Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

doi:10.1016/j.jmb.2005.10.014

J. Mol. Biol. (2005) 354, 1052–1068

Assembly of TbRI:TbRII:TGFb Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent Jorge E. Zu´n˜iga1†, Jay C. Groppe1†, Yumin Cui1, Cynthia S. Hinck1 Vero´nica Contreras-Shannon 1, Olga N. Pakhomova1, Junhua Yang2 Yuping Tang2, Valentı´n Mendoza3, Fernando Lo´pez-Casillas3 LuZhe Sun2 and Andrew P. Hinck1* 1

Department of Biochemistry University of Texas Health Science Center at San Antonio San Antonio, TX 78229-3900 USA 2

Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio TX 78229-3900, USA 3

Instituto de Fisiologı´a Celular Universidad Nacional Auto´noma de Me´xico, Ciudad de Me´xico, Me´xico

Transforming growth factor-b (TGFb) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbRI) and type II (TbRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbs 1, 3) must first bind TbRII to recruit TbRI into the complex, and a third (TGFb2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbRI (TbRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbRII (TbRII-ED) and TGFbs 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbRI requires combined interactions with TbRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbRI-ED, like TbRII-ED, acts as an antagonist of TGFb signaling, indicating that receptor–receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbRII-ED, nor TbRII-ED and TbRI-ED combined, form a complex with TGFb2, showing that receptor–receptor interaction is insufficient to compensate for weak ligand–receptor interaction. However, TbRII-ED does bind with high affinity to TGFb2-TM, a TGFb2 variant substituted at three positions to mimic TGFbs 1 and 3 at the TbRII binding interface. This proves both necessary and sufficient for recruitment of TbRI-ED, suggesting that the three different TGFb isoforms induce assembly of the heterotetrameric receptor complex in the same general manner. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: TGF-beta; TGF-beta type I receptor; TGF-beta type II receptor; TbRI, TbRII cooperative assembly; NMR

Introduction † J.E.Z. & J.C.G. contributed equally to this work. Present addresses: Y. Cui, Hutchinson Medipharma Ltd., Shanghai, China; V. Contreras-Shannon, Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA. Abbreviations used: TGFb, transforming growth factor-b; ED, extracellular domain; BMP, bone morphogenetic protein; FBHE, fetal bovine heart endothelial. E-mail address of the corresponding author: [email protected]

Transforming growth factor beta (TGFb) isoforms regulate cell proliferation, cell differentiation, and expression of extracellular matrix proteins, and are the founding members of a highly diversified superfamily of w25 kDa homodimeric signal ligands.1 These isoforms, and other structurally related proteins of the TGFb superfamily, such as activins, bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), exert their biological effects by binding and bringing together two pairs of structurally similar, single-pass

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

transmembrane receptors, classified as types I and II.2 The ligand-mediated assembly of these two receptor types triggers an intracellular phosphorylation cascade initiated by the serine-threonine kinase domain of the type II receptor, which transphosphorylates the adjacent type I kinase.3 The type I kinase in turn phosphorylates the nucleartranslocating Smad proteins, which, together with various transcriptional coactivators and corepressors,4 regulate transcription of target genes. Three TGFb isoforms (TGFbs 1–3) have arisen in mammals. They share 70–82% sequence identity and are encoded by distinct genes that are expressed in developmentally regulated and tissue-specific fashions.1 Although the phenotypes of isoform-specific null mice are non-overlapping,5–7 indicative of distinct temporal-spatial roles in vivo, the ligand isoforms exhibit overlapping biological activities in tissue culture assays. However, TGFb2 differs from the other two isoforms, in that it binds the signaling receptors in cell-based affinity labeling experiments approximately 10–20-fold more weakly and requires a co-receptor (betaglycan) for comparable levels of cellular response.8,9 In addition to this isoform-dependence, binding of TGFb ligands to their two types of signaling receptors is both sequential and cooperative. Affinity labeling of cell surface receptors with iodinated ligands has shown that TGFbs 1 and 3 bind the type II receptor (TbRII) with high affinity independent of type I (TbRI).10 In contrast, TbRI requires co-expression of TbRII to crosslink radiolabeled ligands efficiently.10,11 Thus, at least two of the isoforms, TGFbs 1 and 3, bind the receptor types sequentially, complexing with TbRII prior to the cooperative recruitment of TbRI into ternary complex. TGFb2 may also bind the two receptor types in this general manner, although less is known owing to the lower affinity of TbRII for this isoform.8 The cooperative nature of TGFb receptor assembly is likely independent of any interactions between the receptor cytoplasmic domains, as cell lines transfected with cytoplasmically truncated TbRII have been shown to bind ligands and recruit TbRI in the same cooperative manner as cells expressing full-length TbRII.12 Cell surface assembly of the TbRI:TbRII:TGFb complex has been shown with differentially tagged receptors to form with a stoichiometry of 2:2:1,13,14 which has been confirmed, in part, through the determination of the structure of the TbRII extracellular domain (TbRII-ED) bound to the TGFb3 homodimer.15 TbRII-ED binds wedge-like between the tips of the two extended fingers on each ligand monomer, protruding into the solvent from the distal ends of the dimer. This distal position precludes the possibility of contact between the receptor pair extracellularly. The structure of TbRII-ED-bound TGFb3 differs from the crystal structure of the free form reported earlier,16 both with respect to the relative orientation of its two monomers and with respect to the fact that both the palm and finger regions are visible in

1053 the structure of the free form, but not the bound form. These differences do not, however, appear to be connected in any way to TbRII-ED binding, as previous NMR studies17,18 have shown that free TGFb3 also adopts a state in which the finger regions are structurally ordered but the palm regions are not. At present, the TbRI extracellular domain (TbRI-ED) remains uncharacterized structurally, although based on the structure of the BMP type Ia receptor extracellular domain (BMPRIa-ED) bound to BMP-2,19 all type I receptors of the TGFb superfamily are proposed to bind their cognate ligands at the dimer interface, simultaneously contacting monomers in a mode similar to the pair of BMPRIa-EDs.20 Two disparate models have been proposed to account for the cooperativity in the TGFb system. In one, TbRI is recruited into the complex by binding a composite interface formed by both TGFb and TbRII-ED.15 This direct interaction model is supported by superposition of the TGFb and BMP ligands in the complex structures, which shows the binding sites for TbRII and BMPRIa as adjacent. In the other, binding of membrane-tethered TbRII induces the formation of the TbRI binding site on the ligand, which is assumed to be unstructured otherwise and incapable of binding TbRI.21 This indirect interaction model was prompted by the dynamic behavior of the monomers of TGFb3,17,18 which can adopt a non-canonical “open” arrangement and no longer pack against one another.15 Like the direct interaction model, this one is based on the assumption that TbRI and BMPRIa bind their ligands in similar ways. The objective of the studies reported here was to investigate the mechanism of assembly of the TGFb signaling complex by analyzing its formation in vitro with highly purified preparations of the extracellular domains of the receptors. Toward that end, it was first necessary to develop a means of producing a structurally homogeneous preparation of the extracellular domain of TbRI (TbRI-ED) to complement the TbRII extracellular domain (TbRIIED) already in hand.15 This was accomplished by refolding Escherichia coli-expressed, monomeric receptor and purifying it to homogeneity based on a native gel-binding assay with the TbRIIED:TGFb3 binary complex. The purified protein was shown to bind with high affinity and 2:1 stoichiometry to complexes of TbRII-ED and TGFbs 1 or 3, but undetectably with either TbRII-ED or the ligands alone. These in vitro studies indicate that recruitment of TbRI-ED requires combined interactions with TbRII-ED and ligand but not membrane attachment, suggesting that most or all of the cooperativity is derived through direct interaction, not indirect interaction. In keeping with the structural paradigm of the BMPRIa:BMP2 complex,19 the TbRI-TGFb interface appears to be comprised of both monomers of the ligand, as revealed by a monomeric form of TGFb3 complexed with TbRII-ED with diminished affinity for TbRI. In addition, we found that interaction between the extracellular domains of the two receptor types is

1054 not sufficiently strong to compensate for the weak interaction between TGFb2 and TbRII, since TbRIED and TbRII-ED combined failed to form a stable complex with this isoform. Nevertheless, TGFb2 appears to induce the assembly of the heterotetrameric complex in the same overall manner as TGFbs 1 and 3, as substitution at three positions to mimic TGFbs 1 and 3 at the TbRII binding interface allowed for high-affinity binding of TbRII-ED and cooperative recruitment of TbRI-ED.

Results Refolded TbRI-ED is cooperatively recruited into ternary complex The models proposed for assembly of the TGFb signaling complex15,21 are based upon results that have emerged from structural analyses of TGFb ligand alone17,18,22 and in complex with TbRII-ED.15 The differences between these models stem, in large part, from the current lack of knowledge regarding the manner by which TbRI binds and is cooperatively recruited into the complex. To investigate the mechanism directly, we sought to isolate a structurally homogeneous form of the TbRI-ED. Type I and type II receptor extracellular domains of the TGFb superfamily are small (100–140 residue) cysteine-rich domains. The four that have been characterized structurally thus far, ActRIIa,23 ActRIIb,21,24 TbRII,25–27 and BMPRIa,19 have been shown to adopt a three finger toxin fold and to share four structurally conserved disulfide bonds. TbRI-ED, which has a predicted extracellular domain 101 residues in length,11 is expected to adopt a similar structure as its ten cysteine residues are positionally conserved relative to those in BMPRIa (Figure 1). Three of these four receptor extracellular domains have been produced by E. coli expression. BMPRIa-ED28,29 and ActRIIb-ED21 were expressed as thioredoxin fusions that remained soluble and yielded active protein upon purification. TbRII-ED has been expressed both fused to thioredoxin25 and alone.30,31 The fusion protein remained soluble and yielded active protein,25 while TbRII-ED expressed alone was insoluble but could be refolded to yield active protein.30,31

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

To prepare TbRI-ED, we tested the two approaches described above. The thioredoxin fusion protein remained soluble, although in contrast to the related receptor extracellular domains produced by this method, TbRI-ED accumulated mainly in the form of disulfide-linked oligomers. Thus, the alternative strategy of refolding was pursued by expression of TbRI-ED with an N-terminal hexahistidine tag. This form of the protein, which predictably formed inclusion bodies, was resolubilized in urea, enriched by NiNTA affinity chromatography and refolded in nondenaturing buffer in the presence of glutathione redox couple at pH 8.0. The protein was isolated from the folding mixture by NiNTA affinity chromatography, eluted with an imidazole gradient to enrich for monomeric species and concentrated for activity assay. The binding activity of the enriched, refolded protein preparation was analyzed by native polyacrylamide gel electrophoresis. At the pH of this buffer system, TbRII-ED migrated near the dye front, whereas TGFb3 failed to enter the resolving gel (Figure 2(a), lanes 1 and 2). Combining TbRIIED with TGFb3 led to the appearance of a complex band with an intermediate migration rate (lanes 3–8). The intensity of the complex band increased as the receptor/ligand ratio approached 2:1, consistent with the established stoichiometry of the TbRIIED:TGFb3 binary complex.13–15 The addition of excess refolded TbRI-ED to the TbRII-ED:TGFb3 complex (Figure 2(b), lane 7) resulted in a discrete new species that migrated more slowly than the binary complex. This was accompanied by the consumption of binary complex, as the band corresponding to this species was of lower intensity in the presence of TbRI-ED (lane 7) than in its absence (lane 6). This production of a likely more massive species, together with the apparent consumption of binary complex, strongly suggested that an active component of the refolded TbRI-ED preparation was recruited by the binary complex to form the TbRI-ED:TbRII-ED:TGFb3 ternary complex. As anticipated, the patterns observed after refolded TbRI-ED was mixed with either TGFb3 or TbRII-ED alone were simply the sum of the individual components (lanes 3 and 5, respectively), indicating that the interactions between

Figure 1. Sequence alignment of the extracellular domains of TbRI and BMPRIa. Boundaries of the TbRI and BMPRIa extracellular domains were defined by the predicted termini of the flanking signal peptide and transmembrane segments.11,52 The five disulfide linkages depicted were determined from the crystal structure of BMPRIa in complex with BMP2,19 as well as the boundary of the folded core (black) and flanking disordered (grey) residues. The extracellular domain of TbRI likely shares a common folded core with BMPRIa, flanked by only five or six disordered residues at each end. Numbering is based on the mature N termini, not the initiator methionine of the signal peptides.

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

1055

Figure 2. TbRI-ED is cooperatively recruited into ternary complex. (a) TbRII-ED:TGFb3 binary complex (BC) was formed by adding 0.5–3 molar equivalents of TbRII-ED (lanes 3–8) to a molar equivalent of TGFb3, then electrophoresed through a native 12% polyacrylamide gel and stained with Coomassie brilliant blue (1.50 mg/eq. TGFb3, 0.90 mg/eq. TbRII-ED). (b) TbRI-ED:TbRII-ED:TGFb3 ternary complex (TC, lane 7) was formed by adding ten equivalents of refolded TbRI-ED (std, lane 2) to the 2:1 TbRII-ED:TGFb3 binary complex (BC std, lane 6) and analyzed as above (1.50 mg/eq. TGFb3, 0.90 mg/eq. TbRII-ED, 0.66 mg/eq. TbRI-ED). (c) Composition of putative binary and ternary complexes with purified TGFb3 and TbRII-ED and refolded TbRI-ED. After staining, protein complexes were dissected from the native gel shown in (b), soaked in SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Lanes 4 and 5 contain the binary (BC) and ternary (TC) complexes. Lanes 1, 2, and 3 contain TbRI-ED, TGFb3, and TbRII-ED standards, respectively, in relative molar equivalents as above. (d) Titration of the 2:1 TbRII-ED:TGFb3 complex (1.80 mg TbRII-ED, 1.5 mg TGFb3) with 1 ml increments of HPLC-purified TbRI-ED (lanes 2–6) to produce the TbRI-ED:TbRII-ED:TGFb3 ternary complex. Lane 1, BC control. After removal of the N-terminal histine tag by thrombin digestion, free TbRI-ED migrated near the dye front, faster than the uncleaved form analyzed after refolding (cf. (b), lane 2). (e) Composition of binary and ternary complexes formed with purified TGFb3, TbRII-ED, and TbRI-ED. After staining, binary complex from lane 1 and ternary complex from lane 4 were dissected from the native gel shown in (d), soaked in SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Lanes 4 and 5 contain the binary (BC) and ternary (TC) complexes. Lanes 1, 2, and 3 contain TbRI-ED, TGFb3, and TbRII-ED standards, respectively, in relative molar equivalents as above.

TbRI-ED and ligand or the extracellular domains of the receptors were indeed too weak to detect by electrophoresis. The composition of the binary and ternary complexes was confirmed by first dissecting the stained bands of protein from a native gel. These were then soaked in SDS sample buffer and the protein components re-electrophoresed on an SDS/ polyacrylamide gel. The protein band containing the binary complex (Figure 2(b), lane 6, BC) was composed of two species that co-migrated with TbRII-ED and TGFb3 standards (Figure 2(c), lane 4). The other one containing putative ternary complex (Figure 2(b), lane 7, TC) consisted of three proteins

that co-migrated with TbRI-ED, TbRII-ED and TGFb3 standards (Figure 2(c), lane 5). Thus, although not structurally homogeneous, the TbRI-ED preparation contained an active component that could be recruited by the binary complex, resulting in the formation of a stable TbRIED:TbRII-ED:TGFb3 ternary complex. In addition, the active TbRI-ED component was not able to interact stably with either TbRII-ED or TGFb3 alone. These in vitro findings, that TbRII-ED binds TGFb3 in a TbRI-ED-independent manner, while TbRI-ED requires TbRII-ED to be pre-bound, mirror observations from cell-based cross-linking experiments in the TGFb system.10,11 This indicates that the

1056 ligand-mediated assembly of TbRI-ED and TbRIIED occurs in the same overall manner as that of the cell-surface TGFb receptors. Purification of refolded TbRI-ED The ability to detect formation of the TbRIED:TbRII-ED:TGFb3 complex by native gel assay was a critical step that enabled subsequent purification of TbRI-ED to apparent homogeneity. This was accomplished by two different methods as described below, although prior to either, the refolded, NiNTA-enriched preparation was digested with thrombin to remove the N-terminal histidine tag. This yielded the 101 residue TbRI-ED flanked N-terminally by a tetrapeptide originating from two residues of the thrombin recognition site (GlySer) and two residues encoded by an NdeI cloning site (HisMet). TbRI-ED was initially purified by passing the thrombin-digested folding mixture over an affinity matrix bearing the 2:1 TbRII-ED:TGFb3 complex. The column was then washed and active bound TbRI-ED was eluted with 8 M urea, which presumably denatured the protein, causing it to be released from the matrix. This denaturation, however, was reversible, as removal of the urea by dialysis yielded protein that exhibited its maximum expected binding activity (see below). The affinity column has the advantage that it enables rapid isolation of active TbRI-ED, although it has the disadvantage that significant effort is needed to prepare the matrix. Therefore, TbRI-ED was also purified by separating active from inactive forms using conventional HPLC-based ionexchange and C18 reversed-phase methods. The reversed-phase step, which was carried out last and used a water-acetonitrile buffer system, presumably also denatured the protein. This denaturation, however, was also shown to be reversible, as upon lyophilization and reconstitution, the water-acetonitrile TbRI-ED eluate exhibited its maximum expected binding activity (see below). The specific activity measurements were made using the native gel assay described above in which a fixed amount of 2:1 TbRII-ED:TGFb3 binary complex was titrated with increasing amounts of affinity or HPLC-purified TbRI-ED. As shown for the HPLC-purified sample (Figure 2(d)), addition of increasing amounts of purified TbRI-ED led to a progressive disappearance of binary complex (BC) concomitant with the appearance of TbRIED:TbRII-ED:TGFb3 ternary complex (TC) (lanes 1 and 2). After saturation of the free binary complex (lanes 4–6), further addition of TbRI-ED led to increasing amounts of free protein. A similar pattern was observed with affinity-purified TbRIED. Affirmation of the binary and ternary complex bands formed was accomplished by dissection and SDS-PAGE as before (Figure 2(e)). The titration results enabled calculation of the concentrations of active TbRI-ED in each preparation, assuming two equivalents of TbRI-ED

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

were recruited into the 2:1 TbRII-ED:TGFb3 complex. These values were then compared to concentrations determined by measuring UV absorbance at 280 nm and using a molar extinction coefficient calculated from the amino acid and disulfide composition of TbRI-ED (1280 MK1 cmK1),32 providing a measure of the total amount of protein. The concentrations determined by measuring UV absorbance were found to be, at most, 14%, higher than those determined by native gel assay, indicating that TbRI-ED, purified by either the affinity or HPLC method, was nearly or fully homogenous. The native gel titrations also enabled an accurate determination of total amount of active TbRI-ED isolated using the two procedures. This was found to be 0.5–1 mg per 100 mg of protein folded, indicating an overall recovery efficiency of 0.5–1%. Purified TbRI-ED is structurally homogeneous Native gel electrophoresis is commonly used to analyze the homogeneity of protein preparations, not just with respect to degree of contamination, but also structural homogeneity or uniformity, which cannot be evaluated by the less rigorous analysis of SDS-PAGE. Thus by this criterion, the purified TbRI-ED appeared to be quite homogeneous, migrating as a single uniform species in the native gels (see Figure 2(d), lane 6). For a second independent assessment of the structural homogeneity of the purified TbRI-ED, two-dimensional NMR spectroscopy was employed. In 2-D 1H–15N shift correlation spectra, peaks arise from backbone and side-chain amide sites and, as in any type of NMR spectrum, the local electronic environment at each site determines their position. In well-structured proteins, especially those rich in hydrogen-bonded b-sheet structure, backbone amide 1H chemical shifts generally range from approximately 6 ppm to about 9.5 ppm. In the 1H–15N shift correlation spectrum of TbRI-ED recorded at pH 6.5 and 20 8C (Figure 3), this overall pattern is observed, consistent with the b-sheet rich, three-finger toxin fold anticipated for this domain. Moreover, the total number of peaks observed (95 backbone amides, six side-chain amides of asparagine and glutamine) corresponded closely with the total number expected (97 backbone amides, six side-chain amides) based on the amino acid sequence of the polypeptide analyzed (see the legend to Figure 3). In addition to these two attributes, several intense peaks were also apparent in the random coil region between 7.8 ppm and 8.2 ppm in the 1H dimension that likely were due to structurally disordered residues of the N and C-terminal segments. As shown by Figure 1, five N-terminal and six C-terminal residues are indeed likely to be disordered, extending beyond the last structurally ordered residues observed in the crystal structure of BMPRIa (Figure 1), as well as the N-terminal tetrapeptide jointly derived from Escherichia coli expression and thrombin-cleavage mentioned above.

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

1057

Figure 3. Purified TbRI-ED is structurally homogeneous. The two-dimensional 1H–15N HSQC NMR spectrum shown was recorded at a 1H resonance frequency of 500 MHz and a sample temperature of 20 8C. The protein concentration was 0.13 mM and the buffer was 25 mM sodium phosphate, 5% 2 H2O, 0.02% (w/v) sodium azide at pH 6.5. Single peaks apparent in the spectrum correspond to signals that arose from backbone amide protons. The total number of single peaks expected (97) is equal to the total number of residues (105) minus the number of proline residues (7) and the N-terminal residue (an amine, not an amide). Pairs of peaks connected by horizontal broken lines correspond to signals that arose from side-chain amide (Asn and Gln) protons.

Complex formation is isoform-dependent In cell-based affinity labeling experiments, the binding affinity of TbRI and TbRII for both TGFbs 1 and 3 is much greater (Kd w 40 pM) than that for TGFb2 (Kdw500 pM).8 Native gel assays of binary and ternary complex formation with TGFb1 and TGFb2 were performed to examine the mechanism underlying these differences (Figure 4). As seen for TGFb3, TbRII-ED also formed a stable 2:1 binary complex with TGFb1 (Figure 4(a), left panel). The addition of purified TbRI-ED to this complex led to the progressive appearance of a more slowly migrating species, the 2:2:1 TbRI-ED:TbRIIED:TGFb1 ternary complex (Figure 4(b), left panel). Although the conversion of this binary to this ternary complex produced only a modest reduction in mobility, TbRI-ED was clearly consumed (lanes 3–6), detected as free protein only after two equivalents had been added (lanes 8 and 9). This uptake was confirmed by subsequent SDSPAGE analysis of the complex bands, which were composed of ligand and receptors in the expected stoichiometries (Figure 4(c), lanes 5 and 6). TGFb2 behaved markedly different in the native gel binding assay. A distinct binary complex as observed with TGFb1 and TGFb3 was not detectable and free TbRII-ED was apparent at all ratios of TbRII-ED:TGFb2 examined, even those where TGFb2 was in excess (Figure 4(a), right panel). Addition of purified TbRI-ED to the 2:1 mixture of

TbRII-ED and TGFb2 had little effect, yielding no distinct band characteristic of a ternary complex, although there was a detectable decrease in the amount of free TbRII-ED (Figure 4(b), right panel). Although the phenomenom responsible for this is not known, it may be that TbRI-ED and TbRII-ED cooperatively bind to form a transient ternary complex that disassociates during the course of electrophoresis. Thus, TGFbs 1 and 3, but not TGFb2, can form binary complexes with TbRII-ED and subsequently recruit TbRI-ED; however, the interactions between the receptors responsible for the cooperative recruitment of TbRI-ED are not sufficiently strong to fully compensate for the weak ligand–receptor interaction of TGFb2 and the type II receptor. On the other hand, these receptor–receptor interactions are of sufficient strength for cooperative binding of homodimeric, Fc-fused TGFb receptors,33 which have presumably enhanced but still unmeasurable affinity for TGFb2 alone. To show that the lack of or diminished activity of TGFb2 in this assay was related to its instrinsic binding properties and not the integrity of the preparation, the potency of this TGFb2 (E. coliexpressed, refolded) was compared against a TGFb2 preparation from a commercial source in a growth inhibition assay with fetal bovine heart endothelial (FBHE) cells. The activities of the two protein preparations were indistinguishable, each producing a 50% inhibition of growth at about

1058

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

Figure 4. Formation of binary and ternary complexes is isoform-dependent. (a) Native gel assay for TbRII-ED:TGFb binary complex (BC) formation with TGFb1 (left) and TGFb2 (right), adding 0.5–3 molar equivalents of TbRII-ED per molar equivalent of ligand (1.25 mg/eq. TGFb1, 1.50 mg/eq. TGFb2, 0.75 mg/eq. (left) or 0.90 mg/eq. (right) TbRII-ED) as before with TGFb3 (see Figure 2(a)). (b) Native gel assay for TbRI-ED:TbRII-ED:TGFb ternary complex (TC) formation with TGFb1 (left) and TGFb2 (right), adding 0.5–3 molar equivalents of TbRI-ED per molar equivalent of ligand (0.55 mg/eq. (left) or 0.66 mg (right) TbRI; equivalents of TGFb1, TGFb2, and TbRII-ED were as in (a) above). Electrophoresis was through a higher percentage (15%) gel with TGFb1, the standard (12%) gel with TGFb2. (c) Composition of putative binary and ternary complexes. After staining, protein complexes with TGFb1 (b left) were dissected from the native gel, soaked in SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Lanes 5 and 6 contain the binary (BC) and ternary (TC) complexes. Lanes 2, 3, and 4 contain 0.55 mg of TbRI-ED, 0.90 mg of TbRII, and 1.25 mg of TGFb1, standards, respectively. Lane 1, molecular mass markers. (d) Growth-inhibition activity of TGFb2 preparation used in binary and ternary complex formation assays. After treatment with picomolar concentrations of TGFb2, FBHE cells were pulsed with 5-[125I]iodo-2 0 -deoxyuridine and their DNA extracted for measurement of deoxyuridine incorporation. Filled circles, commercially obtained TGFb2 standard preparation. Open circles, TGFb2 preparation used in binary and ternary complex formation assays ((a) and (b), right).

35 pM (Figure 4(d)). This potency in the cell-based assay is comparable to activities reported,8 indicating that the inability to form a complex with TbRII-ED must indeed stem from the intrinsic binding properties of TGFb2, not from lack of stability or purity of the preparation. TbRI-ED is an antagonist of TGFb signaling Soluble TbRII-ED has been shown to act as an antagonist of TGFb signaling by sequestering the ligands in solution, preventing their interaction

with the cell-surface receptors.34,35 Because TbRIED binds TGFb1 and TGFb3 with high affinity, albeit in a TbRII-ED-dependent manner, it should also act as a cellular TGFb1 or TGFb3 antagonist by blocking receptor complex assembly. This would be expected to occur at a later step, however, by binding preformed TbRII–ligand complexes in the membrane and thus preventing recruitment of membrane-bound TbRI into the complex. To determine whether TbRI-ED can indeed act as an antagonist, plasminogen activator inhibitor-1: luciferase reporter gene (PAI-LUC)36 assays were

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

performed with cultured mink lung epithelial cells. The reporter gene assay demonstrated that TbRI-ED and TbRII-ED can both antagonize the promoter activity induced by TGFb3 (Figure 4(a)), although the effect is clearly greater with TbRII-ED than with TbRI-ED (59% versus 34% reduction) tested at the same concentration (5 mg mlK1). Due to the concentration dependence of the response (compare 1 and 5 mg mlK1 TbRI-ED) and the further reduction in promoter activity after TbRI-ED and TbRII-ED are combined (89% reduction overall), the antagonistic effects of TbRI-ED addition, although less robust than with TbRII-ED, are unambiguous. The comparative effects of TbRI-ED and TbRIIED on cell signaling were examined in a second independent assay and cell line by analyzing the level of Smad2 phosphorylation37 in human breast epithelial cells (Figure 5(b)). TbRI-ED (lane 7) and TbRII-ED (lane 8) again both antagonized the cellular response induced by TGFb3 at 5 mg mlK1, and as before, TbRII-ED was more potent than TbRI-ED. If TbRI-ED and TbRII-ED were combined at either 2.5 or 5 mg mlK1 (lanes 9 and 10), Smad2

Figure 5. TbRI-ED is an antagonist of TGFb signaling. (a) Antagonistic potential of TbRI-ED and TbRII-ED assayed by a TGFb reporter construct. Mink lung epithelial cells stably transfected with a PAI-luciferase reporter gene were treated with TGFb3 (2 ng mlK1), with or without addition of TbRI-ED and TbRII-ED as indicated. Luciferase activity and accompanying errors represent the mean and standard deviation of triplicate measurements. (b) Antagonistic potential of TbRI-ED and TbRII-ED analyzed by Smad2 phosphorylation assay. Human breast epithelial (MCF-10A) cells were treated with TGFb3 (1 ng mlK1), with or without addition of TbRI-ED and TbRII-ED as indicated. After preparation of cell lysates, phosphorylated Smad2 was detected by Western blotting with an anti-phospho-Smad2 antibody (upper panel). Control analyses of total Smad2 protein and GAPDH are aligned below.

1059 phosphorylation was reduced even further than with 5 mg mlK1 TbRII-ED alone (lane 8). These results were consistent with those of the luciferase reporter assay and demonstrated conclusively that TbRI-ED, like TbRII-ED, acts as a cellular TGFb antagonist. TGFb2-TM variant binds TbRII-ED and recruits TbRI-ED The crystal structure of the 2:1 TbRII-ED:TGFb3 complex15 revealed that residues contacting TbRII are identical in the high-affinity ligands, TGFbs 1 and 3, but are conservatively substituted at three positions (Arg25OLys, Val92OIle, Arg94OLys) in the low-affinity ligand TGFb2 (Figure 6(a)). Thus, despite the conserved nature of the substitutions, these three residues appear responsible for the diminished affinity of TGFb2 for the type II receptor. To test this hypothesis and evaluate the requirements for recruitment of TbRI, a TGFb2 triple mutant (K25R, I92V, K94R; TGFb2-TM) was produced and analyzed by native gel assay. After mixing at a 2:1 molar ratio, TbRII-ED and TGFb2-TM produced a new species that migrated more slowly than TbRII-ED alone (Figure 6(b), lanes 9 and 4, respectively). A similar product was observed with TGFb3 (lane 5), but not with TGFb2 (lane 7), suggesting that the new species was the 2:1 TbRII-ED:TGFb2-TM binary complex. Thus, the three conservative substitutions at the binding interface were indeed sufficient to impart highaffinity binding to TGFb2. In addition, the 2:1 TbRIIED:TGFb2-TM complex was shifted by addition of two equivalents of TbRI-ED (lane 10), similar to the shift observed with TGFb3 (lane 6), indicating that binary complex with TGFb2-TM was also capable of recruiting TbRI-ED to form a stable ternary complex with 2:2:1 stoichiometry. The identity and stoichiometry of complexes formed with TGFb2TM were confirmed by SDS-PAGE analysis and native gel titrations (data not shown) as with TGFbs 1 and 3. Since TGFb2-TM formed stable binary and ternary complexes (lane 10), but TGFb2 did not (lane 8), high-affinity binding of TbRII-ED was therefore both necessary and sufficient for the subsequent recruitment of TbRI-ED. This indicates that the three different TGFb isoforms induce assembly of ternary complex in the same general manner. Monomeric TGFb3:TbRII-ED complex recruits TbRI-ED only weakly The two models proposed for TGFb ternary complex assembly15,21 both arose from the assumption that TbRI binds at the dimer interface of TGFb isoforms, based on the structural paradigm of the BMPRIa:BMP2 complex,19 the only type I receptor: ligand structure determined to date. To test this assumption, a monomeric form of TGFb3 was compared to the dimeric ligand with respect to ternary complex formation in the native gel binding

1060

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

Figure 6. The TGFb2-TM variant binds TbRII-ED and recruits TbRI-ED. (a) Alignment of the amino acid sequences of human TGFb1, and in the regions contacted by TbRII. The residues shown on a black background correspond to those that are contacted by TbRII in the TbRII-ED:TGFb3 crystal structure. The three contact residues conservatively substituted in TGFb2 are shown on a grey background. (b) Native gel assay of binary and ternary complex formation with a TGFb2 triple mutant (TGFb2-TM) conservatively substituted at the TbRII binding site to mimic TGFbs 1 and 3. Molar ratios of ligands and receptor extracellular domains are indicated above (1.50 mg/eq. each ligand, 0.90 mg/eq. TbRII-ED, 0.66 mg/eq. TbRI-ED). Asterisks indicate complexes formed with TGFb2-TM, which migrated more slowly than their binary (BC) and ternary (TC) counterparts formed with TGFb3.

assay. This monomeric form of TGFb3 (TGFb3m) was generated by substituting the cysteine that normally forms the intersubunit disulfide bond with serine (C77S). In a previous NMR study, TbRIIED interacted with TGFb3m in the same overall manner as dimeric TGFb3,38 in keeping with the 2:1 TbRII-ED:TGFb3 complex structure,15 which showed that the type II receptor binds by contacting residues from just one ligand monomer. In contrast, in the BMPRIa:BMP2 complex,19 the type I receptor binds by contacting residues from both monomers. It is expected, therefore, that TbRII-ED would bind with high affinity to both monomeric and dimeric TGFb3, whereas TbRI-ED would not, assuming that it required a dimeric ligand binding site for recruitment into ternary complex. As anticipated, addition of equimolar amounts of TbRII-ED to TGFb3m yielded the monomeric binary complex (Figure 7(a), lane 8) concomitant with the loss of both free TGFb3m (standard, lane 1) and free TbRII-ED (standard, lane 2). The stoichiometry of complex formation was confirmed to be 1:1 by titration, as excess receptor was observed after one equivalent of TbRII-ED was added (not shown). Addition of TbRI-ED to this 1:1 TbRII-ED:TGFb3m complex neither led to a sizeable shift nor significant uptake of TbRI-ED (lane 9). Because TbRI-ED bound the dimeric (lanes 5, 6) but not the monomeric (lane 9) binary complex, clearly the second monomer was required for recruitment of TbRI-ED into a stable ternary complex. Nevertheless, TbRI-ED did appear to interact to some extent with the 1:1 TbRII:TGFb3m complex, as addition of TbRI-ED (lane 9) caused the complex band to smear and lessen in intensity relative to the 2:1 TbRII-ED:TGFb3 complex control (lane 8). To determine whether this effect was an artifact of

electrophoresis of the mixture or actually the result of a diminished yet specific interaction, inactive TbRI-ED was prepared by reduction and alkylation and tested for binding. With the dimeric binary complex, no specific evidence of any binding was observed (lane 7), indicating that the chemically modified TbRI-ED was indeed inactive. Addition of the inactive TbRI-ED to the monomeric binary complex failed to produce the smearing and weakening seen before with active receptor. Thus, loss of the contacts from the second TGFb monomer led to dimunition, but not complete elimination, of the affinity of TbRI-ED for the 1:1 TbRII-ED:TGFb complex. Biological activity of monomeric TGFb3 In a cell-based assay, a monomeric form of TGFb1 (C77S) was previously shown to stimulate a TGFb-responsive promoter with a potency of 20% that of the native dimeric TGFb1.39 It was proposed that part or all of this activity might be due to a propensity of the TGFb1 C77S monomers to exist transiently as a non-covalently bonded dimer, stabilized by hydrophobic interactions at the interface, providing for the assembly of a functional signaling complex and transduction of the signal. In the experiments presented above, we found that TGFb3m binds TbRII-ED with high affinity (Figure 7(a)). In cells bearing both types of TGFb receptors, TGFb3m would therefore be expected to bind with high affinity to TbRII and, in turn, this monomeric binary complex might recruit TbRI with low affinity, resulting in a transient signaling complex. It is concievable, therefore, that the bioactivity of monomeric TGFb1 was initiated not by the assembly of

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

1061

Figure 7. Monomeric TGFb3:TbRII-ED complex recruits TbRI-ED only weakly. (a) Native gel assay of binary and ternary complex formation with a monomeric form of TGFb3 (TGFb3m). Molar ratios of TGFb3m, TGFb3 and the receptor extracellular domains are indicated above (0.63 mg/eq. TGFb3m, 1.25 mg/eq. TGFb3, 0.75 mg/eq. TbRII-ED, 0.66 mg/eq. TbRI-ED). Inactive TbRI-ED refers to active protein that had been treated with DTT and iodoacetamide. BCm marks the 1:1 binary complex with TGFb3m. (b) TGFb-responsive luciferase reporter assay of signaling by monomeric versus dimeric TGFb3. Mink lung epithelial cells stably transfected with a PAI-luciferase reporter gene were treated with dimeric or monomeric (TGFb3m) forms of TGFb3 as indicated. Luciferase activity and accompanying errors represent the mean and standard deviation of measurements performed in triplicate. (c) SDS-PAGE analysis of the crosslinked products formed upon incubation of a crosslinker (BS3) with TbRI-ED, TbRII-ED, and dimeric TGFb3 alone and in combination with one another. Crosslinking mixtures in which both ligand and receptor were present contained a fiveto tenfold molar excess of receptor relative to ligand. Labels shown along the right-hand side of the gel correspond as follows: 1:1 BC and 1:2 BC designate the 1:1 and 2:1 complexes, respectively, formed between TbRII-ED and TGFb3; 1:2:1 and 2:2:1 TC designate the 1:1 and 2:1 complexes, respectively, formed between TbRI-ED and the 2:1 TbRII-ED:TGFb complex. (d) Same as in (c) but with monomeric (C77S) TGFb3 in place of dimeric TGFb3. Labels shown along the righthand side of the gel correspond as follows: 1:1 BCm designates the 1:1 complex formed between TbRII-ED and TGFb3m; 1:1:1 TCm designates the 1:1:1 complex formed between TbRII-ED, TbRI-ED and TGFb3m; 2:1 BC, 1:2:1 TC, and 2:2:1 TC are the same as in (c), except with BS3-crosslinked TGFb3m dimer rather than native dimer.

a non-covalent dimer per se, but instead by assembly of TbRII and TbRI around the monomeric ligand and subsequent dimerization via the interaction of TbRI with both ligand monomers. To examine this in greater detail, we determined whether monomeric TGFb3, like its TGFb1 counterpart, possessed any measurable activity relative to the dimeric form in a cell-based assay. Mink lung epithelial cells, stably transfected with a PAI-LUC reporter gene, were incubated with each form of the ligand over a tenfold concentration range and with a mixture of the two (Figure 7(b)). Based on the relative intensities observed in this assay, the luciferase activity induced by monomeric TGFb3 was estimated at 15% of that induced by dimeric TGFb3 with comparable molar concentrations of both ligand forms (0.5 ng mlK1 monomeric, 1 ng mlK1 dimeric) (Figure 7(b)). Monomeric TGFb3 therefore possesses an inductive activity similar to that of monomeric TGFb1,39 indicating that it may be able to, albeit weakly, assemble

a signaling complex in the cell membrane of TGFbresponsive cells. To assess the propensity of monomeric TGFb3 to dimerize, and to determine the extent to which receptor binding influenced dimerization, we carried out a series of chemical crosslinking studies. To validate the method, we crosslinked TbRI-ED, TbRII-ED, and dimeric TGFb3 to themselves as well as to one another in all possible combinations (Figure 7(c)). TbRII-ED, when combined in excess relative to dimeric TGFb3, yielded a major band at 50 kDa and a minor band at 37 kDa (lane 7). These two bands were not observed when either component was crosslinked to itself (lanes 2 and 3), indicating that the observed major and minor bands correspond to specific protein complexes, presumably the 2:1 TbRII-ED:TGFb3 (53 kDa) and the 1:1 TbRII-ED:TGFb3 (39 kDa) binary complexes, respectively. The addition of TbRI-ED diminished the intensity of the bands corresponding to the two binary complexes and led to the appearance of two

1062 higher molecular mass species, one 75 kDa and another 63 kDa (lane 8). These higher molecular mass bands were absent from the other binary mixtures and therefore are presumed to be the 2:2:1 TbRI-ED:TbRII-ED:TGFb3 (75 kDa) and 1:2:1 TbRIED:TbRII-ED:TGFb3 (64 kDa) ternary complexes, respectively. The fact that the binary (lane 7) and ternary (lane 8) complexes appeared as mixtures in which both one and two equivalents of receptor were bound is likely due to the fact that for each interface there exists a finite probablity that the corresponding site on the ligand, receptor, or both is modified in a way such that complex formation is blocked. The crosslinking approach was next used to determine whether monomeric TGFb3 had any propensity to form transient dimers, either alone, or in the presence of the TbRI and TbRII EDs. The results show that when crosslinked to itself, TGFb3m yields only a 13 kDa species (Figure 7(d), lane 1), suggesting that it has little or no propensity to dimerize (although not shown, similar results were obtained when tenfold larger amouts of TGFb3m were tested). The addition of TbRII-ED led to the appearance of not only a major band at 26 kDa, which presumably corresponds to the 1:1 TbRII-ED:TGFb3m binary complex (27 kDa), but also a minor band at 39 kDa, which presumably corresponds to the binary complex noted above together with an additional molecule of bound TGFb3m (40 kDa) (lane 7). The appearance of this dimeric species is inconsistent with the absence of a dimeric species when TGFb3m was crosslinked to itself, since TbRII-ED binds at a site remote from the dimer interface,15 and thus would not be expected to alter the propensity of TGFb3m to dimerize. The tendency of TGFb3m to form non-specific higherorder aggregates in the absence of receptor binding, as previouly observed,37 might account for this discrepancy. To explore this, we compared the band intensity of crosslinked and non-crosslinked TGFb3m (not shown) and observed that it was indeed much more intense in the absence of crosslinker than in its presence, indicating that TGFb3m does have a strong propensity to form non-specific higher-order aggregates (dimeric TGFb3 was also shown to exhibit this property, which accounts for its absence from Figure 7(c), lane 2). The notion that receptor binding leads to a dimunition, or complete abrogation, of the non-specific aggregation of both monomeric and dimeric TGFb3 follows from the previous finding that complexes of these ligands with TbRII-ED are soluble between pH 6 and pH 9,15,38 whereas the ligands alone are not,38,40 The overall conclusion, therefore, is that the 39 kDa band observed in Figure 7(c), lane 7 is due to the intrinsic propensity of TGFb3m to dimerize. This propensity to dimerize, however, is enhanced when both TbRII-ED and TbRI-ED are present. This is illustrated in Figure 7(d), lane 8, where it is shown that the addition of TbRII-ED and TbRI-ED to TGFb3m leads to at least four

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

high molecular mass species, one at 37 kDa, which presumably corresponds to that of the transient complex formed between TbRII-ED, TbRI-ED, and TGFb3m (38 kDa), but as well three others at 50 kDa, 62 kDa, and 75 kDa. The latter three are identical with the high molecular mass species formed when TbRII-ED and TbRI-ED were crosslinked to dimeric TGFb3 (Figure 7(c), lane 8), and thus presumably correspond to complexes with the same overall composition, but with BS3-crosslinked TGFb3m dimer rather than natural TGFb3 dimer. The fact that such complexes form, particularly those in which TbRI-ED is bound, is presumably driven, in part, by TbRI-ED, which, as shown, likely binds by simultaneously contacting both ligand monomers as well as TbRII-ED. These data account for the residual biological activity of monomeric TGFb3 (and likely that of monomeric TGFb1) by showing that receptor binding, coupled with an intrinsic propensity of the ligands to dimerize, is likely responsible for assembly of a dimeric biologically active signaling complex on the cell surface.

Discussion TGFb isoforms initiate their response by binding and bringing together TbRI and TbRII.3,10 TbRII, the high-affinity receptor for TGFbs 1 and 3, has been shown to bind wedge-like between the fingertips on the distal end of the ligand homodimer.15 Little is known regarding the mechanism by which TbRI is cooperatively recruited into the heterotetrameric receptor signaling complex, although two reports, one by Docagne et al.41 and another by del Re et al.,33 have shown that artificially dimerized, Fc-chimeric TbRI-ED cooperatively binds TGFb isoforms and Fc-chimeric TbRII-ED. On the one hand, these results are significant, since they provide experimental support for models of receptor assembly based on either direct15 or indirect21 interactions. On the other hand, definitive conclusions based on these results are limited, since artificial dimerization of the receptor may well underlie much of the cooperativity observed. An additional compromising aspect of these preparations was their homogeneity, which was not demonstrated by del Re et al.33 and questionable for the receptor produced by Docagne and co-workers,41 since multiple high molecular mass species, likely disulfide-linked oligomers, were apparent in non-reducing SDS/polyacrylamide gels. In addition, this preparation of receptor extracellular domain had TGFb agonist, not antagonist activity as expected.41 To overcome these limitations, we produced a monomeric form of human TbRI-ED by oxidative refolding of E. coli-expressed protein. The purified protein was shown to be structurally homogeneous as judged by its uniformity in native gels and the appearance of the 2-D 1H–15N HSQC spectrum of purified 15N-labeled protein. As anticipated, the TbRI-ED acted as a TGFb antagonist, albeit with

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

a potency 20–30% lower than that of TbRII-ED (Figure 5(a)). This result with our E. coli-expressed, refolded TbRI-ED preparation was in contrast to that with the soluble TbRI-ED-Fc fusion protein produced in tissue culture that was reported to function as a TGFb agonist.41 The origin of this discrepancy is unclear, although residual TGFb from the cell culture medium could account for the agonist activity observed with the secreted receptor preparation. The purified protein bound with high affinity to the binary complex between TGFbs 1 and 3 and TbRII-ED with a 2:1 stoichiometry. This important finding, together with the additional results obtained through semi-quantitative binding studies, have helped us to distinguish between the two fundamentally different assembly models based on biochemical data and structural insights, involving either direct15 or indirect21 interactions (Figure 8(a)). In the direct interaction model, because TbRI-ED and TbRII-ED bind at adjacent sites on the ligand, the cooperative recruitment

1063 of the type I receptor arises from contacts with both TGFb and the TbRII-ED. In the indirect interaction model, binding of the TGFb isoforms by membrane-tethered TbRII alters the conformational state of the ligand, enabling high-affinity binding of TbRI. These models share a common mode of type I receptor binding, homologous to BMPRIa on BMP2 (Figure 8(b)), but differ in that the indirect one requires TbRII to be anchored in the membrane, whereas the direct one does not. Our in vitro binding studies with the extracellular domains of the receptors clearly show that membrane attachment is not required for cooperative assembly, in keeping with the notion that the cooperativity is largely if not entirely derived by direct interaction, not indirect interaction. Direct interaction, but not the indirect interaction, is also consistent with the behavior of TGFb1, which does not undergo rearrangement in solution22,42 yet nonetheless cooperatively recruits TbRI in a manner indistinguishable from TGFb3.

Figure 8. Mechanism of TGFb ternary complex assembly and composite ternary complex models produced by superposition. (a) Mechanism of TGFb ternary complex assembly incorporating the equilibrium between closed and open forms of TGFb3, in both free17,18 and TbRII-bound15 states. The type II receptor site is unaltered by the transition between closed and open forms (left), allowing for high-affinity binding of TbRII to both (center). However, the type I receptor binding site is unstructured and incapable of binding TbRI in the open state (center, below). In the direct interaction model,15 TbRI is recruited into ternary complex in the closed state by binding cooperatively to a composite site formed by the dimer interface of the ligand and a surface of TbRII (upper right). In the indirect interaction model,21 TbRII must be membrane-tethered to induce formation of the closed state and, with that, the type I receptor binding site. (b) Model of the BMP ternary complex produced by alignment of BMP ligands in the experimentally determined BMP2:BMPRIa19 and BMP7:ActRII44 binary complex structures (after Greenwald et al.44). (c) Model of a hypothetical TGFb ternary complex produced by alignment of TGFb and BMP ligands in the experimentally determined TGFb3:TbRII-ED15 and BMP2:BMPRIa-ED19 binary complex structures (after Hart et al.15). In each case, the BMP2 ligand of the common BMP2:BMPRIa core is shown, along with the bound type I and superpositioned type II receptors.

1064 The direct interaction model proposed for the cooperative assembly of TGFb complexes is supported by superposition of the TGFb and BMP ligands in two crystal structures,15 which shows the binding sites for TbRII and BMPRIa as adjacent (Figure 8(c)). However, this hypothetical TGFb ternary complex is based on conservation of the type I receptor binding site within the ligand superfamily. Because the TGFb type II receptor binding site is distinct from the type II sites of BMP and activin ligands, the possibility remains that the type I receptor sites are also non-conserved. For example, an alternative site could be envisioned on the backs of the ligand fingers, the knuckle epitope of BMPs and activin for type II receptors, that is also adjacent to the TbRII binding site. Interactions at this site would be limited to one monomer, unlike the BMPRIa-like site at the dimer interface that would require interactions from both (Figure 8(c)). However, further support for a common mode of type I receptor interaction with ligand emerges from studies with a variant of TGFb3 (Cys77Ser), in which the cysteine residue providing the interchain disulfide bond has been substituted with serine. This monomeric form of TGFb3 was shown by electrophoresis through native gels to form a 1:1 binary complex with TbRII, similar to the 2:1 complex observed with the dimeric ligand. Therefore, the affinity of TGFb3 for TbRII-ED is largely, if not entirely, unaltered by monomerization. This behavior is consistent with the structure of the 2:1 TbRII:TGFb3 complex,15 which shows that each TbRII-ED interacts with only a single TGFb3 monomer. In contrast, the binary complex of TbRII-ED and monomeric TGFb3 showed significantly diminished ability to recruit TbRI-ED relative to the dimeric complex, indicating that both TGFb monomers are required for type I receptor binding. Thus, TbRI appears to bind its ligands in a similar manner as BMPRIa at the dimer interface, interacting with TbRII from this adjacent site, rather than the alternative one at the knuckle epitope. On the other hand, small steric clashes are observed in the superposition model between the TGFb ligand and the type I receptor, which furthermore presents a relatively small surface (edge-on) at the putative TbRII interface. Thus, the precise position and orientation of TbRI in the ternary complex may vary to some extent from that predicted by superposition of BMPRIa onto the TbRII:TGFb3 binary complex. In cell-based assays we observed that monomeric TGFb3 was nearly 15% as potent as native dimeric TGFb3, a potency seen also with the Cys77Ser variant of TGFb1.39 In that study, non-covalent dimerization of monomers was hypothesized to suffice for assembly of a functional signaling complex, which as shown through the work of Weis-Garcia and co-workers,43 is believed to be that of a dimer wherein the cytoplasmic domains of TbRI interact. In order to determine whether the residual activity of monomeric TGFb3 was due to its intrinsic propensity to dimerize, as suggested in the

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

previous study,39 or alternatively whether assembly of the dimer was driven by cooperative binding of the receptors, we carried out a series of crosslinking studies. Interestingly, these revealed that both processes appear to play a role; that is, monomeric TGFb3 possesses an intrinsic propensity to dimerize, and such dimerization is enhanced by cooperative binding of TbRI and TbRII. In summary, we have shown that TGFbs 1 and 3, which have high affinity for TbRII, bind the extracellular domains of the two receptor pairs in vitro in the same stepwise and cooperative manner as the full-length receptors on the cell surface. Recruitment of the low-affinity type I receptor into a heterotetrameric complex appears to result from direct contact between the extracellular domain of TbRI and a composite interface formed by surfaces of both TGFb and the extracellular domain of TbRII. Because high-affinity binding, imparted to TGFb2 by substitution at three positions at the TbRII binding interface, was sufficient for cooperative recruitment of TbRI, all three ligand isoforms share a single mode of initiating the assembly of the signaling complex. The intrinsically weak interaction between TGFb2 and TbRII-ED may be compensated for in vivo by the highly abundant co-receptor betaglycan, allowing for the subsequent recruitment of both receptor types into a functional signaling complex in a passive, membrane-dependent mechanism similar to the mechanism of cooperative assembly proposed for BMPs.44,45 Whether any ligand of the TGFb superfamily requires membrane attachment of one receptor pair to induce the formation of the binding site for the other receptor pair, as hypothesized in the indirect interaction model, remains to be determined. Due to its inherent flexibility, activin has also been proposed to recruit its type I receptor (ALK4) by this model.21 However, our studies with the highly flexible TGFb3 isoform show that these indirect interactions need not be invoked. Because activin, like BMPs, is expected to bind its receptors at nonadjacent sites24,46,47 and does not recruit low-affinity receptor ECD cooperatively,48 the membrane may indeed be required, not to actively induce formation of the type I site, but simply to confine the ligand to the surface of the membrane. This high local concentration of ligand could transform a lowaffinity interaction into one of high affinity, compensating for the lack of direct receptor–receptor interactions as hypothesized for the cooperative assembly of BMP signaling complexes.44,45

Materials and Methods Over-expression and refolding Two DNA fragments encoding the mature human TbRI-ED (101 aa) were PCR-amplified from a cDNA template11 with primers that introduced either KpnI/ BamHI or NdeI/BamHI sites into the termini. After

1065

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

digestion, the KpnI-BamHI fragment was inserted into a modified form of pET32a (Novagen) containing a KpnI site flanking the encoded thrombin cleavage site, resulting in a Trx-His-TbRI-ED construct with a cleavable N-terminal thioredoxin-hexahistidine tag. The NdeIBamHI fragment was inserted into pET15b (Novagen), generating a His-TbRI-ED construct with a cleavable N-terminal hexahistidine tag. The integrity of both constructs was verified by DNA sequencing. Recombinant proteins were expressed in E. coli BL21(DE3) transformants cultured at 37 8C in LB medium containing 200 mg mlK1 of ampicillin. Expression was induced by the addition of 0.8 mM IPTG at mid-log phase (0.6 A600). Cells were harvested 5 h after induction and frozen at K20 8C. Cells from a 1 l culture harboring the Trx-His-TbRI-ED plasmid were resuspended in 20 ml of lysis buffer (50 mM Na2HPO4 (pH 8.0), 300 mM NaCl, 1 mM PMSF) and sonicated. The sonicated preparation was then centrifuged and the supernatant loaded onto a 5 ml NiNTA (Qiagen) column equilibrated with lysis buffer. After washing, TbRI-ED was eluted with lysis buffer containing two units of thrombin per 1 mg of bound fusion protein. The eluate was concentrated by ultrafiltration (Millipore) and multimeric species removed by gel-filtration with a Sephacryl S100 (Amersham Biosciences) column equilibrated with 25 mM sodium phosphate (pH 7.0). Cells from a 1 l culture harboring the His-TbRI-ED plasmid were resuspended in 20 ml of denaturing lysis buffer (8 M urea, 50 mM Na2HPO4 (pH 8.0), 300 mM NaCl) and sonicated. The sonicated preparation was then centrifuged at 40,000g and the supernatant was removed and diluted with denaturing lysis buffer to 40 ml. This preparation was loaded onto a 5 ml NiNTA column equilibrated with ten column volumes of denaturing lysis buffer. After washing, the protein was eluted with a linear (0–300 mM) imidazole gradient. His-TbRI-ED was pooled, reduced with 250 mM DTT and dialyzed for 4 h against 4 l of 20 mM acetic acid five times. The protein was refolded by slowly diluting the dialyzed preparation into 0.1 M Tris–acetate, 1 mM reduced glutathione, and 0.5 mM oxidized glutathione at pH 8.0 to yield a final protein concentration of 50 mg mlK1. The refolding mixture was stirred slowly for 15 h at 4 8C. The insoluble aggregates that formed were removed by filtration and the filtrate loaded onto a 2.5 ml NiNTA column equilibrated with 50 mM Tris–HCl (pH 8.0). The protein was eluted with a linear (0–300 mM) imidazole gradient.

loaded into the well of a 12% Tricine-SDS/12% polyacrylamide gel and electrophoresed. Purification Purification of active TbRI-ED from the folding mixture was achieved using two different methods. Prior to purification by either, the N-terminal histidine tag was removed by digesting at pH 8.0 at 4 8C for 24 h with thrombin at four units per 1 mg of protein. In the purification method based on affinity chromotography, active TbRI-ED was isolated by passing the folding mixture over a TbRII-ED:TGFb3 affinity matrix. This was prepared by combining TbRII-ED with a C-terminal hexahistidine tag and TGFb3 at 2:1 molar ratio. The mixture was dialyzed against 20 mM Tris–HCl (pH 8.0) and bound to NiNTA agarose. To isolate active TbRI-ED, the refolding mixture was loaded onto the affinity column and washed with 20 mM Tris–HCl (pH 8.0). Active TbRI-ED was then eluted with 8 M urea buffered with 20 mM Tris–HCl (pH 8.0). The eluate, containing both TbRI-ED and TGFb3, was dialyzed against 25 mM sodium phosphate (pH 6.5). TGFb3 is not soluble at significant concentrations in the absence of denaturant at this pH, producing a precipitate that was removed by centrifugation to yield active TbRI-ED. In the purification method based on HPLC, active TbRIED was isolated by applying the protein to a 10 ml Source 15Q column (Amersham Biosciences) equilibrated with six volumes of 50 mM Tris–HCl (pH 8.0). Bound TbRI-ED was eluted with a linear gradient (0–30%) of buffer B (50 mM Tris–HCl (pH 8.0), 1 M NaCl) over ten column volumes at 2 ml minK1 at ambient temperature. Fractions (5 ml) with TbRI-ED activity were identified using the native gel assay described above and then pooled, lyophilized, and resuspended in 2 ml of 0.1% (v/v) TFA in water. The enriched mixture was then applied to a 4.6 mm!250 mm C18 reversed-phase HPLC column (Thomson Liquid Chromatography) pre-equilibrated with 5% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (TFA). TbRI-ED was eluted with a linear gradient (22–47%) of buffer B (0.1% TFA in acetonitrile) at 1 ml min K1 over 127 column volumes at ambient temperature. After lyophilization, peak fractions (5 ml) were resuspended in water and tested for binding activity using native gels as described above.

Native gel binding assays

Chemical inactivation of TbRI-ED

TbRII-ED used in all binding assays was an N-terminally truncated variant (15–136) that lacked the first 14 residues following the site of signal peptide cleavage.15 TbRI-ED analyzed after folding and enrichment by NiNTA affinity chromatography was first further concentrated by centrifugal ultrafiltration. Protein samples were mixed under non-reducing conditions with an equal volume of native gel sample buffer (20% glycerol, Trisbuffered at pH 8.4) at room temperature and immediately loaded onto a native polyacrylamide gel. These gels were cast with a short (1 cm) 4% polyacrylamide stacking gel buffered with 0.25 M Tris–HCl (pH 6.8) followed by a long (7 cm) 12%–16% polyacrylamide running gel buffered with 0.38 M Tris–HCl (pH 8.8) and run at 125 V for approximately 2 h. SDS-PAGE analysis of native gel species was performed by dissecting the Coomassiestained proteins from the gel with a razor blade and transferring the slices to 2! Tricine-SDS sample buffer. The gel fragments and accompanying buffer were then

TbRI-ED was dialyzed into 8 M urea buffered with 20 mM sodium phosphate (pH 8.0) and reduced by addition of solid dithiothreitol to a final concentration of 5 mM. After 15 min, free thiols were blocked with 25 mM iodoacetamide and the inactivated protein dialyzed exhaustively against 20 mM sodium phosphate at 4 8C. NMR sample preparation and spectroscopy M9 minimal medium containing 0.1% (w/v) 15NH4Cl was inoculated with E. coli strain BL21(DE3) transformed with the His-TbRI-ED construct. Protein expression, refolding and purification by the affinity column method were carried out in the manner described above. The purified, active [15N]TbRI-ED samples were prepared in a volume of 300 ml and were contained in a 5 mm microcell (Shigemi) at a protein concentration of 0.1–0.2 mM in 25 mM sodium phosphate (pH 6.5), 5% 2H2O, 0.02% (w/v) sodium azide. Two-dimensional 1H–15N HSQC

1066 spectra were then collected at a 1H frequency of 500 MHz using a 1H-[ 13C,15N] X,Y,Z gradient probe and a conventional 1H–15N HSQC pulse sequence with water flipback pulses.49 Data were collected at 20 8C with 32 scans and acquisition times of 102 ms (1024 complex points) and 105 ms (175 complex points) in the 1H and 15N dimensions, respectively.

In vitro Assembly of TbRI-ED:TbRII-ED:TGFb

(BD Transduction), or an anti-GAPDH antibody (Ambion) for 1 h at ambient temperature or overnight at 4 8C. After three washes with TBST, the membrane was incubated with HRP-linked anti-rabbit or anti-mouse antibody (Santa Cruz) for 1 h at ambient temperature and washed again. Bound complexes were visualized with chemiluminescence procedures (NEN Life Science Products) according to the manufacturer’s instructions.

Other proteins Chemical crosslinking Expression, isolation, refolding and purification of TbRII-ED, TGFb3, TGFb2, TGFb2-TM and monomeric TGFb3 were carried out as described.30,38,50 TGFb1 was purchased from a commercial source (R&D Systems). Growth inhibition assay The effect of TGFb isoforms on the proliferation of cultured fetal bovine heart endothelial (FBHE) cells was tested as described.8 Briefly, FBHE cells were seeded in 24-well plates and cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco-BRL) and 100 mg mlK1 endothelial cell mitogen (Biomedical Technologies). Once FBHE cells reached a density of 50,000 cells per well, TGFb isoforms were added at the indicated concentrations in fresh medium without endothelial mitogen, and the cells were then incubated for another 48 h. DNA synthesis was measured during the last 8 h of the TGFb isoform treatment by pulsing the cells with 5-[125I]iodo-2 0 deoxyuridine (Amersham) at 1 mCi mlK1. TGFb2 standard preparation was obtained commercially (R&D Systems, Minneapolis). Reporter gene assay The plasminogen activator inhibitor-1 (PAI-1) reporter gene assay was performed essentially as described.36,51 Briefly, mink lung epithelial cells stably transfected with a PAI-1 promoter-luciferase construct were transferred to a 96-well plate at a density of 3000 cells per well. After growing for three days, the cells were treated with a fixed amount of dimeric TGFb3, with or without TbRII-ED and TbRI-ED (antagonist assay), or increasing amounts of dimeric or monomeric forms and a mixture of the two (monomer assay). HPLC rather than affinity-purified TbRI-ED was used for these experiments to eliminate the risk of contamination with TGFb3. After 16 h, the cells were lysed and their lysates analyzed for luciferase activity. Smad2 phosphorylation assays Exponentially growing MCF-10A human breast epithelial cells were treated with 1 ng mlK1 TGFb3 and 2.5–5 mg mlK1 TbRII-ED and TbRI-ED (HPLC-purified). After 30 min, the cells were rinsed twice with ice-cold PBS and lysed in 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Nonidet P-40, containing protease (Roche) and phosphatase (1 mM NaVO3 and 1 mM NaF) inhibitors. Samples containing 20 mg of total protein were then separated by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with TBST (100 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween-20) containing 5% (w/v) non-fat dried milk and incubated with a rabbit polyclonal anti-phospho-Smad2 (Ser-465/467) antibody (Upstate Biotechnology), an anti-Smad2/3 antibody

Chemical crosslinking reactions were carried out by combining an excess of the purified receptor EDs (9.7 mg TRI-ED or 11.0 mg TRII-ED) with either monomeric (C77S) (1.5 mg) or dimeric (1.5 mg) TGFb3. The pH was then adjusted to 6.5 by adding an equal volume of conjugation buffer (500 mM Hepes, pH 6.5) and the crosslinking reaction was initiated by adding a water-soluble bisfunctional crosslinker (bis(sulfosuccinimidyl) suberate or BS3) (Pierce) to a final concentration of 55 mM. The reaction was incubated for 1 h at 25 8C and then quenched by adding an excess of Tris–HCl. The excess salt was removed by adding trichloroacetic acid to a final concentration of 5% (w/v) followed by centrifugation. The protein precipitate was resuspended in 20 ml of water, mixed with 5 ml of SDS sample buffer, and then loaded onto a Tricine SDS-PAGE (12% (w/v) acrylamide) gel.

Acknowledgements Financial support was provided by the NIH (GM58670 and RR13879 to A.P.H.; CA75253 to L.S.; CA54174 to the Macromolecular Structure Shared Resource of the San Antonio Cancer Institute), the Robert A. Welch Foundation (AQ1431 to A.P.H.), and an International Research Scholar Grant from the Howard Hughes Medical Institute (F.L.-C.) and the Consejo National de Ciencia y Tecnologı´a (37749N to F.L.-C.).

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Edited by J. Karn (Received 13 July 2005; received in revised form 1 October 2005; accepted 5 October 2005) Available online 27 October 2005