ligand-induced dimerization of the extracellular domain of the tgf-β receptor type ii

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 224, 709–716 (1996) ARTICLE NO. 1088 Ligand-Induced Dimerization of the Extracellular Domain of the TGF-b Receptor Type II Odile Letourneur, 1 Jean-Franc ¸ois Goetschy, Michel Horisberger, and Markus G. Gru ¨tter Core Drug Discovery Technologies, Pharmaceutical Division, Ciba Geigy Ltd., CH-4002 Basel, Switzerland Received June 7, 1996 Transforming growth factor-b (TGF-b) signals by mediating the association of two distinct transmem- brane serine/threonine kinase receptors, the type I (TbRI) and II (TbRII). Here, we took advantage of recombinant human TbRII extracellular domain (TbRII-ED) to analyze TGF-b/TbRII complex formation which is the initial event in the construction of a signaling complex. We found that recombinant TbRII- ED binds TGF-b3 more efficiently than TGF-b2 and therefore maintains the native TbRII binding selectivity for the different TGF-b isoforms. Biochemical analysis showed that free TbRII-ED is expressed as a monomer. Upon ligand binding, both TGF-b3 and -b2 isoforms induce homodimerization of TbRII-ED, each TGF-b subunit being able to bind one TbRII-ED molecule. These results suggested that ligand dependent receptor dimerization may be an important early step in the TGF-b signaling complex formation. q 1996 Academic Press, Inc. Member molecules of the transforming growth factor-b (TGF-b) family have important functions in growth regulation, differentiation, migration, and adhesion of various cell types. The three mammalian TGF-b isoforms: TGF-b1, -b2 and -b3 are 25 kDa disulfide-linked homodimers sharing 71-76% amino acid identities and nearly identical properties (1). TGF-bs exert their pleiotropic effects through binding to specific cell surface receptors and binding proteins, which include type I receptor (TbRI), type II receptor (TbRII) and betaglycan (TbRIII) (2- 4). TbRI and TbRII are two transmembrane serine/threonine kinases which mediate the multiple effects of TGF-b, while TbRIII may be involved in presentation of the ligand to the signaling receptors (2, 5-7). The activation mechanism of the TGF-b receptors has been described (8). TGF-b binds directly to TbRII which is a constitutively active kinase and bound TGF-b is then recognized by TbRI which is recruited into a very stable ternary complex (8, 9). At this point, TbRI becomes phosphorylated by TbRII and phosphorylation allows TbRI to propagate the signal to downstream substrates (8). TbRII binds ligand indepen- dently of TbRI but cannot signal in the absence of type I receptors while TbRI requires TbRII to bind TGF-b (10). The sequence of events in the formation of the heteromeric signaling receptor complex as well as the stoichiometry of TbRI and TbRII involved in this complex remains uncertain. Indeed, it has been recently shown that TbRI and TbRII already have an inherent affinity for each other in the absence of ligand and therefore that TGF-b could activate a preformed heteromeric complex (11). A complex in which two molecules each of TbRI and TbRII is formed after binding of TGF-b has been proposed (9, 12). This is supported by the fact that a constitutive homodimerization of TbRII, independent of ligand binding, has been reported (12, 13). 1 Correspondence: Fax:/41 61 696 93 01. Abbreviations: TGF-b(s), transforming growth factor b(s); TbR, TGF-b receptor; TbRII-ED, TbRII external domain; TbRII-EDi, TbRII-external domain produced from baculovirus infected insect cells; TbRII-EDm, TbRII- external domain produced from a mouse myeloma cell line; DSS, disuccinimidyl suberate. 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 709

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Page 1: Ligand-Induced Dimerization of the Extracellular Domain of the TGF-β Receptor Type II

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 224, 709–716 (1996)ARTICLE NO. 1088

Ligand-Induced Dimerization of the ExtracellularDomain of the TGF-b Receptor Type II

Odile Letourneur,1 Jean-Francois Goetschy, Michel Horisberger, and Markus G. Grutter

Core Drug Discovery Technologies, Pharmaceutical Division, Ciba Geigy Ltd., CH-4002 Basel, Switzerland

Received June 7, 1996

Transforming growth factor-b (TGF-b) signals by mediating the association of two distinct transmem-brane serine/threonine kinase receptors, the type I (TbRI) and II (TbRII). Here, we took advantage ofrecombinant human TbRII extracellular domain (TbRII-ED) to analyze TGF-b/TbRII complex formationwhich is the initial event in the construction of a signaling complex. We found that recombinant TbRII-ED binds TGF-b3 more efficiently than TGF-b2 and therefore maintains the native TbRII binding selectivityfor the different TGF-b isoforms. Biochemical analysis showed that free TbRII-ED is expressed as amonomer. Upon ligand binding, both TGF-b3 and -b2 isoforms induce homodimerization of TbRII-ED,each TGF-b subunit being able to bind one TbRII-ED molecule. These results suggested that liganddependent receptor dimerization may be an important early step in the TGF-b signaling complex formation.q 1996 Academic Press, Inc.

Member molecules of the transforming growth factor-b (TGF-b) family have importantfunctions in growth regulation, differentiation, migration, and adhesion of various cell types.The three mammalian TGF-b isoforms: TGF-b1, -b2 and -b3 are 25 kDa disulfide-linkedhomodimers sharing 71-76% amino acid identities and nearly identical properties (1).

TGF-bs exert their pleiotropic effects through binding to specific cell surface receptors andbinding proteins, which include type I receptor (TbRI), type II receptor (TbRII) and betaglycan(TbRIII) (2- 4). TbRI and TbRII are two transmembrane serine/threonine kinases whichmediate the multiple effects of TGF-b, while TbRIII may be involved in presentation of theligand to the signaling receptors (2, 5-7). The activation mechanism of the TGF-b receptorshas been described (8). TGF-b binds directly to TbRII which is a constitutively active kinaseand bound TGF-b is then recognized by TbRI which is recruited into a very stable ternarycomplex (8, 9). At this point, TbRI becomes phosphorylated by TbRII and phosphorylationallows TbRI to propagate the signal to downstream substrates (8). TbRII binds ligand indepen-dently of TbRI but cannot signal in the absence of type I receptors while TbRI requires TbRIIto bind TGF-b (10).

The sequence of events in the formation of the heteromeric signaling receptor complexas well as the stoichiometry of TbRI and TbRII involved in this complex remains uncertain.Indeed, it has been recently shown that TbRI and TbRII already have an inherent affinityfor each other in the absence of ligand and therefore that TGF-b could activate a preformedheteromeric complex (11). A complex in which two molecules each of TbRI and TbRIIis formed after binding of TGF-b has been proposed (9, 12). This is supported by the factthat a constitutive homodimerization of TbRII, independent of ligand binding, has beenreported (12, 13).

1 Correspondence: Fax:/41 61 696 93 01.Abbreviations: TGF-b(s), transforming growth factor b(s); TbR, TGF-b receptor; TbRII-ED, TbRII external

domain; TbRII-EDi, TbRII-external domain produced from baculovirus infected insect cells; TbRII-EDm, TbRII-external domain produced from a mouse myeloma cell line; DSS, disuccinimidyl suberate.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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A better understanding of the interaction between TGF-b and its receptors could help inthe design of clinically useful TGF-b antagonists. Here, we analyzed the association betweenTGF-b and recombinant TbRII-ED produced in baculovirus-infected insect cells or secretedby a mouse myeloma cell line, as a step towards structure studies of TGF-b /TbRII-EDcomplexes (14-16). The current report documents that TGF-b3 and -b2 effects dimerizationof TbRII-ED in solution.

MATERIALS AND METHODS

Materials. Recombinant human TGF-b2 (CGP 46 613) and TGF-b3 (CGP 46 614) were produced by Ciba GeigyLtd, Basel. Human recombinant external domain of TbRII produced by a mouse myeloma cell line, NSO (TbRII-EDm) was purchased from R&D Systems, Abingdon, UK (Cat. Nr. 241-R2-025) (17).

Radio-labeling of TGF-b3. Recombinant human TGF-b3 was iodinated using the Iodo-beads (Pierce) according tothe manufacturer’s procedure with a specific activity of 750 000 cpm/pmole TGF-b3. [125I] TGF-b3 was stored in 20mM acetic acid, pH 3.4 containing 20 % isopropanol and 0.1 % bovine serum albumin.

Preparation of polyclonal anti-TbRII antibodies. Rabbits were immunized with a synthetic peptide, correspondingto amino acid residues Lys30-Asn41 of the human TbRII, synthesized as a multiple antigenic peptide resin MAP8(Neosystem S.A., Strasbourg). The resulting specific polyclonal anti-peptide antibodies were affinity purified using acolumn of peptide coupled to Affigel 10 beads (BioRad).

Cell culture. Insect cells (H5) were grown in suspension in Ex-Cell 400TM medium supplemented with 5% fetalcalf serum. The cells were infected with recombinant baculovirus at a multiplicity of infection of 2-3 and harvestedat 66 hours post-infection.

BALB/c 3T3 mouse fibroblasts (provided by D. Cox, Ciba Geigy Ltd) were grown in Dulbecco‘s Modified EagleMedium (DMEM) supplemented with 10 % fetal calf serum.

Cloning, expression and purification of the extracellular domain of the human TbRII. The cDNA encoding the fullextracellular domain of the human TbRII-ED (Ile24-Asp159) without the N-terminal signal sequence and followedby six consecutive histidine residues at the C-terminal of the protein (6 1 His affinity tag) was cloned into thebaculovirus transfer vector (pVL1392) downstream of the polyhedrin promotor (Goetschy et al., in preparation).Recombinant baculovirus carrying the TbRII-ED cDNA were generated as described (18). TbRII-EDi accumulatedin the cytoplasmic compartment of recombinant baculovirus infected-insect cells and was affinity purified on a Nickelcolumn (Ni-NTA), from the cell lysates made in presence of 100 mM iodoacetamide, following manufacturer’sinstructions (Qiagen).

Binding studies. [125I] TGF-b3 binding studies were as described (19). Briefly, near confluent BALB/c 3T3 fibroblastsmonolayers were incubated with [125I] TGF-b3 (2 1 10011 M) and unlabelled TGF-b3 or TbRII-ED. After a 45 minincubation at 377C, cell-associated radioactivity was measured in a gamma counter after solubilization of the cellmonolayers in a 1% Triton X-100 containing buffer. Radioactivity was determined by subtracting from the totalamount of radioligand bound, the amount bound in the presence of 250-500 fold excess of the corresponding unlabelledligand.

Covalent cross-linking. TbRII-EDm (2 mg, Ç8 mM) or TbRII-EDi (2 mg, Ç10 mM) in 10 ml of buffer (50 mMHepes, 100mM NaCl, pH 7.5, containing 0.05% Chaps and 0.1% bovine serum albumin) was added to tubes containing[125I] TGF-b3 (5 ng, 20 nM) with or without unlabelled TGF-b3 or TGF-b2 (2 mg, 8 mM) stored in 20 mM aceticacid, 20 % isopropanol and previously dried in a speed vac. After an overnight incubation at 47C, samples were cross-linked by incubation at 47C for 30 min with 0.162 mM disuccinimidyl suberate (DSS, Pierce). The reaction wasquenched by addition of Laemmli sample buffer. Bovine serum albumin was added in all steps to avoid unspecificcross-linking reaction between TGF-b and TbRII-ED.

Two-dimensional non-reducing/reducing SDS–PAGE and immunoblotting. Cross-linked proteins were mixed withLaemmli sample buffer, and resolved under non- reducing conditions on a 13 % poly-acrylamide gel. After the firstdimension, individual lanes of the gel were cut out and incubated with 0.5% dithiothreitol and 0.1% SDS in 125 mMTris-HCl buffer pH 6.8 for 1 h at room temperature and then laid horizontally on the stacking 13% poly-acrylamide geland subjected to the second dimension. The separated proteins were electrophoretically transferred onto nitrocellulosemembrane HybondTM (Amersham Life Science) and analyzed by immunoblotting or autoradiography. After transfer,nitrocellulose membranes were blocked in phosphate buffered saline, pH 7.5, containing 0.1% tween 20 (PBS-tween)and 5% non-fat milk for one hour at room temperature and then incubated for one hour in PBS-tween, 0.5% milk,containing 0.45 mg/ml of purified anti-TbRII antibodies. After washing, membranes were incubated for 45 min inPBS-tween containing donkey anti-rabbit antibodies horseradish peroxydase linked F(ab*)2 at the dilution 1/4000(Amersham Life Science) and developed using enhanced chemiluminescence according to the manufacturer’s instruc-tions (Amersham Life Science).

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FIG. 1. Cross-competition binding of [125I] TGF-b3 on native TGF-b receptors found on 3T3 fibroblasts. Bindingof [125I] TGF-b3 (2 1 10011 M) to native receptors was competed by increasing amounts of either unlabelled TGF-b3 (l), TbRII-EDm (h), or TbRII-EDi (s). Bound radioactivity was measured in duplicate. Relative specific bindingis plotted as percent of specific binding of [125I] TGF-b3 (A). Scatchard analysis of TGF-b3 binding to native TbR(s)(B).

RESULTS AND DISCUSSION

Recombinant TbRII-ED Inhibits TGF-b3 Binding onto Cell Surface Receptors

To analyze TGF-b/TbRII-ED complex formation in solution we made use of purified recom-binant human external domain of TbRII (TbRII-ED) produced by baculovirus-infected insectcells (TbRII-EDi) or secreted by a mouse myeloma cell line (TbRII-EDm). First, to assessthe activity of both recombinant TbRII-EDs, we tested whether they could inhibit the bindingof TGF-b3 onto native receptors. As already described, TGF-b3 closely resembles TGF-b1in its binding characteristics to cell surface receptors and TbRII-ED, while TGF-b2 is poorlyrecognized by TbRI and TbRII (1-4, 17, 20). 3T3 fibroblasts were incubated with [125I]TGF-b3 and increasing amounts of unlabelled TGF-b3 or recombinant TbRII-ED (Fig. 1A).

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Scatchard analysis (Fig.1B) indicated that the mouse 3T3 fibroblast cell line has a single classof binding sites with a dissociation constant (Kd) between 2-5 1 10011 M and Ç 40-50,000TGF-b3 binding sites/cell which is consistent with previously published value (21). It wasexpected that a large excess of soluble receptor might be needed to inhibit the labeling of thecell surface receptors (TbRI, II, and III) found on 3T3 fibroblasts as native receptors couldcooperate together to bind TGF-b with higher affinity (5, 22). This expectation was confirmedsince we found that a molar excess of Ç380 TbRII-EDm and Ç16700 TbRII-EDi overmembrane receptor was required to inhibit 50% of the TGF-b3 binding. Indeed, TbRII-EDmwas able to inhibit the TGF-b3 association with native TGF-b receptors with an IC50 Ç8 nM(200 ng/ml) and TbRII-EDi with an IC50 Ç350 nM (7 mg/ml) (Fig. 1A). Similar values wereobtained whether ligand and soluble receptor were incubated with the cells for 4 hours at 47Cor 45 min at 377C which indicate that ligand internalization and down-regulation of TbRs didnot interfere with the interpretation of our data (23 ). These results suggest that TbRII-EDmmore efficiently binds TGF-b3 than TbRII-EDi does.

Ligand-Induced Dimerization of TbRII-ED

Next, we investigated the formation of complexes between TbRII-ED and [125I] TGF-b3after covalent cross-linking with the chemical cross-linking agent disuccinimidyl suberate(DSS) and two-dimensional nonreducing/reducing SDS-PAGE analysis followed by autoradi-ography or immunoblotting with anti-TbRII antibodies (Fig. 2).

Cross-linked TbRII-EDm was identified by immunoblotting with anti-TbRII antibodies asa 22-32 kDa heterogeneous monomer resolved mainly into two bands of apparent molecularmass (m) 22 and 29 kDa (Fig. 2G). The TbRII-EDm heterogeneity appeared mainly due toglycosylation as deglycosylation with N-glycanase yields a molecular mass specie close to thepredicted m (15.4 kDa) of the core protein (results not shown). When TbRII-EDm was incu-bated with [125I] TGF-b3 (20nM) and unlabelled TGF-b3 (8 mM, TGF-b3/TbRII-EDm ratio1:1) before cross-linking, complexes were observed after autoradiography (Fig. 2E) and recog-nized by anti-TbRII antibodies (Fig. 2H). According to their respective migration, the 48-63kDa unreduced complexes (Fig. 2D and E) correspond to one TGF-b3 dimer associated withone receptor, while the 74-100 kDa unreduced complexes are composed of one TGF-b3 dimerassociated with two receptor molecules. After reduction of the disulfide bonds in the TGF-b3dimers, part of the 48-63 kDa complexes was separated into one TGF-b3 subunit cross-linkedto one receptor molecule (m 35-43 kDa) and one free TGF-b3 subunit, while part of the 74-100 kDa complexes was separated into two complexes, each composed of one TGF-b3 subunitcross-linked with one receptor molecule (Fig. 2D, E, H). The heterogeneity of each complexspecies reflects the heterogeneity of the TbRII-EDm monomer.

When TbRII-EDm was incubated with [125I] TGF-b3 (20nM) and unlabelled TGF-b2 (8mM), the complexes between TbRII-ED and [125I] TGF-b3 were still observed and immunoblot-ting revealed the presence of small amount of complexes between TGF-b2 and TbRII-EDm(Fig. 2F, I). These results suggest that TbRII-EDm recognizes TGF-b2 albeit not with thesame efficiency as TGF-b3 (compare Fig. 2I and H).

At high concentration, TGF-b is weakly soluble and precipitates in neutral or mildly basicbuffer. As a consequence, the amount of [125I] TGF-b3 cross-linked resolved by two dimen-sional gels appeared weaker in presence of unlabelled TGF-b3 or -b2 than without (compareFig. 2A, B and C). However, the amount of [125I] TGF-b3 associated with TbRII-EDm wasalmost the same in the presence or absence of unlabelled TGF-b3 or TGF-b2 (see Fig. 2D,E, F). This suggests that the interaction between TGF-b and TbRII-EDm allows tsolubilizationof the ligand.

To determine whether the different TGF-b/TbRII-ED complexes observed were specific toTbRII-EDm or could correspond to a general characteristic of the TbRII extracellular domain,

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FIG. 2. Two-dimensional non-reducing/reducing SDS–PAGE analysis of TGF-b/TbRII-EDm complexes. TbRII-EDm (2 mg, Ç8 mM) was incubated with [125I] TGF-b3 (5 ng, 20 nM) alone (D, G) or in presence of unlabelledTGF-b3 (E, H) or TGF-b2 (2mg, 8 mM) (F, I) and followed by cross-linking with DSS. Proteins were then resolvedby two dimensional non-reducing/reducing (NR/R) SDS–PAGE (first and second dimension 13% acrylamide), trans-ferred to nitrocellulose and visualized using anti-TbRII antibodies (G, H, I) or by autoradiography (D, E, F). Controlexperiments without TbRII-ED are shown (A, B, C). The positions of the molecular weight markers expressed as10031 Mr are indicated to the right of the gels as well as the positions of TGF-b3 dimer crosslinked (Tdi), TGF-b3mono-unit (Tmo), TbRII-ED (R), complexes between one TGF-b3 mono-unit and one TbRII-ED molecule (Tmo-R), complexes between one TGF-b3 dimer and one TbRII-ED molecule (Tdi-R), complexes between one TGF-b3dimer and two TbRII-ED molecules (Tdi-2R). Note that TGF-b3 mono-units migrate with the electrophoresis gelfronts and were off the gels in (B), (D), and (E).

we further analyzed after cross-linking, the two dimensional SDS-PAGE pattern of complexesbetween TGF-b and TbRII-ED produced by insect cells. Immunoblotting indicated that TbRII-EDi without ligand was found after cross-linking as a monomeric and homogeneous 20 kDaprotein (Fig. 3D) not sensitive to glycosydase (not shown). Based on the TGF-b/TbRII-EDicomplexes migration pattern (Fig. 3A), we interpreted the 44-54 and 64-90 kDa unreducedcomplexes as being one [125I] TGF-b3 dimer associated with one or two TbRII-EDi moleculeswhich could be reduced in 35-41 kDa complexes corresponding to one TGF-b3 subunit associ-ated with one TbRII-EDi molecule. Unlabelled TGF-b3 (8 mM) could compete with theformation of [125I] TGF-b3/TbRII-EDi complexes (compare Fig. 3B and 3A) while TGF-b2(8 mM) only partially competed with the formation of [125I] TGF-b3/TbRII-EDi complexes(compare Fig. 3C and 3A). Immunoblotting with anti-TbRII was not sensitive enough to detectTGF-b3 or -b2 /TbRII-EDi complexes, suggesting that only a small amount of TbRII-EDi

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FIG. 3. Two-dimensional non-reducing/reducing SDS–PAGE analysis of TGF-b/TbRII-EDi complexes. TbRII-EDi (2mg, 10 mM) was incubated with [125I] TGF-b3 alone (5 ng, 20 nM) (A, D) or in presence of unlabelled TGF-b3 (B, E) or TGF-b2 (2mg, 8 mM) (C, F) and followed by cross-linking with DSS. Proteins were then analyzed bytwo dimensional non-reducing/reducing (NR/R) SDS-PAGE followed by autoradiography (A, B, C) or immunoblottingwith anti-TbRII antibodies (D, E, F). See Fig. 2 for interpretation of the spots.

(1% or less) was able to bind the ligand and be cross-linked under our experimental conditions(see Fig. 3D, E and F).

Taken together, cross-linking experiments and competition of [125I] TGF-b binding on mem-brane receptors by TbRII-EDs suggest that TbRII-EDi binds less efficiently TGF-b thanTbRII-EDm. At the contrary to TbRII-EDi, TbRII-EDm is glycosylated as observed for thenative receptor in mammalian cells (17). It has been suggested that the glycosylation sitescould participate in the structure of the TGF-b receptor binding site (16). Alternatively, glyco-sylation could play a role during conformational maturation of TbRII-ED as described forother receptors (24, 25) and consequently, unglycosylated TbRII-EDi could be partly mis-folded. However, both TbRII-EDm and TbRII-EDi present a preferential binding for the TGF-b3 isoform over TGF-b2. This is in agreement with previously published observations for themembrane and soluble receptors (3, 17, 20). Both TGF-b2 and TGF-b3 are able to induceTbRII-ED dimerization upon binding (Fig. 2 and 3). The high efficiency of DSS to cross-link complex composed of one TGF-b dimer and two receptors suggest that this componentcorresponds to the preferred ligand-receptor association that occurs in solution (see Fig. 2D-I and Fig. 3A). Complexes containing more than two receptors and one TGF-b3 or -b2 werenot found (Fig. 2 and 3).

Previous reports (13) have shown that on cells cotransfected with two differently epitope-tagged type II receptors, homo-oligomers of TbRII exist on the cell surface even prior tothe addition of ligand. The present study has not given any proof of the existence of inter-receptor binding after cross-linking in the absence of ligand but indicates that TGF-b is

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the bridge linking two receptor extracellular domains. Therefore, TbRII transmembraneand/or cytoplasmic domain could be involved in the TbRII oligomerization process ob-served on the cell surface (12, 13). It would be interesting to know if dimerization of theextracellular domain of the TbRII upon ligand binding induces conformational changes ofthe preexistent homodimeric TbRII found at the cell surface as suggested for the EpidermalGrowth Factor receptor (26).

Ligand-induced receptor dimerization has been demonstrated as an essential mechanism ofsignal transduction for members of the tyrosine kinase receptor family. Typically, occupationof the receptors by ligands induces dimerization of adjacent extracellular receptor domainsand leads to interactions and rapid transphosphorylation between the cytoplasmic receptordomains (27-29). As a result, a cascade of signaling events is activated and leads to the variousligand-stimulated biological responses. In the case of TGF-b receptors, which are part of theserine/threonine kinase receptor family, it has been shown that TbRII is already autophosphory-lated before ligand binding and that TbRI has to be recruited into the preformed TGF-b/TbRIIcomplex to be phosphorylated by TbRII and induce signaling (8). Therefore, it would beinteresting to assess whether dimerization of TbRII upon ligand binding is a prerequisite tothe formation of a stable TGF-b/TbRII/TbRI signaling complex or in contrast if the formationof TGF-b/TbRII homodimer could prevent further recruitment of TbRI. In both cases thedimerization of TbRII could modulate the signaling capacity of the complexes. Future studieson the interactions of TbRI with TbRII and TGF-b will be required to address these issues.

ACKNOWLEDGMENT

We thank John Priestle for his assistance in revising the manuscript.

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