A Pivotal Role for the Transmembrane Domain in Transforming Growth Factor-beta Receptor Activation*

Hong-Jian ZhuDagger and Andrew M. Sizeland

From the Ludwig Institute for Cancer Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta ) delivers diverse growth and differentiation signals by binding two distantly related transmembrane serine/threonine kinase receptors: the type I receptor (Tbeta RI) and the type II receptor (Tbeta RII). In an attempt to establish the role of the transmembrane domain in receptor signaling, two chimeric TGF-beta receptors, Tbeta RI-II-I and Tbeta RII-I-II, containing the opposite transmembrane domain were generated. When transfected into a mutant mink lung epithelial cell line R1B, which lacks functional Tbeta RI, Tbeta RI-II-I restored TGF-beta 1-induced transcriptional activation of a TGF-beta reporter p3TP-Lux to ~25% of the levels restored by wild-type Tbeta RI. In the mutant mink lung epithelial cell line DR26, which contains a truncated, nonfunctional Tbeta RII, wild-type receptor Tbeta RII restored the TGF-beta responsiveness, while the Tbeta RII-I-II cDNA was inactive. When both Tbeta RI and Tbeta RII were transfected into R1B, DR26, or Mv1Lu cells, a low level of constitutive p3TP-Lux activity was observed. However, cotransfection of both transmembrane chimeric receptors, Tbeta RI-II-I and Tbeta RII-I-II, or the wild-type Tbeta RI with the transmembrane chimeric Tbeta RII-I-II resulted in high levels of ligand-independent receptor activation. These results suggest that the transmembrane domains of both TGF-beta receptors are essential and play a pivotal role in receptor activation. To investigate the role of the transmembrane domain further, four type II transmembrane mutants were generated: Tbeta RIIDelta -1, Tbeta RIIDelta -2, Tbeta RIIDelta -3, and Tbeta RIIDelta -4, which have one, two, three, or four amino acids deleted at the N terminus of the transmembrane domain, respectively. Interestingly, co-expression of Tbeta RIIDelta -1 with the wild-type Tbeta RI in DR26 cells resulted in high levels of constitutive activation, while only low levels of the activation were observed when Tbeta RIIDelta -2, Tbeta RIIDelta -3, or Tbeta RIIDelta -4 were co-expressed with the wild-type Tbeta RI. However, Tbeta RIIDelta -1 restored very little the TGF-beta responsiveness in DR26cells. Expression of Tbeta RIIDelta -2, Tbeta RIIDelta -3, and Tbeta RIIDelta -4 resulted in a progressive increase in TGF-beta responsiveness, with Tbeta RIIDelta -4 reaching the level of activity of the wild-type Tbeta RII. Furthermore, like Tbeta RII-I-II, co-expression of Tbeta RIIDelta -1 with Tbeta RI-II-I also resulted in high levels of constitutive activation. These results are consistent with an important role for the transmembrane region of the receptors. We further propose a model of receptor activation in which receptor activation occurs via relative orientational rotation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta )1 is a member of a superfamily of secreted signaling molecules that play important roles in intercellular communication (1-3). TGF-beta superfamily members regulate proliferation, differentiation, migration, and adhesion of many cell types and affect a wide range of biological functions, including embryonic development, hematopoiesis, and immune and inflammatory cell responses (4). Alterations in the activity of these growth factors or their receptors have been implicated in fibrosis, immunosuppression, cancer, and other disorders (4-6).

Recently, receptors for TGF-beta superfamily members have been identified and cloned (1, 3, 7-9). In mammalian cells, most responses to TGF-beta are mediated by the type I and II cell surface receptors, which are expressed in most cell types and tissues (2, 8-10). Molecular cloning of Tbeta RI and Tbeta RII (11-13) has shown that they are members of a family of proteins with a small extracellular region, a single transmembrane domain, and a cytoplasmic region with a serine/threonine kinase domain. There is 40% homology between the Tbeta RI and Tbeta RII kinase domains (3). Genetic evidence from mutant cells resistant to the action of TGF-beta suggests that both type I and type II receptors are required for TGF-beta signaling (10, 14). Furthermore, loss of functional Tbeta RII by deletion or mutation results in abnormal cell overproliferation and is thought to contribute to the malignant phenotype of certain types of cancers (5, 15). Tbeta RII is a constitutively active kinase and is autophosphorylated (16). While TGF-beta binds directly to Tbeta RII, it is currently believed that TGF-beta signaling occurs through Tbeta RI (17). Early studies have shown that TGF-beta binds directly to Tbeta RII; Tbeta RI is then recruited into the complex and becomes phosphorylated by Tbeta RII. Tbeta RI then propagates a signal to downstream substrates (17-21).

While the overwhelming majority of studies are consistent with the above model of TGF-beta receptor activation and signaling (17-21), more recent studies (22-25) suggest that some fundamental questions concerning the molecular mechanism of receptor activation have yet to be answered. 125I-TGF-beta affinity labeling and cross-linking followed by immunoprecipitation using Tbeta RI- and Tbeta RII-specific antisera and analyses by two-dimensional gel electrophoresis under nonreducing and reducing conditions, demonstrates that TGF-beta induces the formation of heteromeric receptor complexes (22). This complex is most likely a heterotetramer containing two molecules each of Tbeta RI and Tbeta RII (22). Further double immunoprecipitation analyses using lysates from metabolically labeled cells cotransfected with differentially epitope-tagged type II receptors have demonstrated that type II receptors form a homomeric complex both in the presence and absence of TGF-beta (23). It has also shown that both the extracellular and intracellular domains of type II receptor can interact with each other and form homomeric associations (23, 24). These results indicate that both type II extracellular and intracellular domains have an inherent affinity for each other in the absence of TGF-beta . In addition, experiments using a yeast two-hybrid interaction assay and double immunoprecipitation analyses (25) have also shown that type I and type II receptors form heteromeric complexes even in the absence of TGF-beta . Also, the type I and type II receptor extracellular domains alone or the intracellular domains alone form heteromeric complexes, again in the absence of ligand. Taken together, these results suggest that both the extracellular and intracellular type I and type II receptors domains have inherent affinities for each other in the absence of TGF-beta and form preexisting, heterooligomeric receptor complexes. More recently, it has been shown that after ligand binding, Tbeta RII formed a heteromeric complex with Tbeta R-2.1 (26), a chimeric receptor containing the extracellular and transmembrane domains of type II receptor and the intracellular domain of type I receptor. The Tbeta RII·Tbeta R-2.1 failed to signal any TGF-beta response in R1B cells, which lack a functional type I receptor. These results suggest that the receptor heterooligomerization may be necessary but not sufficient for TGF-beta signaling.

Most recent studies on chimeric granulocyte-macrophage colony-stimulating factor (GM-CSF)/TGF-beta receptors (27) and chimeric erythropoietin (Epo)/TGF-beta receptors (28) have shown some surprising results. GM-CSF induces the TGF-beta signaling through chimeric receptors alpha I and beta II or alpha II and beta I, which consist of the extracellular domain of GM-CSF alpha  or beta  receptor fused to the transmembrane and cytoplasmic domain of TGF-beta receptor type I or II, but chimeric receptors alpha I and beta I or alpha II and beta II are signaling-incompetent. Similarly, Epo signals the TGF-beta activity by binding to chimeric Epo/TGF-beta receptors E-RI and E-RII, consisting of the extracellular domain of Epo receptor and the cytoplasmic TGF-beta receptor type I and II, respectively. These studies confirmed that homodimerization of the receptor cytoplasmic domains of either type I or type II receptors does not activate the receptor complex and that heterooligomerization of the receptor cytoplasmic domains is required for receptor activation. However, given that the extracellular domain of the type II TGF-beta receptor may also homodimerize (23-25), it is surprising that the Tbeta RII·Tbeta R-2.1 complex failed to signal (26), yet the Epo or GM-CSF chimeric receptors were signaling-competent. In addition, the intensity of E-RI and E-RII signaling depends on the combination of transmembrane domains in the chimeric receptors (28). In cells expressing E-RI and E-RII chimeras where the transmembrane domains are Epo receptor in one construct and TGF-beta receptor in the other, the maximum extent of growth inhibition was lower, compared with cells expressing E-RI and E-RII chimeras with either Epo receptor's transmembrane domains or TGF-beta receptor's transmembrane domains (28). Taking together the observations in these studies (26-28), we propose that the transmembrane domain may play a role in receptor activation.

Given that the type I and II receptors form signaling-incompetent receptor complexes in the absence of ligand, we need to understand what change occurs in the receptor complex following ligand binding. The observations that (i) Tbeta R-2.1 fails to reconstitute signaling despite heterodimerization with the type II receptor (26), (ii) heterodimerization of the chimeric GM-CSF/TGF-beta or Epo/TGF-beta receptors results in active signaling (27, 28), and (iii) the transmembrane domain plays a role in E-RI and E-RII signaling (28) indicate that more work is required to define the role of the transmembrane domain in receptor activation. In particular, the role of the transmembrane domain in transmitting a signal following ligand binding is not clear. In order to investigate these questions, we have generated two TGF-beta transmembrane chimeric receptors, in which the transmembrane domains of the type I and II receptors were interchanged, and four type II transmembrane mutants with a series of deletions in the transmembrane domain. The signaling activity of these chimeric and mutant receptors in mutant mink lung cells has been examined, and our results identify an important role played by the transmembrane domain in receptor activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of C-terminal Tagged Wild-type, Transmembrane Chimeric, and Transmembrane Mutant TGF-beta Receptors-- Polymerase chain reaction (PCR) and human cDNAs ALK-5 (Tbeta RI) (11) and H2-3FF (Tbeta RII) (12) were used to generate transmembrane chimeric and mutant TGF-beta receptors. The following primers were used (where a single underline indicates the cDNAs of the type I receptor, a double underline indicates the type II receptor, lowercase type indicates restriction sites, boldface type indicates stop and start codes, and italic type indicates the M2-flag coding sequence): RI-1s, GGTaagcttGCCACCATGGAGGCGGCGGTCGCTGCTCCGCGT; RI-Ss, ACTgcatgcCTGCTcccgggGGCGACGGCGTT; RI-2s, GTGGAgctagcAGCTGTCATTGCTGGACCA; RI-3s, CACAttcgaaCTGTCATTCACCATCGAGTG; RI-1a, AATGactagtAGCAGTTCCACAGGACCAAGGCCAGGTGATGA; RI-2a, AGatcgatTAACGCGGATATAGACCATCAACATGAG; RI-3a, gcatgcTCACTTGTCATCGTCGTCCTTGTAGTCCATTTTGATGCCTTCCTGTTG; RII-1s, GCAaagcttGCCACCATGGGTCGGGGGCTGCTCAGGGGCCTGT; RII-2s, TTGTTactagtCATATTTCAAGTGACAGGC; RII-3s, GTTAggcgccAGCAGAAGCTGAGTTCAACC; RII-1a, TAGCtctagaTCAGGATTGCTGGTGTTATATTCTTC; RII-2a, CTatcgatTGTGGCAGTAGCAGTAGAAGATGATGAT; RII-3a, gaattcTTTGGTAGTGTTTAGGGAGCC; RIIDelta -1a, GactagtAAGTCAGGATTGCTGGTGTT; RIIDelta -3a, gacgtcAGGATTGCTGGTGTT; RIIDelta -3s, gacgtcATATTTCAAGTGACAGGC; RIIDelta -4a, gatatcAGGATTGCTGGTGTTATA; RIIDelta -4s, gatatcTTTCAAGTGACAGGCATC.

The above primers were designed to exchange the type I receptor transmembrane domain (22 amino acids, positions 126-147) (11) with the type II receptor transmembrane domain (30 amino acids, positions 160-189) (12). PCR products of extracellular, transmembrane, and cytoplasmic domains of type I and II were obtained using a Perkin-Elmer DNA thermal cycler with Taq DNA polymerase (BIOTECH), ALK-5 (11), or H2-3FF (12) as templates and primers as indicated in Fig. 1. The PCR products were first ligated to a linearized pCRII vector (Invitrogen). Deletion of about 20 nucleotides in the signal sequence region of Tbeta RI was observed frequently in the resulting PCR product, Tbeta RI-E. A similar deletion has been reported in other work (26). A NcoI-XhoI fragment of ALK-5 (nucleotides 1-236) was used to replace the deleted portion of Tbeta RI-E-pCRII by ligating at NcoI and XhoI sites. Three fragments, Tbeta RI-E, Tbeta RII-T, and Tbeta RI-C, were ligated to pcDNA I/Amp (Invitrogen) at HindIII and SphI sites to form the Tbeta RI-II-I(-M2) cDNA construct. To create HA3-tagged transmembrane chimeric type II receptor Tbeta RII-I-II(-HA3), a EcoRI-XbaI fragment consisting of three repeats of HA coding sequence was ligated to pcDNA I/Amp at corresponding sites; then, at its HindIII and EcoRI sites, fragments Tbeta RII-E, Tbeta RI-T, and Tbeta RII-C were ligated to the pcDNA I/Amp vector containing HA3. By replacing the XhoI-BamHI portion in Tbeta RI-II-I(-M2) with a corresponding fragment in ALK-5, tagged wild-type Tbeta RI(-M2) was created. Similarly, by replacing MluI-BglII cDNAs in Tbeta RII-I-II(-HA3) with those in H2-3FF, Tbeta RII(-HA3) was constructed.


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Fig. 1.   Schematic illustration of construction of TGF-beta wild-type, chimeric, and type II transmembrane residue deletion receptors. Tbeta RI-E, Tbeta RI-T, and Tbeta RI-C are type I receptor extracellular, transmembrane, and cytoplasmic domains; Tbeta RII-E, Tbeta RII-T, and Tbeta RII-C are the corresponding type II receptor domains. All of the six domains were generated by PCR, and primers used are indicated with arrows. Tbeta RI(-M2) is the wild-type type I receptor with a C-terminal M2-FLAG tag; Tbeta RII(-HA3) is type II with a C-terminal three repeats of hemagglutinin epitope HA3 tag. Tbeta RI-II-I(-M2) contains the extracellular and cytoplasmic domains of type I and the transmembrane domain of type II, and Tbeta RII-I-II(HA3) is a wild-type type II with type I transmembrane domain. Tbeta RIIDelta -1 contains a deletion of one transmembrane residue Leu160 in Tbeta RII. Tbeta RIIDelta -2 deletes Leu160 and Leu161, Tbeta RIIDelta -3 deletes Leu160, Leu161, and Leu162; and Tbeta RIIDelta -4 deletes Leu160, Leu161, Leu162, and Val163. All four deletion mutants are C-tagged with HA3.

Similar PCR strategy has been used to generate the type II transmembrane mutant receptors (Fig. 1). Ligation of the extracellular fragment generated using primers RII-1s and RIIDelta -1a with the transmembrane-cytoplasmic fragment generated by primers RII-2s and RII-3a at their SpeI sites into the pcDNA I/Amp vector containing HA3 forms C-terminal HA3-tagged Tbeta RIIDelta -1. This resulted in Tbeta RIIDelta -1 containing a Leu160 deletion in the transmembrane domain of the type II receptor. Replacing RIIDelta -1a by RII-1a and ligating the two fragments at their XbaI/SpeI sites, Tbeta RIIDelta -2 was generated, which results in a deletion of two residues (Leu160 and Leu161). To construct Tbeta RIIDelta -3, which contains a deletion of three residues (Leu160, Leu161, and Leu162), the extracellular fragment generated by primers RII-1s and RIIDelta -3a and the transmembrane-cytoplasmic fragment generated by primers RIIDelta -3s and RII-3a were ligated at their AatII sites. Primers RIIDelta -4a and RIIDelta -4s were used to produce corresponding fragments, which were then ligated at their EcoRV sites to generate Tbeta RIIDelta -4. It resulted in a deletion of four residues (Leu160, Leu161, Leu162, and Val163) in the transmembrane domain of the type II receptor (Fig. 1).

Cell Culture and Transient Transfection-- COS-1 cells were obtained from the American Type Culture Collection. Mutant mink lung epithelial (Mv1Lu) cells R1B and DR26 (10) were gifts from A. B. Roberts (National Institutes of Health). The cells were grown in a 5% CO2 atmosphere at 37 °C in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (CSL, Melbourne, Australia), 60 µg/ml penicillin, and 100 µg/ml streptomycin. Transient transfections were performed using a DEAE-dextran protocol (29), and transfected cells were assayed 48 or 72 h later.

Binding and Affinity Cross-linking-- 125I-TGF-beta was purchased from Amersham Corp. Binding and affinity cross-linking assays using bis(sulfosuccinimidyl) suberate (Pierce) were performed as described previously (22). Briefly, 2 days after transient transfection with TGF-beta receptor constructs, COS-1 cells in six-well plates were washed with binding buffer (phosphate-buffered saline containing 0.9 mM CaCl2, 0.49 mM MgCl2, and 1 mg/ml bovine serum albumin), and incubated on ice for 3 h with 0.4 µCi of 125I-TGF-beta /well in 200 µl of binding buffer. After incubation, the cells were washed with the binding buffer without bovine serum albumin and cross-linked with 0.5 ml of 0.28 mM bis(sulfosuccinimidyl) suberate (3) (in binding buffer without bovine serum albumin) for 15 min on ice. The cells were then washed with phosphate-buffered saline and lysed in 100 µl of lysis buffer consisting of 25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N',-tetraacetic acid, 10% glycerol, 1% Triton X-100, and 1.5% Trasylol (Bayer). The cell lysates were immunoprecipitated using anti-M2 FLAG antibody conjugated beads (IBI; Eastman Kodak Co.) followed by SDS-gel electrophoresis using 10% polyacrylmide and autoradiography.

Luciferase Assay-- The p3TP-Lux (10) TGF-beta -inducible luciferase reporter construct, containing a region of the human plasminogen activator inhibitor-1 gene promoter and three repeats of 12-O-tetradecanoylphorbol-13-acetate-responsive elements upstream of the luciferase gene (10), was obtained from A. B. Roberts. p3TP-Lux (6 µg) was co-transfected into mutant Mv1Lu cells together with 6 µg TGF-beta receptor construct(s). The cells in a 10-cm dish were divided into six wells in six-well plates 24 h after transfection. At 48 h post-transfection, the media were changed to Dulbecco's modified Eagle's medium, 0.2% bovine serum albumin, and three wells of each transfected cell line were stimulated with TGF-beta (2 ng/ml). Thereafter, cells were lysed in 100 µl/well lysis buffer and assayed for luciferase activity using the luciferase assay system (Promega). Cell lysates (20 µl/well) were used to measure the total light emission in 10 s using a luminometer (ML 3000 Microtiter Plate Luminometer). The rest of the cell lysates were directly analyzed by SDS-gel electrophoresis using 12% polyacrylamide followed by Western blotting. Anti-M2 monoclonal antibody was purchased from IBI-Kodak. Anti-HA3 polyclonal antibody was obtained from D. Bowtell (Peter MacCallum Cancer Institute, Melbourne).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of C-terminal Tagged TGF-beta Receptors and Receptor-Receptor Complex Formation-- In order to identify and monitor the expression of TGF-beta receptors, an octapeptide FLAG marker M2 was tagged to the C terminus Tbeta RI and Tbeta RI-II-I. A triple hemagglutinin epitope tag HA3 was fused to the C termini of Tbeta RII and Tbeta RII-I-II. The M2-tagged Tbeta RI(-M2) and Tbeta RI-II-I(-M2) receptors were transfected into COS-1 cells, and Western blot analysis using a monoclonal anti-M2 antibody showed that both were expressed (Fig. 2A). The HA3-tagged Tbeta RII, Tbeta RII-I-II, and Tbeta RIIDelta -1, Tbeta RIIDelta -2, Tbeta RIIDelta -3, and Tbeta RIIDelta -4 receptor cDNAs were transfected into Mv1Lu cells and analyzed using Western blotting by a polyclonal anti-HA3 antibody (Fig. 2B). The receptor cDNA constructs transfected into COS-1 cells were used to investigate the ligand binding properties of the wild-type and chimeric receptors. Binding of 125I-TGF-beta 1 was detected by affinity cross-linking followed by immunoprecipitation with anti-M2 antibody (Fig. 2C). Both the wild-type Tbeta RI and the transmembrane chimeric Tbeta RI-II-I bind TGF-beta in the presence of either the wild type Tbeta RII or the transmembrane chimeric Tbeta RII-I-II (Fig. 2C). The fact that the type II receptors (wild-type, chimeric, or transmembrane mutant) were present after immunoprecipitation with anti-M2 (type I receptor) antibody confirmed the formation of type I-type II complexes. These results indicate that the tagged wild-type, chimeric, and transmembrane mutated receptors are transported to the cell surface, bind TGF-beta , and form receptor complexes.


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Fig. 2.   Expression and ligand binding properties of wild-type, chimeric, and transmembrane deletion TGF-beta receptors. A, detection of expression of the wild-type and the transmembrane chimeric type I receptors in COS-1 cells by Western blotting. Tbeta RI(-M2) and Tbeta RI- II-I(-M2) were transfected into COS-1 cells using the DEAE-dextran method and lysed 48 h later. The cell lysates were subjected to SDS-gel electrophoresis, transferred onto nitrocellulose membrane, and Western blotted (29) using a monoclonal anti-M2 antibody. B, detection of expression of wild-type, chimeric, and transmembrane residue de- letion type II receptors in Mv1Lu cells by Western blotting with a polyclonal anti-HA3 antibody. C, 125I-TGF-beta cross-linking to receptor. As described under "Experimental Procedures," COS-1 cells were transfected with receptor constructs, and 125I-TGF-beta was added and cross-linked to receptors using bis(sulfosuccinimidyl) suberate. Cell lysates were immunoprecipitated using anti-M2 FLAG antibody-conjugated beads followed by SDS-gel electrophoresis using 10% polyacrylmide and autoradiography.

Tbeta RI(-M2) and Tbeta RII(-HA3) Are Functional TGF-beta Receptors-- Activation of the plasminogen activator inhibitor promoter has been frequently used as a marker for TGF-beta signaling activity (10). A reporter gene construct, p3TP-Lux (16), in which the plasminogen activator inhibitor-1 promoter drives expression of luciferase, is cotransfected with the TGF-beta receptor into mutant Mv1Lu cells. The luciferase activity was used as a measure of the activation of TGF-beta receptor. As shown above, Tbeta RI(-M2) and Tbeta RII(-HA3) can be successfully expressed in COS-1 and Mv1Lu cells and form heteromeric receptor complexes with TGF-beta . It was necessary to first confirm that the M2 and HA3 tags did not interfere with the receptor functions. When Tbeta RI(-M2) was transfected into mutant Mv1Lu cells (R1B), which lack functional Tbeta RI and do not normally respond to TGF-beta stimulation, the p3TP-Lux transcriptional response to TGF-beta was restored (Fig. 3A). In DR26 cells, which are mutant Mv1Lu cells lacking functional Tbeta RII and therefore cannot transduce TGF-beta induced signals, transfection of Tbeta RII(-HA3) restored TGF-beta responsiveness (Fig. 3B). These results demonstrate that the C-terminal tags, i.e. M2 and HA3, do not interfere the function of the TGF-beta receptors.


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Fig. 3.   Signaling from wild-type and chimeric TGF-beta receptors. cDNA constructs for TGF-beta receptors and p3TP-Lux were transiently transfected into mutant Mv1Lu R1B cells (A), which lack functional Tbeta RI, or DR26 cells (B), which contain a truncated, nonfunctional Tbeta RII. Luciferase activity was assayed as described under "Experimental Procedures." The results are representative of three separate experiments. C, Western blots of the expression levels of Tbeta RII and Tbeta RII-I-II in DR26 cells using the same cell lysates for luciferase assay in B.

Tbeta RI-II-I Restores TGF-beta Responsiveness in R1B Cells, but Tbeta RII-I-II Cannot Functionally Substitute for Tbeta RII in DR26 Cells-- Tbeta RI-II-I expression restored TGF-beta responsiveness in R1B cells. However, the degree of TGF-beta -stimulated luciferase activity was less than that resulting from transfection of the wild-type receptor Tbeta RI (Fig. 3A). In contrast, expression of Tbeta RII-I-II in DR26 cells did not restore the TGF-beta responsiveness, whereas the expression of the wild-type Tbeta RII did restore TGF-beta responsiveness (Fig. 3B). The expression level of the transmembrane chimeric Tbeta RII-I-II was similar to the wild-type Tbeta RII (Fig. 3C) in DR26 cells. Interestingly, transfection of particularly Tbeta RI, but also Tbeta RII, into DR26 cells increased the basal level of luciferase activity significantly (2.6-fold; Fig. 3B).

Expression of both the Wild-type Tbeta RI and Tbeta RII Results in a Low Level of Constitutive Activation, while Expression of both Transmembrane Chimeras Tbeta RI-II-I and Tbeta RII-I-II Results in a Very High Level of Constitutive Activity-- Expression of Tbeta RI or Tbeta RII in DR26 cells resulted in some constitutive activation of the p3TP-Lux reporter in the absence of ligand (Fig. 3B). To further evaluate the effect of receptor overexpression on TGF-beta receptor activation, Tbeta RI and Tbeta RII were cotransfected into R1B cells. This contransfection resulted in not only the restoration of TGF-beta responsiveness in the cells but also an elevated basal level of activity in the absence of TGF-beta (Fig. 4A), indicating some level of constitutive receptor activation following overexpression. To investigate whether expression of transmembrane chimeric receptors results in a change in this constititutive activation, Tbeta RI-II-I and Tbeta RII-I-II were cotransfected into R1B cells. A significantly high basal level/constitutive activity was observed (Fig. 4A). The activity marginally increased upon TGF-beta stimulation. The level of the constitutive activity was compatible with TGF-beta stimulation of Tbeta RI-R1B cells (Fig. 3A). Given that Tbeta RI-II-I alone restored TGF-beta responsiveness to only ~25% and Tbeta RII-I-II failed to restore the TGF-beta responsiveness in DR26 cells (Fig. 3A), the high level of ligand-independent p3TP-Lux activation suggests that the receptor transmembrane domain plays a significant role in receptor activation. Transfection of both chimeric receptor constructs into DR26 cells also resulted in a high level of constitutive activity, confirming the results obtained in R1B cells (Fig. 4B).


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Fig. 4.   Constitutive activation of wild-type and chimeric TGF-beta receptors. cDNA constructs for TGF-beta receptors and p3TP-Lux were transiently transfected into R1B cells (A) or DR26 cells (B). Luciferase activities were assayed as described under "Experimental Procedures." The results are representative of at least three separate experiments. C, Western blot of the expression levels of Tbeta RII and Tbeta RII-I-II in DR26 cells using the same cell lysates for luciferase assay in B.

Expression of Tbeta RI with Tbeta RII-I-II Results in a High Level of Constitutive Activity, but Expression of Tbeta RI-II-I with Tbeta RII Gives Low Level Constitutive Activity-- Given that expression of both wild-type receptors Tbeta RI and Tbeta RII results in a low level receptor constitutive activity while expression of both transmembrane chimeric receptors Tbeta RI-II-I and Tbeta RII-I-II gives rise to a very high constitutive activity (Fig. 4), we undertook further experiments to clarify the function of transmembrane domain on receptor activation. Cotransfection of the wild-type Tbeta RI and the transmembrane chimeric Tbeta RII-I-II in R1B cells gave rise to a relatively high constitutive activity (Fig. 4A), which was higher than that due to Tbeta RI/Tbeta RII but lower than that following Tbeta RI-II-I/Tbeta RII-I-II cotransfection. The level of activation was not further enhanced by the addition of TGF-beta , although the wild-type Tbeta RI was transfected, perhaps because the constitutive activity was already close to a maximal level of receptor (type I) activation. The high constitutive activity resulting from cotransfection of Tbeta RI and Tbeta RII-I-II also suggests that the transmembrane chimeric receptor Tbeta RII-I-II is as capable of signaling as the wild-type type II receptor Tbeta RII. When the transmembrane chimeric Tbeta RI-II-I and the wild-type Tbeta RII were cotransfected into R1B cells, a low constitutive activity was observed (compatible with Tbeta RI/Tbeta RII constitutive activity) (Fig. 4A). Little TGF-beta stimulation was observed, only to a level similar to that obtained by ligand stimulation following Tbeta RI-II-I transfected in R1B cells. Similar results were obtained when DR26 (Fig. 4B) or the wild-type Mv1Lu (data not shown) cells were used, except that in Mv1Lu cells the p3TP-Lux activity was slightly stimulated by TGF-beta , probably due to the cells expressing wild-type receptors. Western blotting analysis (Fig. 4C) shows that the expression level of type II receptors and chimeras was similar in all experiments.

Receptor Complex Formation Parallels Receptor Complex Activation-- To investigate the correlation between TGF-beta receptor complex formation and activation, various combinations of receptor constructs were transfected into COS cells. Similar levels of expression of the wild-type or chimeric receptors were achieved (Fig. 5, B and C). In co-immunoprecipitation experiments, immunoprecipitation with anti-M2 (type I receptor) antibody and the Western blotting with anti-HA3 (type II receptor) antibody demonstrated that the receptor complex formation exactly paralleled the high level of ligand-independent constitutive receptor activation (Fig. 5A). These results demonstrate that by simply changing the transmembrane regions, a drastic effect is observed on receptor complex formation.


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Fig. 5.   Receptor-receptor complex formation. Indicated cDNA constructs were transfected into COS-1 cells using the DEAE-dextran method, and the cells were lysed 72 h later. The cell lysates were then immunoprecipitated using anti-M2 FLAG antibody-conjugated beads. The precipitates were subjected to SDS-gel electrophoresis using 10% polyacrylmide, transferred onto nitrocellulose membrane, and Western blotted using a polyclonal anti-HA3 antibody (A), without immunoprecipitation (B), and without immunoprecipitation, blotted with a monoclonal anti-M2 antibody (C).

Tbeta RIIDelta -1 Is Almost Inactive, Tbeta RIIDelta -2 and Tbeta RIIDelta -3 Are Partially Active, and Tbeta RIIDelta -4 Is Fully Active in DR26 Cells-- As demonstrated above, exchanging the transmembrane domains of the type I and II receptors had a dramatic impact on the activities of the receptor. To further identify the importance of the transmembrane domain in receptor activation, the signaling activity of type II transmembrane deletion mutants was examined in DR26 cells. Expression of Tbeta RIIDelta -1, which has a Leu160 deletion in the transmembrane domain of the type II receptor, restored very little of the TGF-beta responsiveness in DR26 cells (Fig. 6A). This observation is similar to the expression of Tbeta RII-I-II. However, transfection of the cDNAs of the type II receptor containing a two- or three-residue deletion in the transmembrane domain (Tbeta RIIDelta -2 and Tbeta RIIDelta -3) restored about half of the wild-type Tbeta RII activity (Fig. 5A). Moreover, the expression of the four-residue deletion type II receptor, Tbeta RIIDelta -4, fully restored the activity of the wild-type receptor (Fig. 5A). Western blotting analysis (Fig. 6B) demonstrates similar expression levels of the wild-type and the mutant type II receptors, and all were found to bind TGF-beta (Fig. 2C).


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Fig. 6.   Signaling activity of type II receptors with transmembrane residue deletion in DR26 cells. A, cDNA constructs for TGF-beta receptors and p3TP-Lux were transiently transfected into DR26 cells. Luciferase activities were assayed as described under "Experimental Procedures." The results are representative of four separate experiments. B, Western blot of the expression levels of Tbeta RII and type II transmembrane residue deletion mutants in DR26 cells using the same cell lysates for luciferase assay in A.

Expression of Tbeta RIIDelta -1 with Tbeta RI or Tbeta RI-II-I Resulted in a High Constitutive Activation-- As shown earlier, Tbeta RII-I-II was inactive in DR26 cells (Fig. 3B) but exhibited high levels of constitutive activation when co-expressed with Tbeta RI or Tbeta RI-II-I (Fig. 5, A and B). To investigate whether Tbeta RIIDelta -1 and Tbeta RII-I-II share other signaling properties, the transcriptional activation of p3TP-Lux was examined following the co-expression of Tbeta RIIDelta -1 with either Tbeta RI or Tbeta RI-II-I. In DR26 cells, a very high ligand-independent activation was obtained when Tbeta RIIDelta -1 was co-expressed with the wild-type Tbeta RI, while only a moderate activation was exhibited by Tbeta RIIDelta -2, Tbeta RIIDelta -3, or Tbeta RIIDelta -4 with Tbeta RI (Fig. 7A). Similarly, co-expression of Tbeta RIIDelta -1 and Tbeta RI-II-I in DR26 cells also resulted in a high constitutive activation. However, Tbeta RIIDelta -2 with Tbeta RI-II-I showed little constitutive activation, while Tbeta RIIDelta -3 or Tbeta RIIDelta -4 with Tbeta RI-II-I resulted in a moderate activation (Fig. 7B). This trend of constitutive activation was also obtained in R1B cells (data not shown). These results demonstrated that the relative constitutive activity among the type II transmembrane residue deletion mutants differs from their TGF-beta -induced activity, and more importantly, Tbeta RIIDelta -1 and Tbeta RII-I-II share similar signaling activity.


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Fig. 7.   Constitutive activation of type II receptors with transmembrane residue deletion in DR26 cells. cDNA constructs for TGF-beta type II receptors and p3TP-Lux were transiently transfected into DR26 cells together with the wild-type Tbeta RI (A) or the transmembrane chimeric Tbeta RI-II-I (B). Luciferase activities were assayed as described under "Experimental Procedures." The results are representative of at least three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies on TGF-beta signaling have suggested a clearly postulated mechanism of TGF-beta receptor activation (17-21). TGF-beta binds directly to the type II receptor, which is a constitutively active kinase and is autophosphorylated. The TGF-beta /type II receptor then recruits and phosphorylates the type I receptor, which is activated and signals to downstream substrates (21). This mechanism is consistent with a model of heteromeric association of the type I and II receptors that follows TGF-beta binding. Subsequent double immunoprecipitation and yeast two-hybrid studies have shown that the type I and II receptors form a heteromeric complex even in the absence of TGF-beta (25). The formation of a homomeric type II-type II complex has also been detected (23). Interactions between Tbeta RI and Tbeta RII or between Tbeta RII and Tbeta RII occur between both the extracellular domains and the intracelluar domains without ligand stimulation (23, 24). Given that the receptors preexist in a heteromeric complex but only are activated by TGF-beta binding, the ligand may perform functions in addition to simply bringing the receptors together. We hypothesize that the binding of ligand to the extracellular domains of the type I and type II receptors also results in specific changes in the intracellular region of the receptor complex that lead to signaling. Such changes are transmitted from the extracellular to the intracellular region of the receptor through the transmembrane domains. Indeed the fact that Tbeta R2.1, a chimeric receptor containing the extracellular and the transmembrane domains of the type II receptor and the intracellular domain of the type I receptor, fails to reconstitute signaling despite the fact that the wild-type type II receptor heterodimerizes with it (26) confirms that receptor heterooligomerization is not sufficient for TGF-beta signaling. The data presented in our study establish that by simply changing the hydrophobic transmembrane domain, the signaling ability of the receptor is radically altered.

Expression of the wild-type Tbeta RI restores the TGF-beta responsiveness in R1B cells (Ref. 10; Fig. 3A). Changing the transmembrane domain of Tbeta RI from type I to type II reduces the signaling activity of the receptor to ~25% (Fig. 3A). Expression of Tbeta RII restores the TGF-beta responsiveness in DR26 cells (10, Fig. 3B). Simply changing the Tbeta RII transmembrane domain from type II to type I all but abolishes the signaling activity of the receptor (Fig. 3B) despite high levels of expression, complex formation, and ligand binding (Fig. 2, B and C). Our results further suggest that the transmembrane domains play an important role in receptor activation. Overexpression of Tbeta RI and Tbeta RII in cells results in some ligand-independent constitutive activation of the receptor in our studies (Fig. 4, A and B) and works by others (30). However, by simply swapping the transmembrane domains of Tbeta RI and Tbeta RII we see a marked increase in constitutive activity to a maximal level (Fig. 4, A and B). A slightly lower level of activation is seen if both receptors have type I transmembrane domains, while if both receptors have the type II transmembrane domains only a modest constitutive activation is seen (Fig. 4, A and B). This suggests that the transmembrane domain may mediate a change in the Tbeta RI-II-I·Tbeta RII-I-II or Tbeta RI·Tbeta RII-I-II receptor complex resulting in activation and that this change is similar to that induced by ligand binding to the wild-type receptor.

The type I receptor consists of 22 amino acids in its transmembrane region (Leu126-Ile147) (11), and the type II transmembrane region contains 30 amino acids (Leu160-Tyr189) (12). Given that the transmembrane domain folds in a predicted alpha -helical structure (29), exchanging the type I and type II transmembrane domains has two consequences: (i) increasing the distance between the extracellular and intracellular domains of the type I receptor but decreasing the distance between these domains in the type II receptor and (ii) altering the relative orientation between the extracellular and intracellular domains about 100° in the type I receptor in one direction but 100° in the other direction in the type II receptor. If the signaling incompetence of the type II transmembrane chimeric receptor Tbeta RII-I-II in DR26 cells (Fig. 3B) is due to the shortening of the distance between the extracellular and the intracellular domains or if the low activity of the type I transmembrane chimeric receptor Tbeta RI-II-I in R1B cells (Fig. 3A) is due to the lengthening of the distance between the extracellular and intracellular domains, then coexpression of Tbeta RI-II-I and Tbeta RII-I-II would not be expected to have any TGF-beta signaling activity. However, coexpression of Tbeta RI-II-I and Tbeta RII-I-II results in a very high constitutive activation of the receptors in either R1B or DR26 cells (Fig. 4, A and B), indicating that the distance between the extracellular and intracellular domains may not be critical for achieving receptor activation. Perhaps the relative orientation between the extracellular and intracellular domains in the receptors and the relative orientation between the Tbeta RI and Tbeta RII receptors in the receptor complexes are more important. We propose that TGF-beta binding to the preexisting receptor complex results in relative rotation between the extracellular domains of the type I and type II receptors, which, through the transmembrane domains, results in a change in orientation between the Tbeta RI and Tbeta RII intracellular domains. This relative reorientation of the intracellular kinase domains results in a productive interaction between the type I and type II kinase (Fig. 8).


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Fig. 8.   A general model for TGF-beta receptor activation via relative rotation. TGF-beta receptors form a receptor complex in the absence of ligand. To the wild-type receptor complex, TGF-beta binds to the receptor extracellular domains, resulting in relative rotation between receptors and therefore the receptor activation and signaling. Exchanging the type I and type II receptor transmembrane domains in the receptors results in relative rotation between the extracellular and intracellular domains in the receptor. The interaction between extracellular domains brings the cytoplasmic domains of the transmembrane chimeric receptors into a productive interaction orientation, leading to constitutive activation and signaling in the absence of the ligand.

By changing the transmembrane domains, the resulting reorientation between the type I and II intracellular domains may be similar to that normally induced by TGF-beta resulting in receptor activation. Hence, coexpression of Tbeta RI-II-I and Tbeta RII-I-II or Tbeta RI and Tbeta RII-I-II results in constitutive activity in the absence of ligand (Fig. 4, A and B). These data suggest that a 100-200° (approximately) reorientation between the kinase domains of Tbeta RI and Tbeta RII results in activation. Since the constitutive activity resulting from coexpression of Tbeta RI with Tbeta RII-I-II is similar to that induced by ligand when Tbeta RI and Tbeta RII are expressed in cells (Fig. 4, A and B), we postulate that TGF-beta may induce approximately 100° of relative rotation between the type I and type II kinase domains in the wild-type receptor complex. The addition of TGF-beta to the Tbeta RI·Tbeta RII-I-II complex has no effect on the activation level (Fig. 4, A and B) possibly because the complex has assumed a rotated confirmation and ligand is no longer capable of inducing further rotation. Introducing the type II transmembrane domain to Tbeta RI results in 100° (approximately) rotation in the opposite direction. The kinase domains may then be in a less favorable orientation in the resulting Tbeta RI-II-I·Tbeta RII complex, and therefore little constitutive activation is seen (Fig. 4, A and B). Ligand binding to the Tbeta RI-II-I·Tbeta RII receptor complex changes the orientation between the kinase domains to provide some receptor activation but less than that following ligand binding to the wild-type receptor complex (Fig. 3A).

Since the type I and type II extracellular domains interact with each other in the absence of ligand, it is possible that they may provide a constraint preventing the receptor intracellular kinase domains from interacting in an active conformation in the preexisting wild-type receptor complex. In support of this possibility, a recent paper suggests that overexpression of the extracellular domain-truncated type I and type II receptors results in a high level of ligand-independent receptor activation and signaling (30). Further consistent with this is the supposition that the extracellular interactions work against the intracellular kinase interactions in the receptor complex. Ligand binding to the receptor complex may change the orientation of the receptor-receptor interactions in the extracellular domain, which in turn results in more favorable, or even enhanced, interactions between the kinase domains (activation). In the Tbeta RI-II-I·Tbeta RII-I-II and Tbeta RI·Tbeta RII-I-II complexes, the extracellular interactions strengthen the kinase interactions in the absence of ligand, resulting in constitutive receptor activation. Accordingly, the interactions in the Tbeta RI-II-I·Tbeta RII-I-II and Tbeta RI·Tbeta RII-I-II complexes are expected to be stronger than those in the Tbeta RI·Tbeta RII and Tbeta RI-II-I·Tbeta RII complexes. The immunoprecipitation and Western blotting results in Fig. 5 confirm the above prediction. Furthermore, this result demonstrates an important role played by the receptor transmembrane domains in the formation of receptor complex.

To further elucidate the critical role of the transmembrane domain in receptor activation, we constructed four TGF-beta type II receptor mutants with consecutive residue deletions in the transmembrane domain (Fig. 1). Tbeta RIIDelta -1 has a deletion of Leu160; Tbeta RIIDelta -2 has a deletion of Leu160 and Leu161; Tbeta RIIDelta -3 has a deletion of Leu160, Leu161, and Leu162; and Tbeta RIIDelta -4 has a deletion of Leu160, Leu161, Leu162, and Val163. Since the transmembrane domain is alpha -helical and 3.4-3.7 residues constitute one alpha -helical turn, each residue deletion in the transmembrane domain may result in approximately 100° of relative rotation between the extracellular and intracellular domains. A deletion of four residues in the transmembrane region should return the receptor close to the original wild-type orientation. As described before, changing the transmembrane domain of type II receptor to type I receptor may result in 100° relative rotation. Therefore, the relative orientations between the extracellular and the intracellular domains of Tbeta RII-I-II and Tbeta RIIDelta -1 are similar. Expression of the Tbeta RII one-residue deletion construct Tbeta RIIDelta -1 in DR26 cells failed to restore TGF-beta -induced plasminogen activator inhibitor-1 activation as assayed by luciferase activity using p3TP-Lux reporter construct (Fig. 5A), similar to the results obtained by expression of Tbeta RII-I-II (Fig. 3B). However, expression of a transmembrane two- or three-residue deletion construct (Tbeta RIIDelta -2 or Tbeta RIIDelta -3) resulted in some restoration of TGF-beta responsiveness in DR26 cells. More importantly, the four-residue transmembrane deletion receptor Tbeta RIIDelta -4 fully restored the TGF-beta responsiveness (Fig. 6A) in DR26 cells. This would be predicted, since Tbeta RIIDelta -4 retains a relative orientation close to that of the wild-type Tbeta RII. These results demonstrate a pivotal role for the transmembrane domain in receptor activation, and further support our model (Fig. 8) in which relative orientations between the extracellular and intracellular receptor domains and hence of the receptor kinase domains are critical in activation of the TGF-beta receptor complex.

The TGF-beta -induced signaling activity of both Tbeta RII-I-II and Tbeta RIIDelta -1 has been shown to be severely impaired (Figs. 3B and 6A). The constitutive activation observed when Tbeta RII-I-II was co-expressed with Tbeta RI or Tbeta RI-II-I (Fig. 4, A and B) indicates that the transmembrane chimeric receptor Tbeta RII-I-II can be as potent as the wild-type Tbeta RII. It is expected that the one-transmembrane residue deletion mutant Tbeta RIIDelta -1 exhibits similar signaling potency, since the relative orientations between the extracellular and intracellular domains of Tbeta RII-I-II and Tbeta RIIDelta -1 are expected to be similar. The constitutive activation resulting from cotransfection of Tbeta RIIDelta -1 with Tbeta RI or Tbeta RI-II-I (Fig. 7, A and B) meets this expectation. It should be noted that the trend of TGF-beta -induced activity exhibited in Fig. 6A among the type II receptors differs dramatically from the trend of the constitutive activity observed when cotransfected with the wild-type Tbeta RI (Fig. 7A). In addition, these trends are different from the one of constitutive activity exhibited in Fig. 7B when the transmembrane chimeric Tbeta RI-II-I was contransfected. The orientation of the type I intracellular domain (relative to the type II receptors) in each case differs from the other. These results provide further evidence supporting our conclusion that the relative orientation between receptors determines whether the receptor complex is activated or not.

The type I and the type II receptors for the TGF-beta superfamily are related, with high conservation in the kinase domain (3). Since the transmembrane domains play a pivotal role in TGF-beta receptor activation as demonstrated above, it is instructive to compare the length and primary sequence of the domain in the superfamily. Using a BLAST program (31), the transmembrane sequences of the TGF-beta receptor superfamily were retrieved and aligned (Fig. 9). The length and the primary sequence of the domain vary between subfamily receptors. However, the length within each subfamily is strictly conserved, although the primary sequence may not be. The conservation of the length of the transmembrane domain within each subfamily may contribute to the ligand-specific activation of the receptor complex.


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Fig. 9.   Sequence alignment of the transmembrane domain of TGF-beta receptor superfamily. The sequences were retrieved and aligned using a BLAST program (31). The transmembrane residues are highlighted. Five residues before and after the domain in the type I receptors are included as well as three residues in the type II. BMPR, BMP receptor; ActR, activin receptor; ALK, activin receptor-like kinase; k, mink; b, bovine; h, human; r, rat; m, mouse; c, chicken; x, Xenopus; f, fugu rubripes; z, zebrafish.

In conclusion, we have demonstrated that the transmembrane region of the TGF-beta receptors is not just a passive hydrophobic membrane anchor but is critically important in receptor association and activation. We propose that TGF-beta activates the type I and II receptor complex through induction of relative rotation between the receptors (Fig. 8). We are currently analyzing various mutants of the receptor transmembrane regions in order to gain even further insights into the regulatory role played by the transmembrane region of the TGF-beta receptor and indeed how generally this mechanism may be applied in the activation of other transmembrane receptors.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. Tel: 61-3-93413155; Fax: 61-3-93413104; E-mail: Hong.Jian.Zhu{at}ludwid.edu.au.

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; Tbeta R, TGF-beta receptor; GM-CSF, granulocyte/macrophage colony-stimulating factor; Epo, erythropoietin; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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