Extracellular and Cytoplasmic Domains of Endoglin Interact with the Transforming Growth Factor-β Receptors I and II
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m111991200
ISSN1083-351X
AutoresMercedes Guerrero-Esteo, Tilman Sanchez‐Elsner, Ainhoa Letamendı́a, Carmelo Bernabéu,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoEndoglin is an auxiliary component of the transforming growth factor-β (TGF-β) receptor system, able to associate with the signaling receptor types I (TβRI) and II (TβRII) in the presence of ligand and to modulate the cellular responses to TGF-β1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TβRI or TβRII. This study shows that full-length endoglin interacts with both TβRI and TβRII, independently of their kinase activation state or the presence of exogenous TGF-β1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TβRI interaction was different from that of endoglin/TβRII. TβRII interacts with the amino acid region 437–558 of the extracellular domain of endoglin, whereas TβRI interacts not only with the region 437–558 but also with the protein region located between amino acid 437 and the N terminus. Both TβRI and TβRII interact with the cytoplasmic domain of endoglin, but TβRI only interacts when the kinase domain is inactive, whereas TβRII remains associated in its active and inactive forms. Upon association, TβRI and TβRII phosphorylate the endoglin cytoplasmic domain, and then TβRI, but not TβRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TβRII, TβRI, and downstream Smad proteins as well as a modulation of TGF-β signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-β responses. Endoglin is an auxiliary component of the transforming growth factor-β (TGF-β) receptor system, able to associate with the signaling receptor types I (TβRI) and II (TβRII) in the presence of ligand and to modulate the cellular responses to TGF-β1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TβRI or TβRII. This study shows that full-length endoglin interacts with both TβRI and TβRII, independently of their kinase activation state or the presence of exogenous TGF-β1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TβRI interaction was different from that of endoglin/TβRII. TβRII interacts with the amino acid region 437–558 of the extracellular domain of endoglin, whereas TβRI interacts not only with the region 437–558 but also with the protein region located between amino acid 437 and the N terminus. Both TβRI and TβRII interact with the cytoplasmic domain of endoglin, but TβRI only interacts when the kinase domain is inactive, whereas TβRII remains associated in its active and inactive forms. Upon association, TβRI and TβRII phosphorylate the endoglin cytoplasmic domain, and then TβRI, but not TβRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TβRII, TβRI, and downstream Smad proteins as well as a modulation of TGF-β signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-β responses. transforming growth factor-β glutathioneS-transferase hemagglutinin plasminogen activator inhibitor 1 TGF-β receptor type I TGF-β receptor type II Members of the transforming growth factor-β (TGF-β)1 superfamily (1Massagué J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3010) Google Scholar, 2Letterio J.J. Roberts A.B. Annu. Rev. Immunol. 1998; 16: 137-161Crossref PubMed Scopus (1686) Google Scholar) exert their biological effects through binding to a heteromeric complex containing two different transmembrane serine/threonine kinases known as type I and type II signaling receptors (3Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar, 4Miyazono K. ten Dijke P. Heldin C.H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar). Upon ligand binding to the type II receptor, the association between type I and type II receptors is induced, leading to phosphorylation and activation of the type I receptor by the constitutively active type II receptor (4Miyazono K. ten Dijke P. Heldin C.H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar, 5Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1658) Google Scholar). Then, activated type I receptor propagates intracellular signal to the nucleus by phosphorylating members of the Smad family of proteins (4Miyazono K. ten Dijke P. Heldin C.H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar,6Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar, 7Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). The TGF-β receptor complex also contains two auxiliary co-receptors named endoglin and betaglycan (8Cheifetz S. Bellón T. Calés C. Vera S. Bernabeu C. Massagué J. Letarte M. J. Biol. Chem. 1992; 267: 19027-19030Abstract Full Text PDF PubMed Google Scholar, 9López-Casillas F. Cheifetz S. Doody J. Andres J.L. Lane W.S. Massagué J. Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (553) Google Scholar, 10Wang X.F. Lin H.Y. Ng-Eaton E. Downward J. Lodish H.F. Weinberg R.A. Exp. Cell. 1991; 67: 797-805Abstract Full Text PDF Scopus (543) Google Scholar). These are transmembrane proteins with large extracellular domains and serine/threonine-rich cytoplasmic regions without consensus signaling motifs. Endoglin binds TGF-β1, TGF-β3, activin-A, BMP-2, and BMP-7 in the presence of the signaling receptor types I and II (8Cheifetz S. Bellón T. Calés C. Vera S. Bernabeu C. Massagué J. Letarte M. J. Biol. Chem. 1992; 267: 19027-19030Abstract Full Text PDF PubMed Google Scholar, 11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 12Barbara N.P. Wrana J.L. Letarte M. J. Biol. Chem. 1999; 274: 584-594Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar) and modulates TGF-β1-dependent cellular responses (11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 13Lastres P. Letamendı́a A. Zhang H. Rius C. Almendro N. Raab U. Lopez L.A. Langa C. Fabra A. Letarte M. Bernabéu C. J. Cell Biol. 1996; 133: 1109-1121Crossref PubMed Scopus (283) Google Scholar, 14Li C. Hampson I.N. Hampson L. Kumar P. Bernabeu C. Kumar S. FASEB J. 2000; 14: 55-64Crossref PubMed Scopus (226) Google Scholar). It is highly expressed on endothelial cells and several lines of evidence support an important role for endoglin in cardiovascular development and vascular remodeling. Thus, endoglin expression is regulated during heart development in humans and chicken (15Qu R. Silver M.M. Letarte M. Cell Tissue Res. 1998; 292: 333-343Crossref PubMed Scopus (57) Google Scholar, 16Vincent E.B. Runyan R.B. Weeks D.L. Dev. Dyn. 1998; 213: 237-247Crossref PubMed Scopus (21) Google Scholar), and genetic inactivation of endoglin in the mouse shows that embryos homozygous for mutant endoglin die at 10–10.5 days postcoitum due to vascular and cardiac anomalies (17Li D.Y. Sorensen L.K. Brooke B.S. Urness L.D. Davis E.C. Taylor D.G. Boak B.B. Wendel D.P. Science. 1999; 284: 1534-1537Crossref PubMed Scopus (723) Google Scholar, 18Bourdeau A. Dumont D.J. Letarte M. J. Clin. Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (390) Google Scholar, 19Arthur H.M. Ure J. Smith A.J. Renforth G. Wilson D.I. Torsney E. Charlton R. Parums D.V. Jowett T. Marchuk D.A. Burn J. Diamond A.G. Dev. Biol. 2000; 217: 42-53Crossref PubMed Scopus (383) Google Scholar). Furthermore, genes encoding endoglin and ALK-1 (a type I TGF-β receptor) are targets for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia (20Guttmacher A.E. Marchuk D.A. White R.I.J. N. Eng. J. Med. 1995; 333: 918-924Crossref PubMed Scopus (907) Google Scholar, 21Shovlin C.L. Letarte M. Thorax. 1999; 54: 714-729Crossref PubMed Scopus (353) Google Scholar, 22Marchuk D.A. Curr. Opin. Hematol. 1998; 5: 332-338Crossref PubMed Scopus (63) Google Scholar). Despite the data supporting an important role for endoglin in the TGF-β system, the molecular basis of endoglin function is still poorly understood. Several lines of evidence suggest that endoglin is not a receptorper se: (a) in endothelial cells, only a small percentage of endoglin molecules is capable of binding TGF-β; (b) endoglin does not bind ligand on its own but requires the presence of the signaling receptors (11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 12Barbara N.P. Wrana J.L. Letarte M. J. Biol. Chem. 1999; 274: 584-594Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar), (c) even in the presence of the signaling receptors, ligand association with endoglin can only be visualized in the presence of a cross-linking agent (12Barbara N.P. Wrana J.L. Letarte M. J. Biol. Chem. 1999; 274: 584-594Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar); and (d) cross-linking of endoglin transfectants with radiolabeled TGF-β showed the existence of ligand-free endoglin associated with TGF-β-loaded signaling receptors (11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). All of these data suggest that endoglin association with the signaling receptors is critical for endoglin access to ligand and for its modulation of the ligand-induced cellular responses. Here, we have investigated the domains involved in the association between endoglin and the signaling receptors as well as the consequences of this interaction on the phosphorylation status of the proteins involved. The monkey kidney COS-7 and the rat myoblast L6E9 cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 5% CO2 at 37 °C in a humidified atmosphere. COS-7 cells were transiently transfected with expression vectors encoding wild type and mutant constructs of endoglin, TβRI, or TβRII using LipofectAMINE Plus as indicated by the manufacturer (Invitrogen). Functional assays were carried out 48 h after transfection. Treatment of cells with recombinant human TGF-β1 (Peprotech) was performed at a concentration of 10 ng/ml for 30 min. Wild type human endoglin cDNA subcloned into the pCMV5 vector (11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) was used to derive the truncated endoglin constructs by PCR amplification. The resulting PCR fragments were cloned into pDisplay (Invitrogen) expression vector, which allows expression of proteins on the cell surface. All endoglin constructs expressed from pDisplay contain the influenza hemagglutinin epitope HA at the NH2 terminus. The oligonucleotides used to prime the PCR synthesis of endoglin fragments were as follows: TMCT-Endo (amino acids 573–658), GAAGATCTAACATCATCAGCCCTGAC and TCCCCGCGGGGCTATGCCATGCTGCT; EC-Endo (amino acids 26–586), GGGGCCCAGCCGGCCGAAACAGTCCATTGT and TCCCCGCGGGGCCTTTGCTTGT; ECTM-Endo (amino acids 26–614), GGGGCCCAGCCGGCCGAAACAGTCCATTGT and TCCCCGCGGTCAGTAGATGTACCA; 558-Endo (amino acids 26–558), GGGGCCCAGCCGGCCGAAACAGTCCATTGT and TCCCCGCGGCCCGGTCTTGGG; 437-Endo (amino acids 26–437), GGGGCCCAGCCGGCCGAAACAGTCCATTGT and TCCCCGCGGTTTCCGCTGTGG; 437/586-Endo (amino acids 437–586), GGGGCCCAGCCGGCCGAAAAAGGTGCACTGC and TCCCCGCGGGCCTTTGCTTGT. The oligonucleotides were designed to introduce a SfiI restriction site at the 5′-end (except for the TMCT-Endo mutant, which contains a BglII restriction site) and a SacII site at the 3′-end of the endoglin DNA fragments. The reverse primers for TMCT-Endo and ECTM-Endo include a stop codon. All primer sequences for this study are given from 5′ to 3′. Full-length endoglin was also subcloned into the pDisplay vector to generate an HA epitope-tagged endoglin. The construct was engineered by PCR using forward primer that included a SfiI restriction site (GGGGCCCAGCCGGCCGAAACAGTCCATTGT) and reverse primer that included a stop codon and a SacII site (TCCCCGCGGGGCTATGCCATGCTGCT). The pCMV5 expression constructs containing cDNAs for TβRII/HA (tagged at the COOH terminus with the influenza hemagglutinin (HA) epitope), HA/TβRII (K277R) (tagged at the NH2 terminus with the HA epitope), TβRI/HA, TβRI (T204D)/HA, and TβRI (K232R)/HA have been described (23Labbe E. Letamendı́a A. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8358-8363Crossref PubMed Scopus (383) Google Scholar) and were a generous gift from Dr. Liliana Attisano (University of Toronto, Canada). To generate the GST-Ecyt fusion protein, the cytoplasmic domain of endoglin was amplified by PCR, using forward (5′-GAATTCTGGTACATCTACTCGCACACGC-3′) and reverse (5′-GGCTATGCCATGCTGCTGGTGG-3′) primers, and the product was cloned into pGEX-1λT (Amersham Biosciences). To generate the GST-GS/TβRI fusion protein, the juxtamembrane and GS domains of TβRI (amino acids 146–207) were amplified by PCR, using forward (5′-CGGGATCCATCTGCCACAACCGC-3′) and reverse (5′-CGGAATTCTTGTAACACAATAGTTCTCGC-3′) primers, and the product was cloned into pGEX4T-3 (Amersham Biosciences). Plasmids encoding GST-Smad2, GST-Smad3, and GST-Smad4 were kindly provided by Dr. Liliana Attisano (University of Toronto). Fusion proteins were expressed and purified according to the manufacturer's instructions. The endoglin-specific monoclonal antibodies P4A4 and P3D1 have been previously described, and they recognize epitopes contained within the fragments Tyr227–Gly331and Glu26–Gly230 of human endoglin, respectively (24Pichuantes S. Vera S. Bourdeau A. Pece N. Kumar S. Letarte M. Tissue Antigens. 1997; 50: 265-276Crossref PubMed Scopus (62) Google Scholar). For recognition of TβRII and TβRI, specific polyclonal antibodies were used (C16 and V22, respectively; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Proteins tagged with the influenza virus HA epitope were detected with the monoclonal antibody 12CA5 (Roche Molecular Biochemicals). Cells were lysed at 4 °C for 30 min with lysis solution (50 mmTris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 1% digitonin, 1 mm NaF, 10 mm NaVO4, 1 mmMo2VO4, and a mixture of protease inhibitors). Aliquots of total cell lysates containing equivalent amounts of total protein were precleared for 4 h with protein G or protein A coupled to Sepharose (Amersham Biosciences) at 4 °C. Specific immunoprecipitations of the precleared lysates were carried out in the presence of the appropriate antibody, using protein G- or protein A-Sepharose. After overnight incubation at 4 °C, immunoprecipitates were isolated by centrifugation and washed three times with lysis buffer. When required, immunoprecipitates were incubated with alkaline phosphatase (Roche Molecular Biochemicals) at 37 °C for 1 h. Total lysates and the precipitated proteins were separated by SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane for immunodetection with the indicated antibody. Immunodetection was performed with the SuperSignal chemiluminescent substrate (Pierce) according to the manufacturer's instructions. COS cells were transiently transfected with the indicated HA-tagged endoglin constructs. After 48 h, cells were incubated with the mouse monoclonal antibody 12CA5 (against the influenza virus HA epitope; Roche Molecular Biochemicals) for 30 min at 4 °C. After two washes with phosphate-buffered saline, fluorescein isothiocyanate-labeled F(ab′)2 rabbit anti-mouse Ig was added, and incubation proceeded for an additional period of 30 min at 4 °C. Finally, cells were washed twice with phosphate-buffered saline, and their fluorescence was estimated with an EPICS-CS (Coulter Cientifica), using logarithmic amplifiers. Results are expressed as an expression index calculated as the percentage of marker-positive cells multiplied by their mean fluorescence intensity. Immunoprecipitates of TβRI or TβRII were washed twice with lysis buffer, washed once with kinase buffer (50 mm Tris-HCl, pH 7.5, 5 mmMnCl2, 5 mm MgCl2, 1 mmCaCl2), and resuspended in 40 μl of kinase buffer with 5 μm ATP, 1 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences), and 2 μg of recombinant GST, GST-Ecyt, GST-GS/TβRI, GST-Smad2, GST-Smad3, or GST-Smad4. The reaction was incubated for 30 min at 37 °C. Phosphorylation was stopped by the addition of Laemmli sample buffer, and the products were resolved by SDS-PAGE. The incorporation of [32P]phosphate was visualized by autoradiography. Assays for TβR-II kinase activity were performed as described above but without adding GST proteins. COS-7 cells were transfected with TβRI, TβRII, and endoglin, as indicated. After 48 h, the cells were washed in phosphate-free medium (ICN Biomedicals) and metabolically labeled with 0.5 mCi/ml [32P]orthophosphate for 3 h. Cells were washed with phosphate-buffered saline, lysed with lysis buffer, and immunoprecipitated with the appropriate antibodies. Immunoprecipitates were analyzed on SDS-polyacrylamide gels, and radiolabeled bands were detected by autoradiography and quantified by densitometry using a PhosphorImager 410 and ImageQuant software. The pARE-lux reporter vector, encoding the activin response element of Xenopus mix.2 promoter fused to the luciferase reporter gene, and the TGF-β transcriptional co-activator Fast-1 have been previously described (25Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (634) Google Scholar, 26Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (494) Google Scholar, 27Watanabe M. Whitman M. Development. 1999; 126: 5621-5634Crossref PubMed Google Scholar). L6E9 myoblasts were selected for these studies, because they do not express endoglin or its homologue betaglycan, and they have proved to be a useful model system to analyze the function of endoglin and betaglycan in TGF-β responses (11Letamendı́a A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabéu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 28López-Casillas F. Wrana J.L. Massagué J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Activity of the TGF-β reporter construct was determined by transient transfection of L6E9myoblasts using Superfect (Qiagen). Briefly, cells in 24-well plates were transfected with the pARE reporter and the endoglin or Fast-1 expression vectors at densities of 5 × 104cells/well. The amount of DNA in each transfection was normalized by using the corresponding insertless expression vector as carrier. After 24 h, cells were washed with Dulbecco's modified Eagle's medium before incubation with 5 ng/ml TGF-β1 for an additional 24-h period. Relative luciferase units from triplicate samples were determined in a TD20/20 luminometer (Promega, Madison, WI). Each transfection experiment was performed at least four times with different DNA preparations. Correction for transfection efficiency was made by cotransfection with the β-galactosidase expression vector pCMV-β-galactosidase, and the corresponding enzymatic activity was determined using the Galacto-Light kit (Tropix). The mean and S.D. were calculated and experimental results of the promoter constructs were displayed either as arbitrary units of luciferase activity, or as a -fold induction with respect to the corresponding untreated sample. To study the interaction of endoglin with TβRI or TβRII, COS cells were transfected with the corresponding expression vectors in the absence of exogenous ligand, and cell lysates were subjected to specific immunoprecipitation, followed by Western blot analysis. As shown in Fig.1, endoglin co-immunoprecipitates with either TβRI or TβRII as revealed by immunodetection with antibodies to TβRI and TβRII, respectively (lanes 3 and6). Although the transfected COS cells express low levels of signaling receptors, no association between endoglin and the endogenous receptors could be detected (lanes 1 and4). These results demonstrate that endoglin interacts with TβRI and with TβRII in the absence of exogenous TGF-β and that the formation of the endoglin-TβRI or endoglin-TβRII complexes does not require the presence of TβRII or TβRI, respectively. To analyze whether the complex formation of endoglin with TβRI or TβRII was affected by the activation state of the signaling receptors, experiments were performed in the presence of exogenous ligand or signaling receptors with varying catalytic activities. First, COS cells were transfected with endoglin and TβRI or TβRII and cultured with different concentrations of fetal calf serum, either in the absence or in the presence of 10 ng/ml exogenous TGF-β1. Fetal calf serum is a minor source of TGF-β, and preliminary experiments indicated that 0.2% is the minimal concentration of fetal calf serum required for cell viability. As shown in Fig.2 A, the amount of TβRI (left upper panel) or TβRII (right upper panel) co-immunoprecipitated by anti-endoglin antibodies is similar for all treatments, suggesting that the addition of exogenous ligand does not affect the formation of endoglin-TβRI or endoglin-TβRII complexes. Since the active TGF-β receptor signaling complex requires both TβRI and TβRII, endoglin association was analyzed when both signaling receptors were cotransfected (Fig. 2 B). Again, exogenous TGF-β did not affect the formation of endoglin complexes with TβRI or TβRII. Next, COS cells were transfected with expression vectors for endoglin as well as wild type and different kinase mutant versions of the signaling receptors. As shown in Fig.2 C, the constitutively active TβRI (T204D) mutant, the constitutively inactive TβRI (K232R) mutant, and the wild type TβRI construct were able to associate with endoglin (upper left panel). Similarly, the kinase-inactive TβRII (K277R), and the wild type TβRII constructs showed interaction with endoglin (upper right panel). Taken together, these results suggest that endoglin association with TβRI and TβRII is independent of the activation state of the signaling receptors. To identify the region(s) involved in the association with the signaling receptors, several truncated versions of endoglin were generated (Fig. 3). Construct HA-TMCT-Endo lacks the extracellular domain, HA-EC-Endo lacks the cytoplasmic and transmembrane regions, and HA-ECTM-Endo only lacks the cytoplasmic domain. Additional mutants encoding only part of the extracellular domain (558-Endo, 437-Endo, and 437/586-Endo) were also analyzed. Constructs 437-Endo and 437/586-Endo were generated around the arginine at position 437, because several lines of investigation suggest that this residue might define a protein domain: (a) it is located at a putative protease cleavage site of the protein; (b) artificial constructs truncated at position 437 can be expressed upon transfection of mammalian cells; and (c) its codon is located in the border between exons 9b and 10 (29). In order to facilitate the analysis of the mutants, all constructs contained an epitope tag of HA at the amino terminus. Transfection of these constructs in COS cells confirmed that all mutant versions of endoglin were expressed as evidenced by Western blot analysis (Fig.3 B). Since the wild type endoglin is a disulfide-linked dimer (30Gougos A. Letarte M. J. Biol. Chem. 1990; 265: 8361-8364Abstract Full Text PDF PubMed Google Scholar), we analyzed whether the truncated forms were also disulfide-linked by subjecting the lysates to electrophoresis under reducing or nonreducing conditions. As shown in Fig. 3 B, all of the constructs were expressed in a dimeric form. Interestingly, the smallest construct, TMCT-Endo, contains only one cysteine residue at position 582 of the extracellular domain, suggesting its involvement in the dimerization process. This was further demonstrated by generating the mutant construct TMCT-Endo/C582G, where the cysteine at 582 has been replaced by a glycine. Transfection of this mutant yielded a monomeric form of endoglin, as opposed to the dimer obtained with the TMCT-Endo plasmid (Fig. 3 C). Furthermore, similar to the wild type endoglin, all of the truncated forms were also expressed at the cell surface as demonstrated by flow cytometry analysis (Fig.3 D). To investigate whether the extracellular domain of endoglin was involved in the association with the signaling receptors, HA-ECTM-Endo, TβRI, and TβRII were expressed upon transfection in COS cells. Immunoprecipitation analysis with anti-TβRI and anti-TβRII demonstrated that HA-ECTM-Endo was co-precipitated only when TβRI or TβRII were expressed (Fig.4 A), indicating that the extracellular domain of endoglin interacts with both signaling receptors. Parallel studies with HA-EC-Endo construct, which does not contain the transmembrane domain of endoglin, revealed similar levels of truncated protein co-precipitated with anti-TβRI and anti-TβRII (Fig. 4 A), further confirming the involvement of the extracellular domain and suggesting a nonrelevant role for the transmembrane region in the interaction with the signaling receptors. Next, the interactions of different truncations of the extracellular domain of endoglin with TβRI and TβRII were analyzed. Immunoprecipitation studies with anti-endoglin, anti-TβRI, and anti-TβRII demonstrated that HA-558-Endo, HA-437-Endo, and HA-437/586-Endo co-precipitated with TβRI, whereas only HA-558-Endo and HA-437/586-Endo co-precipitated with TβRII (Fig. 4, Band C). These results suggest that (a) TβRII interacts with residues 437–558 of the extracellular domain of endoglin, a region proximal to the transmembrane domain and (b) TβRI interacts not only with residues 437–558 but also with a second region comprised between amino acids 26 and 437 of the extracellular domain of endoglin. When studying the interaction between two different transmembrane proteins, it is important to assess both the involvement of their extracellular domains and that of their cytoplasmic domains. Endoglin is constitutively phosphorylated in Ser/Thr residues (31Lastres P. Martı́n-Perez J. Langa C. Bernabeu C. Biochem. J. 1994; 301: 765-768Crossref PubMed Scopus (69) Google Scholar, 32Yamashita H. Ichijo H. Grimsby S. Morén A. Dijke P. Miyazono K. J. Biol. Chem. 1994; 269: 1995-2001Abstract Full Text PDF PubMed Google Scholar), and both signaling receptors are Ser/Thr kinases (33Wrana J.L. Attisano L. Cárcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar, 34Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (508) Google Scholar), thus providing a hint for the participation of their cytoplasmic domains in protein association. To study the interaction between endoglin cytoplasmic domain and TβRI or TβRII, the HA-TMCT-Endo construct (lacking the extracellular domain) was co-transfected with wild type or kinase mutants of TβRI and TβRII (Fig.5). Immunoprecipitation analysis with anti-TβRI and anti-TβRII demonstrated that the cytoplasmic domain of endoglin associates with TβRI when the kinase domain is inactive (K232R) but not with the constitutively active (T204D) form (Fig.5 A). In the same experiment, a weak band of endoglin was found associated with the wild type TβRI. This association is probably due to the TβRI activation induced by oligomerization of the signaling receptors that occurs upon transfection (35Wrana J.L. Attisano L. Weiser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2120) Google Scholar, 36Ventura F. Doody J. Liu F. Wrana J.L. Massagué J. EMBO J. 1994; 13: 5581-5589Crossref PubMed Scopus (79) Google Scholar). In addition, the cytoplasmic domain of endoglin interacts with both the active (wild type) and inactive (K277R) forms of TβRII (Fig.5 B). These results demonstrate that the endoglin cytoplasmic domain interacts with TβRI and with TβRII. A detailed analysis of the bands separated by SDS-PAGE, corresponding to the endoglin cytoplasmic domain, revealed that the electrophoretic mobility of the HA-TMCT-Endo associated with the constitutively active TβRII (wild type) was lower than that of the HA-TMCT-Endo associated with the kinase-inactive (K277R) form of TβRII (Fig. 5, B and C; upper panels). Similar electrophoretic differences were observed when analyzing the endoglin cytoplasmic domain in total lysates derived from cells overexpressing either the wild type or the constitutively inactive form of TβRII, respectively (Fig. 5, B andC; lower panels). It is well known that phosphorylated proteins migrate more slowly that the unphosphorylated forms, suggesting that the electrophoretic differences observed above might be due to different phosphorylation states of
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