cell. Biol. 1995; 15: 3479-3486Crossref PubMed Scopus (524) Google Scholar, 7Nohno T. Ishikawa T. Saito T. Hosokawa K. Noji S. Wolsing D.H. Rosenbaum J.S. J. Biol. Chem. 1995; 270: 22522-22526Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (479) Google Scholar). Receptors of both types are needed to form a functional complex to initiate further signaling events. Activated BMP type I receptors phosphorylate Smad1, Smad5, and Smad8 (R-Smads), which then assemble into heteromeric complexes with Smad4 (Co-Smad) and translocate into the nucleus to regulate transcription of target genes (9Massague J. Chen Y.G. Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 10Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar). In addition, BMP receptors initiate other signaling pathways, distinct from the Smad pathway, resulting in the activation of p38 MAPK and JNK (11Kimura N. Matsuo R. Shibuya H. Nakashima K. Taga T. J. Biol. Chem. 2000; 275: 17647-17652Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Iwasaki S. Iguchi M. Watanabe K. Hoshino R. Tsujimoto M. Kohno M. J. Biol. Chem. 1999; 274: 26503-26510Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 13Piek E. Heldin C.H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (754) Google Scholar). We have shown recently that the oligomerization pattern of the BMP receptors is very different from that of the related TGF-β receptors and is more flexible and susceptible to modulation by ligand (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). Multiple BMP receptor oligomers are present at the cell surface prior to ligand binding, with heterocomplexes of BRII with BRI-a and BRI-b being the most prominent (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). This subpopulation of preformed heteromeric receptor complexes is so far unique for the BMP receptors within the TGF-β receptor superfamily; it implies that, if such complexes transduce specific ligand-induced signals, they should be mediated by conformational changes within the subunits in the complex upon ligand binding. BMP-2 binding mediates a significant elevation in the amount of the heteromeric BMP receptor complexes, as well as in that of homomeric BRI complexes (singly expressed BRII cannot bind ligand). Thus, there are two modes of BMP receptor hetero-oligomerization: one, which occurs prior to ligand binding, and the other, which is ligand-mediated. To study whether these different complexes also activate different signaling pathways, we generated BRII receptor mutants that were defective in their ability to associate with BRI subtypes into preformed complexes. These mutants enabled us to dissect two different pathways, which are initiated by two different modes of BMP receptor oligomerization. Preformed complexes trigger the Smad pathway, whereas BMP-2-induced signaling complexes trigger an independent pathway resulting in the induction of alkaline phosphatase (ALP) activity. The latter pathway is mediated through activation of p38 MAPK. Recombinant human BMP-2 was prepared as described previously (15Ruppert R. Hoffmann E. Sebald W. Eur. J. Biochem. 1996; 237: 295-302Crossref PubMed Scopus (477) Google Scholar). 9E10 (α-myc, directed against the myc tag (16Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2356) Google Scholar)) mouse ascites was purchased from Harvard Monoclonals. HA.11 rabbit serum directed against the influenza hemagglutinin (HA) tag (17Wilson I.A. Niman H.L. Houghten R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (748) Google Scholar) and 12CA5 mouse ascites against this tag (α-HA) were from BAbCO. The IgG fractions were purified from mouse ascites using standard protocols (18Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1988Google Scholar). Anti-phospho-Smad1/5/8 antibody recognizing the phosphorylated form of Smad1, -5, and -8 was a gift from P. ten Dijke. Polyclonal antisera (rabbit) were raised against specific peptides from the BMP receptors (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). The anti-human transferrin receptor antibody (mouse monoclonal antibody B3/25) was purchased from Roche Molecular Biochemicals. Peroxidase-goat anti-mouse IgG or peroxidase-goat anti-rabbit IgG were obtained from Dianova, protein A-Sepharose and protein G-Sepharose CL-4B from Sigma Chemical Co., and disuccinimidyl suberate from Pierce. Cy3-GαR and fluorescein isothiocyanate (FITC)-GαM IgG were from Jackson ImmunoResearch. The anti-phospho p38 antiserum was from Promega. The cell lines COS7 (CRL 1651) and C2C12 (CRL 1772) were purchased from American Type Culture Collection. MC3T3 cells were obtained from P. ten Dijke. The construct pCDTR1, containing as insert the entire human transferrin receptor (TfR) cDNA, was a gift from F. H. Ruddle (Yale University, New Haven, CT). The BMP-receptors were epitope-tagged as described by us earlier (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). To generate truncated versions of BRII we used recombinant PCR mutagenesis by introducing stop codons at the indicated position (TC3 at 1500, BRII-SF at 1587, TC4 at 1911, TC5 at 2133, and TC6 at 2238; nucleotide numbers are given according to a previous study (5Kawabata M. Chytil A. Moses H.L. J. Biol. Chem. 1995; 270: 5625-5630Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar)) and an XbaI site. All mutagenesis oligonucleotides, therefore, had the same 5′ sequence: 5′-GCA ACG TCT AGA TCA TGA TCA-3′. The obtained fragments were digested with DraI/XbaI and subcloned into the vector with either the HA-epitope-tagged or myc-epitope-tagged BRII (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar).DraI cuts at position 1004 according to the sequence used in a previous study (5Kawabata M. Chytil A. Moses H.L. J. Biol. Chem. 1995; 270: 5625-5630Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The cDNAs encoding the truncations TC7 and TC8 were generated in the same way except that the fragments were digested with BglII instead of DraI (stop codons: TC7 at 2946, TC8 at 3064). TC1 was cloned by digestion of the PCR fragment of TC3 with HindIII/XbaI. The fragment was subcloned in pcDNA1 (Invitrogen) resulting in 560 amino acids from BRII and five additional amino acids from pcDNA1 at the C terminus (EGPIL). All constructs were verified by DNA sequencing. The generated BRII truncation mutants resulted in proteins with the following length: TC1 (207 amino acids), TC3 (500 amino acids), BRII-SF (529 amino acids), TC4 (637 amino acids), TC5 (711 amino acids), TC6 (746 amino acids), TC7 (982 amino acids), and TC8 (1021 amino acids). All the constructs contained either the HA or myc tag at the N terminus, because they were generated from the tagged BRII forms described by us earlier (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). The previously described myc- or HA-tagged versions of the full-length BMP receptors (including BRI-a and BRI-b) were also employed in the studies. The expression of the TC mutants of BRII was tested in transfected COS7 cells (see Figs. Figure 2, Figure 3, Figure 4, Figure 5) as well as in C2C12 and MC3T3 cells.Figure 3TC3 forms preformed complexes with BRI-a but not with BRI-b. In vivo labeling with [35S]cysteine and [35S]methionine was performed in COS7 cells co-transfected with HA-tagged TC3, BRII-SF, or TC4 plus tagged BRI-b or BRI-a. After cell lysis immunoprecipitation was performed using α-HA or α-myc antibodies as indicated above each lane. Lanes 1, 2, 4, and5 show preformed receptor complexes of HA-TC4 and HA-BRII-SF with BRI-a or BRI-b, lane 6 shows complex formation between BRI-a and TC3. As shown in lane 3 there is no detectable preformed complex of BRI-b and TC3 in the absence of ligand. BRI-b or BRI-a appear as double bands at around 55 kDa. Lanes 7,8, and 9 are controls.View Large Image Figure ViewerDownload (PPT)Figure 4Immunofluorescence co-patching to explore heteromeric complexes of BRII, TC3, and TC1 with BRI-a or BRI-b at the cell surface. COS7 cells were co-transfected transiently with pairs of receptors carrying different epitope tags as indicated below for each panel. Live cells were labeled in the cold consecutively by a series of antibodies to mediate patching and fluorescent labeling, as detailed under “Experimental Procedures.” This labeling protocol results in HA-tagged receptors labeled by Cy3 (red), whereas myc-tagged receptors or the TfR (control) labeled by FITC (green); mutual patches containing bothred- and green-labeled receptors appearyellow when the two fluorescent charge-coupled device images are overlapped. Bar, 20 μm. A, HA-BRII (red) and myc-BRI-a (green) show a significant level of co-patching above the control (14% over the background level;p < 0.001 according to Student’s t test).B, HA-BRI-b (red) and myc-BRII (green) exhibit a significant co-patching level (23% above the control;p < 0.001). C, HA-BRI-a (red) and myc-TC3 (green) exhibit a co-patching level similar to that of wt BRII with BRI-a, which is significantly higher than the control (17% above the background level; p < 0.001).D, HA-BRI-b (red) and myc-TC3 (green) co-patching is only slightly above the background level (7%;p < 0.001). E, HA-BRI-a (red) and myc-TC1 (green) co-patching is only slightly above the background co-patching level (7%; p < 0.001).F, HA-BRI-b (red) and myc-TC1 (green) do not co-patch significantly above the control (p > 0.1). G, HA-BRI-a (red) and TfR (green) show a low degree of co-patching. This level (18%; see panel H) is the background percentage of co-patching when unrelated receptors are employed. Similar levels were obtained for co-patching of the TfR with other receptors (BRII or the type II TGF-β receptor). H, quantification of the co-patching data. The pairs of co-transfected receptors are designated by theletters corresponding to the representative panels (A–G). Superimposed red and greenimages were analyzed by counting the numbers of green,red, and yellow patches (counting at least 100 patches per cell on 10–15 cells in each case). The % co-patching (percentage of a given tagged receptor in mutual patches with the other receptor) is given by: 100 × [yellow/(yellow + red)] for the red-labeled receptors and 100 × [yellow/(yellow + green)] for the green-labeled receptors (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). Because these values were very close, a single value is depicted for each pair. The values shown are mean ± S.E. of the % co-patching of the different receptor pairs.View Large Image Figure ViewerDownload (PPT)Figure 5Detection of cell surface BMP-receptor hetero-complex formation by ligand binding and cross-linking. COS7 cells were co-transfected with pairs of epitope-tagged receptors. After binding and cross-linking of 125I-BMP-2 (5 nm), immunoprecipitation was performed with 12CA5 (α-HA). Immunoprecipitates derived from the same amount of cell lysates (one 10-cm dish) were loaded on each lane of the gel and analyzed by SDS-PAGE and autoradiography. A, lanes 1–8depict complexes of HA-BRI-a (black arrow) and HA-tagged truncation mutants of BRII (triangle). Binding to all receptors can be detected. B, lanes 1–8 depict complexes of HA-BRI-b (black arrow) and HA-tagged BRII truncation mutants or BRII (triangle). HA-BRI-b complexes with HA-BRII are shown in lane 9. To confirm binding of TC3 in the presence of BRI-b to BMP-2, co-transfection of TC3 with untagged BRI-b and immunoprecipitation of TC3 were performed. Lane 10as well as lane 2 depict complexes of TC3 and BRI-b. The BRII truncation mutants and BRII alone show no binding to 5 nm125I-BMP-2 (not shown).View Large Image Figure ViewerDownload (PPT) COS7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and transfected by the DEAE-dextran method (19Aruffo A. Seed B. EMBO J. 1987; 6: 3313-3316Crossref PubMed Scopus (121) Google Scholar). For immunoprecipitation or ligand cross-linking, transfections were performed on cells grown in 10-cm plates, using 7–10 μg of DNA per construct. For immunofluorescence co-patching experiments, cells were grown on glass coverslips in 30-mm dishes and transfected with 0.25–0.5 μg of DNA per construct. All experiments were performed 44–48 h after transfection. Transfected COS7 or untransfected MC3T3 or C2C12 cells were washed and incubated with ligand (BMP-2) in KRH buffer (50 mm Hepes, pH 7.5, 128 mm NaCl, 1.3 mm CaCl2, 5 mm MgSO4, 5 mm KCl) at 4 °C as described under “Ligand Binding and Cross-linking.” Control dishes were incubated with buffer alone. They were washed twice in ice-cold PBS and solubilized in lysis buffer (PBS, pH 7.4, containing 0.5% Triton X-100, 1 mm EDTA, and 10 μg/ml each of leupeptin, aprotinin, soybean trypsin inhibitor, benzamidine-HCl, pepstatin, and antipain, 1 mm PMSF) at 4 °C for 40 min. Epitope-tagged receptors were immunoprecipitated from extracts of transfected COS7 cells by 12CA5 monoclonal antibodies (α-HA; 20 μg/ml, 4 °C, 2 h) followed by incubation with protein A-Sepharose (30 μl of 1:1 suspension in PBS, 1 h, 4 °C) or by 9E10 (α-myc; 20 μg/ml) antibodies followed by protein G-Sepharose. Endogenous receptors from MC3T3 or C2C12 cells were precipitated using specific rabbit anti-peptide antisera (FB15 for BRI-a, FB63 for BRI-b, and FB60 for BRII; 1:500 dilution, 4 °C, 2 h) followed by protein A-Sepharose. As control the corresponding preimmune sera were used. The beads were washed three times with PBS. For immunoprecipitations, the bound protein was eluted by heating the beads in SDS-PAGE sample buffer containing mercaptoethanol (3 min, 95 °C) and subjected to 10–12% SDS-PAGE. Western blotting was done according to standard protocols. After electrotransfer and blocking (10 mm Tris, pH 7.9, 150 mm NaCl, 0.1% Tween 20, 3% BSA, room temperature, 1 h), the blot was incubated with the monoclonal antibodies 9E10 (20 μg/ml), 12CA5 (10 μg/ml) for 12 h at 4 °C. Detection of adsorbed antibodies was performed by ECL (Amersham Biosciences, Inc.), employing Peroxidase-GαM diluted 1:10,000. For detection of Smads the membrane was blocked with 10 mm Tris, pH 7.9, 150 mm NaCl, 0.5% Tween 20, 3% dry milk at room temperature for 1 h, followed by incubation with α-Phospho Smad1/5/8 (1:1000 dilution) or α-Smad1/5/8 (1:1000 dilution) overnight at 4 °C (20Ishisaki A. Yamato K. Hashimoto S. Nakao A. Tamaki K. Nonaka K. ten Dijke P. Sugino H. Nishihara T. J. Biol. Chem. 1999; 274: 13637-13642Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Detection of adsorbed antibodies was performed by ECL (Amersham Biosciences, Inc.), employing Peroxidase-GαR diluted 1:25,000 in blocking buffer. The antibody for the phosphorylated form of p38 (Promega) was used according to manufacturer's protocol. Blocking was done in TBS/1% BSA, overnight at 4 °C, followed by incubation with the antibody (diluted 1:2000 in TBS/0.05% Tween 20/0.1% BSA) and detection with the secondary antibody Peroxidase-GαR diluted 1:10,000 in TBST/0.1% BSA. BMP-2 was labeled by125I using the chloramine T method as described previously (21Cheifetz S. Bassols A. Stanley K. Ohta M. Greenberger J. Massague J. J. Biol. Chem. 1988; 263: 10783-10789Abstract Full Text PDF PubMed Google Scholar). Iodination efficiency was 99%. Before binding and cross-linking, C2C12 and MC3T3 cells were starved for 12 h in DMEM supplemented with 0.2% FCS. Confluent 10-cm plates of transfected COS cells or 70% confluent 14-cm plates of C2C12 or MC3T3 cells were incubated for 2–6 h at 4 °C with 5 nm125I-BMP-2 in KRH buffer containing 0.5% fatty acid-free BSA. For MC3T3 and C2C12 cells, 0.01% Tween 20 was added. Cross-linking was performed with disuccinimidyl suberate as described previously for TGF-β (22Moustakas A. Lin H.Y. Henis Y.I. Plamondon J. O'Connor M.M. Lodish H.F. J. Biol. Chem. 1993; 268: 22215-22218Abstract Full Text PDF PubMed Google Scholar). Cross-linking was stopped by adding sucrose to a final concentration of 7% in KRH. Cell lysis and immunoprecipitation was performed as described above. Metabolic labeling was performed as described previously (23Chen Y.G. Liu F. Massague J. EMBO J. 1997; 16: 3866-3876Crossref PubMed Scopus (293) Google Scholar). 48 h after transient transfection COS7 cells were rinsed twice with and then starved in serum-free DMEM minus cysteine and methionine for 120 min at 37 °C. The medium was then replaced with medium supplemented with 0.2 mm oxidized glutathione (Roche Molecular Biochemicals) and a mixture (1 mCi/ml) of [35S]methionine and [35S]cysteine (DuPont) and incubated for 3 h at 37 °C. Cells were rinsed twice with PBS and solubilized in lysis buffer (0.5% Triton X-100, 1 mm EDTA in PBS) supplemented with protease inhibitors (10 μg/ml each of leupeptin, aprotinin, soybean trypsin inhibitor, benzamidine-HCl, pepstatin and antipain, 1 mm PMSF) for 1 h at 4 °C. Lysates were immunoprecipitated as indicated and proteins analyzed by SDS-PAGE. To measure oligomerization of BMP receptor mutants directly at the cell surface, we employed antibody-mediated immunofluorescence co-patching of epitope-tagged receptors, described by us recently (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar, 24Henis Y.I. Moustakas A. Lin H.Y. Lodish H.F. J. Cell Biol. 1994; 126: 139-154Crossref PubMed Scopus (179) Google Scholar, 25Gilboa L. Wells R.G. Lodish H.F. Henis Y.I. J. Cell Biol. 1998; 140: 767-777Crossref PubMed Scopus (124) Google Scholar). COS7 cells grown on glass coverslips were co-transfected transiently using DEAE-dextran by pairs of receptors carrying different epitope tags (for example, myc-BRI-a together with HA-BRII, HA-BRI-b together with myc-TC1, or an epitope-tagged BMP receptor together with the unrelated TfR as a control) in mammalian expression vectors (pcDNA1, except for the TfR, which was in pCD). After 44–48 h, the cells were blocked with normal goat IgG (200 μg/ml, 45 min, 4 °C, in Hanks' balanced salt solution containing 20 mm Hepes, pH 7.4, and 1% BSA) and labeled in the cold (to avoid internalization and enable exclusive cell surface labeling) in the same buffer with primary anti-tag IgG (20 μg/ml, 45 min): rabbit HA.11 against the HA tag (BAbCO) together with either mouse α-myc (BAbCO) or mouse B3/25 against the TfR (Roche Molecular Biochemicals). This was followed by labeling/patching with secondary IgG-Cy3-GαR and FITC-GαM (20 μg/ml, 45 min, 4 °C). After washing, the cells were fixed in methanol (5 min, −20 °C) followed by acetone (2 min, −20 °C) and mounted in SlowFade (Molecular Probes). Fluorescence digital images were recorded using a charge-coupled device camera as described (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). The FITC and Cy3 images were exported in TIFF format to Photoshop (Adobe) and superimposed. The numbers of red, green, and yellow (superimposed red and green) patches were counted on the computer screen, counting at least 100 patches per cell on 10–15 cells in each case. MC3T3 cells were transfected with the indicated BMP receptor constructs. 24 h (for Smad phosphorylation) or 44 h (for phosphorylation of p38) post-transfection, cells were starved for 24 h (Smad) or 5 h (p38 phosphorylation) in DMEM supplemented with 0.5% FCS. The cells were then incubated with 20 nmBMP-2 in low serum for 30 min (Smad) or 90 min (p38) and solubilized in TNE lysis buffer (20 mm Tris, pH 7.4, 150 mmNaCl, 1% Triton X-100, 1 mm EDTA) supplemented with protease inhibitors (Boehringer complete mixture and 1 mmPMSF) and phosphatase inhibitors (50 mm NaF, 10 mm Na4P2O7, and 25 mm Na3VO4) for 10 min on ice. Equal amounts of protein were subjected to 12.5% SDS-PAGE. C2C12 and MC3T3 cells (1 × 106 cells per well of 6-well dish) were transfected by electroporation using pSBE-luc (26Jonk L.J. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar) and the indicated BMP receptor constructs as described by us earlier (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). After 7 h in complete medium, cells were starved in medium with 0.5% FCS (4–6 h) and incubated with or without 10 nm BMP-2 in low serum for additional 24 h. Luciferase activity was measured using a dual-luciferase assay system (Promega). C2C12 cells (1 × 106 cells per well of 6-well dish) were transfected by electroporation using 8 μg of pRL-Tk (Promega) as reference for transfection efficiency and 10 μg of BMP receptor constructs (in double-receptor transfections, 5 μg of each receptor; in transfections with a single BMP receptor, 5 μg of pcDNA1-βGal replaced the second BMP receptor construct). After 7 h in complete medium, cells were starved in medium with 0.2% FCS (4–6 h). They were incubated with or without 50 nm BMP-2 in low serum for 3 days. Renilla luciferase activity was measured using the luciferase assay system from Promega. Lysates were normalized to protein content. Alkaline phosphatase was measured using standard protocols (27Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1314) Google Scholar). To study hetero-complex formation between BRII and BRI-a or BRI-b, we performed co-immunoprecipitation studies on COS7 cells co-transfected with a truncated HA-tagged BRII (HA-TC1, -TC3, -BRII-SF, -TC4, -TC5, -TC6, -TC7, -TC8; Fig.1) and myc-tagged BRI-a or BRI-b. After cell lysis, BRI-a or BRI-b were immunoprecipitated with α-myc antibodies or with polyclonal antisera recognizing the receptors specifically (14Gilboa L. Nohe A. Geissendorfer T. Sebald W. Henis Y.I. Knaus P. Mol. Biol. Cell. 2000; 11: 1023-1035Crossref PubMed Scopus (253) Google Scholar). The immunoprecipitates were subjected to SDS-PAGE and Western blotting. Blots were analyzed with α-HA followed by peroxidase-goat-anti-mouse IgG. Whereas the BRII truncation mutants HA-TC8, -TC7, -TC6, -TC5, and -TC4 were able to be expressed in preformed complexes with myc-BRI-b (Fig.2 A, lanes 1–5), HA-TC3 was not (Fig. 2 A, lane 8). The expression of HA-TC3 was shown in cells transfected with HA-TC3 and myc-BRI-b, immunoprecipitated and blotted with the same antibody (α-HA) (Fig.2 A, lane 6). Similar experiments using BRI-a inst
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