Identification of important regions in the cytoplasmic juxtamembrane domain of type I receptor that separate signaling pathways of transforming growth factor-beta.

Proteins in the transforming growth factor-β (TGF-β) superfamily exert their effects by forming heteromeric complexes of their type I and type II serine/threonine kinase receptors. The type I and type II receptors form distinct subgroups in the serine/threonine kinase receptor family based on the sequences of the kinase domains and the presence of a highly conserved region called the GS domain (or type I box) located just N-terminal to the kinase domain in the type I receptors. Recent studies have revealed that upon TGF-β binding several serine and threonine residues in the GS domain of TGF-β type I receptor (TβR-I) are phosphorylated by TGF-β type II receptor (TβR-II) and that the phosphorylation of GS domain is essential for TGF-β signaling. Here we investigated the role of cytoplasmic juxtamembrane region located between the transmembrane domain and the GS domain of TβR-I by mutational analyses using mutant mink lung epithelial cells, which lack endogenous TβR-I. Upon transfection, wild-type TβR-I restored the TGF-β signals for growth inhibition and production of plasminogen activator inhibitor-1 (PAI-1) and fibronectin. A deletion mutant, TβR-I/JD1(Δ150-181), which lacks the juxtamembrane region preceding the GS domain, bound TGF-β in concert with TβR-II and transduced a signal leading to production of PAI-1 but not growth inhibition. Recombinant receptors with mutations that change serine 172 to alanine (S172A) or threonine 176 to valine (T176V) were similar to wild-type TβR-I in their abilities to bind TGF-β, formed complexes with TβR-II, and transduced a signal for PAI-1 and fibronectin. Similar to TβR-I/JD1(Δ150-181), however, these missense mutant receptors were impaired to mediate a growth inhibitory signal. These observations indicate that serine 172 and threonine 176 of TβR-I are dispensable for extracellular matrix protein production but essential to the growth inhibition by TGF-β.


Proteins in the transforming growth factor-␤ (TGF-␤) superfamily exert their effects by forming heteromeric complexes of their type I and type II serine/threonine kinase receptors. The type I and type II receptors form distinct subgroups in the serine/threonine kinase receptor family based on the sequences of the kinase domains and the presence of a highly conserved region called the GS domain (or type I box) located just N-terminal to the kinase domain in the type I receptors. Recent studies have revealed that upon TGF-␤ binding several serine and threonine residues in the GS domain of TGF-␤ type I receptor (T␤R-I) are phosphorylated by TGF-␤ type II receptor (T␤R-II) and that the phosphorylation of GS domain is essential for TGF-␤ signaling.
Here we investigated the role of cytoplasmic juxtamembrane region located between the transmembrane domain and the GS domain of T␤R-I by mutational analyses using mutant mink lung epithelial cells, which lack endogenous T␤R-I. Upon transfection, wild-type T␤R-I restored the TGF-␤ signals for growth inhibition and production of plasminogen activator inhibitor-1 (PAI-1) and fibronectin. A deletion mutant, T␤R-I/JD1(⌬150 -181), which lacks the juxtamembrane region preceding the GS domain, bound TGF-␤ in concert with T␤R-II and transduced a signal leading to production of PAI-1 but not growth inhibition. Recombinant receptors with mutations that change serine 172 to alanine (S172A) or threonine 176 to valine (T176V) were similar to wild-type T␤R-I in their abilities to bind TGF-␤, formed complexes with T␤R-II, and transduced a signal for PAI-1 and fibronectin. Similar to T␤R-I/JD1(⌬150 -181), however, these missense mutant receptors were impaired to mediate a growth inhibitory signal. These observations indicate that serine 172 and threonine 176 of T␤R-I are dispensable for extracellular matrix protein production but essential to the growth inhibition by TGF-␤.
The cell growth and differentiation in a multicellular organism are critically regulated by members of transforming growth factor-␤ (TGF-␤) 1 superfamily including TGF-␤, activin/inhibin, bone morphogenetic protein (BMP), Mü llerian inhibiting substance, and glial cell line-derived neurotrophic factor. TGF-␤ is a prototype in this superfamily of structurally related molecules and regulates cell proliferation, extracellular matrix formation, migration, adhesion, and many other cellular functions important for development and homeostasis (reviewed in Refs. [1][2][3][4]. Certain members of the TGF-␤ superfamily exert their biological actions through heteromeric complexes of two types (type I and type II) of transmembrane receptors with a serine/ threonine kinase domain in their cytoplasmic region (5)(6)(7)(8). To date, more than 15 receptor serine/threonine kinases have been cloned in flies through humans (reviewed in Refs. 4 and 9 -12). Among them six different type I receptors have been identified in mammals (5,8,(13)(14)(15)(16)(17)(18)(19)(20), including one TGF-␤ type I receptor (T␤R-I), two activin type I receptors (ActR-I and ActR-IB), two BMP type I receptors (BMPR-IA and BMPR-IB), and one additional type I receptor called activin receptor-like kinase-1 (also termed TGF-␤ superfamily receptor type I or R3) that has recently been shown to mediate certain signals in response to BMP-7 (osteogenic protein-1). 2 The type I receptors have similar sizes (502-532 amino acid residues) and 60 -90% amino acid sequence identities to each other in their kinase domains. In addition, type I receptors contain a conserved sequence known as the GS domain (also called type I box) in their cytoplasmic juxtamembrane region (10,11). Type I receptors are more similar to each other than they are to the known type II receptors, including TGF-␤ type II receptor (T␤R-II) and two activin type II receptors (ActR-II and ActR-IIB), and thus form a subgroup of mammalian type I receptors in the family of receptor serine/threonine kinases.
TGF-␤ initiates the signaling of its multiple responses through formation of a heteromeric complex of T␤R-I and T␤R-II. TGF-␤ binds directly to T␤R-II that is a constitutively active kinase, which then recruits T␤R-I into the complex. T␤R-II in the complex then phosphorylates the GS domain of T␤R-I, resulting in propagation of further downstream signals (21,22). The catalytic activities of the kinases of T␤R-I and T␤R-II are indispensable for signaling (22)(23)(24)(25). Mutational analyses altering serine and threonine residues in the T␤R-I GS domain have revealed that phosphorylation of certain serines and threonines by T␤R-II is essential for TGF-␤ signaling, although its signaling activity does not appear to depend on the phosphorylation of any particular serine or threonine residue in the TTSGSGSG sequence of the GS domain (22,26,27). In addition, recent identification of a constitutively active form of T␤R-I that does not require T␤R-II and TGF-␤ for signaling suggested that T␤R-I acts as a downstream signaling molecule of T␤R-II (27).
Despite the functional importance of the GS domain for initiating intracellular signals, little is known about how the signals are propagated after phosphorylation of the GS domain. Based on the knowledge of receptor tyrosine kinases, one possible mechanism could be that the phosphorylated serine and/or threonine residues in the GS domain may act as the binding sites for the intracellular substrate to be activated by the T␤R-I kinase. This hypothesis is attractive to explain the signaling mechanism for certain common effects induced by the members of TGF-␤ superfamily because the GS domain of the known type I receptors is highly conserved (10 -12). On the other hand, amino acid sequences of the GS domain of the type I receptors might be too similar to each other to confer specificities to the signals that mediate a wide variety of responses induced by the TGF-␤ superfamily. In fact, a T␤R-I chimeric receptor substituting the GS domain of ActR-I for that of T␤R-I still transduces the TGF-␤-induced antiproliferative signal, which is not mediated through intact ActR-I (27,28). Thus, certain region(s) other than the GS domain in the type I receptors may also be important for diverse signaling activities of the proteins in the TGF-␤ superfamily.
In the present study we focused on the role of the T␤R-I juxtamembrane region preceding the GS domain, and serine 172 and threonine 176 within this region were found to be essential for signaling a TGF-␤ antiproliferative response but not plasminogen activator inhibitor-1 (PAI-1) and fibronectin induction. Identification of such cytoplasmic regions important only for a limited response may suggest that at least two different signals are specified through different cytoplasmic parts of T␤R-I. cDNA Constructions-Stable expression vectors of wild-type T␤R-I and its mutant derivatives were prepared by subcloning the polymerase chain reaction (PCR)-generated cDNA fragments into pMEP4 vector, a Zn 2ϩ -inducible mammalian expression vector (25). To construct wildtype T␤R-I-pMEP4, primer RIS0-hind and primer RIAS-not were used to amplify the coding region of T␤R-I cDNA. Reaction conditions were 1 min at 94°C, 1 min at 48°C, and 2 min at 72°C for 30 cycles. The PCR products were digested with HindIII and NotI and subcloned into the pMEP4 vector. To construct the deletion mutant T␤R-I/JD1(⌬150 -181), the primers RIS0-hind and RIASdel1 were used to amplify the 5Ј part of T␤R-I cDNA fragment, and the primers RISdel5 and RIAS-not were used for the 3Ј fragment. The two primary PCR products were gelpurified, mixed, and subjected to reamplification with primers RIS0hind and RIAS-not. The secondary PCR products were digested with HindIII and NotI and subcloned into the pMEP4 vector. Likewise, for the constructions of single missense mutants T␤R-I/JM1(S165A), T␤R-I/JM2(S172A), and T␤R-I/JM3(T176V), primer RIS0-hind and the mutant antisense primer (AS-1, AS-2, and AS-3, respectively) were used to amplify the 5Ј fragments, and the mutant sense primer (S-1, S-2, and S-3, respectively) and primer RIAS-not were used to amplify the 3Ј fragments. PCR products were mixed in respective combinations and reamplified with primers RIS0-hind and RIAS-not. For T␤R-I/ JM123(S165A/S172A/T176V), PCR was performed using T␤R-I/JM1 as a template for the 5Ј fragment with primers RIS0-hind and AS-2 and using T␤R-I/JM3 as a template for the 3Ј fragment with primers S-2 and RIAS-not. The two PCR fragments were mixed and reamplified with primers RIS0-hind and RIAS-not. The SmaI-XbaI fragments of the mutant PCR products were swapped for the corresponding region of wild-type T␤R-I plasmid.
Expression vectors for bacterial expression of wild-type T␤R-I glutathione S-transferase (GST) fusion protein (GST-WT), its deletion mutant GST-JD1(⌬150 -181), and missense mutants GST-JM1(S165A), GST-JM2(S172A), GST-JM3(T176V), and GST-JM123(S165A/S172A/ T176V) were obtained by insertion of PCR-generated fragments of the corresponding cytoplasmic regions of T␤R-I into pGEX-4T-1 (Pharmacia) using their stable expression plasmids as templates with RIS1-sma or RISdel2-sma as sense primers and RIAS-not as an antisense primer. PCR conditions were 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C for 25 cycles. The resulting PCR products for the GST fusion protein constructs were digested with SmaI and NotI and ligated in-frame into pGEX-4T-1. The structures of PCR-amplified region of the recombinants were all confirmed by sequencing using a Sequenase DNA sequencing kit (U. S. Biochemical Corp.).
Cell Culture and Transfection-The Mv1Lu mink lung epithelial cells (CCL-64; American Type Culture Collection) and the R mutant Mv1Lu cells (clone 4 -2; R4 -2) (29, 30) were maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin. To generate stable transfectants expressing the various mutant forms of T␤R-I, R4 -2 cells were transfected by the calcium phosphate precipitation method using a eukaryotic transfection kit (Promega). Selection of transfected cells was performed in the presence of 120 units/ml of hygromycin B (Wako Chemicals). Resistant cell colonies were examined for the expression of T␤R-I and its mutants by the receptor affinity labeling assays using 125 I-TGF-␤1 after induction of the recombinant proteins by ZnCl 2 . More than two independent clones for each of the transfectants were subjected to the following experiments.
Receptor Binding Assay of T␤R-I Mutants-Recombinant human TGF-␤1 (Kirin Brewery Company) was iodinated using the chloramine T method as described (31). Affinity cross-linking experiments were performed as described previously with minor modifications (25). Briefly, cells were pretreated in DMEM containing 0.2% FBS with or without 100 M ZnCl 2 for 6 h, and the binding of 125 I-TGF-␤1 was allowed in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin for 3 h at 4°C. After washing the cells with PBS three times, the ligand-receptor complexes were cross-linked with 0.27 mM of disuccinimidyl suberate (Pierce). Cells were washed once with 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 10% glycerol and solubilized by incubation in TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing 1.5% of aprotinin for 20 min at 4°C. For immunoprecipitation of the cross-linked complexes, cell lysates were then incubated with an antiserum against T␤R-II (8) for 60 min at 4°C. Immune complexes were bound to protein A-Sepharose (Kabi-Pharmacia) for 45 min at 4°C, washed once with TNE buffer, and eluted by boiling in the SDS sample buffer (100 mM Tris, pH 8.8, 0.01% bromphenol blue, 36% glycerol, 4% SDS) in the presence of 10 mM dithiothreitol (DTT). The samples were analyzed by SDS-8.5% polyacrylamide gel electrophoresis and a Fuji BAS 2000 Bio-Imaging Analyzer (Fuji Photo Film).
Cell Proliferation Assay-Cells were plated into 24-well plates at 5 ϫ 10 4 cells/well in DMEM containing 10% FBS, grown overnight, and placed in DMEM containing 0.2% FBS in the presence or the absence of 100 M ZnCl 2 for 5 h. The cells were then added with TGF-␤1, incubated for additional 16 h, and pulsed with 1 Ci/ml [ 3 H]thymidine (6.7 Ci/ mmol, Amersham Corp.) for 2 h. They were fixed on ice with 12.5% trichloroacetic acid and lysed with 1 N NaOH, and the [ 3 H]thymidine incorporation into the DNA was determined by a liquid scintillation counter.
Protein Kinase Assay-25 l of glutathione-Sepharose beads that attached GST fusion proteins were washed once with kinase buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MnCl 2 , 0.5 mM DTT, 0.05% Triton X-100) and added with 25 l of kinase buffer containing 1 Ci of [␥-32 P]ATP (Amersham Corp.). The beads were incubated for 15 min at 4°C. Proteins were resolved on SDS-10% polyacrylamide gel under reducing conditions and analyzed by Bio-Imaging Analyzer.

Generation of a T␤R-I Deletion Mutant and Its Binding
Ability to TGF-␤-Based on the sequence comparison among the type I receptors, the cytoplasmic juxtamembrane region preceding the kinase domain of T␤R-I can be divided into two subregions (Fig. 1). The C-terminal half of the juxtamembrane region (leucine 177 to valine 206), which is composed of 30 amino acids and rich in serine and threonine residues (three serines and four threonines), contains the highly conserved SGSGSG core sequence and other conserved amino acids among the type I receptors. Therefore, this region was previously designated GS domain (or type I box). Recent findings revealed that phosphorylation of several serine and threonine residues in the GS domain are essential for the TGF-␤ signaling (22). The N-terminal half remnant (asparagine 150 to threonine 176) of the juxtamembrane region is of interest because this region is also rich in serine and threonine residues (two serines and three threonines out of 27 amino acids), and in contrast to the GS domain, the sequences of the corresponding region in the type I receptors are very divergent (Fig. 1A), so that this region might be involved in the specification of the downstream substrates that mediate diverse responses triggered by the proteins in the TGF-␤ superfamily. If this region is essential in T␤R-I, its deletion should prevent signaling. We deleted the 32 amino acids of T␤R-I in this region, yielding T␤R-I/JD1(⌬150 -181) (Fig. 1B). The wild-type T␤R-I and mutant T␤R-I/JD1(⌬150 -181) in pMEP4, a Zn 2ϩ -inducible vector, were stably transfected into a T␤R-I-defective Mv1Lu cell line, R4 -2 (29,30,33). The expression of the exogenous receptors and their complex formation with the endogenous T␤R-II were tested by affinity cross-linking of the cells using 125 I-TGF-␤1 followed by immunoprecipitating the ligand-receptor complexes with anti-T␤R-II antiserum. Fig. 2 shows that the T␤R-I/JD1(⌬150 -181) (65-kDa component), like the wild-type T␤R-I (70-kDa component), was able to bind TGF-␤ in a Zn 2ϩ -inducible manner and formed a physiological complex with T␤R-II (90-kDa component). Migration of the affinity labeled T␤R-I/ JD1(⌬150 -181) that was slightly faster than that of the wildtype T␤R-I complex was observed (Fig. 2) as expected from its shortened structure. A faint band observed in the uninduced wild-type T␤R-I-transfectant may be ascribed to the leaky expression of the pMEP4 vector (27).
Signaling Activity of T␤R-I/JD1(⌬150 -181)-The signaling activities of T␤R-I/JD1(⌬150 -181) were determined by testing its ability to rescue biological responses to TGF-␤ in R4 -2 cells. We first examined the induction of PAI-1 and fibronectin because these responses in the parent Mv1Lu cells are well characterized, and they were the representatives among the various matrix proteins induced by TGF-␤ (34). In Mv1Lu cells, synthesis of PAI-1 was increased by the treatment with TGF-␤ but not in R4 -2 cells transfected with the vector alone (Fig. 3A). When R4 -2 cells were transfected with the wild-type T␤R-I or T␤R-I/JD1(⌬150 -181), the cells produced PAI-1 upon treatment with TGF-␤ in the presence of ZnCl 2 (Fig. 3A). Fibronectin production by TGF-␤ was restored also in R4 -2 cells transfected with the wild-type T␤R-I but much less potently in the cells transfected with T␤R-I/JD1(⌬150 -181) (Fig. 3B). PAI-1 and fibronectin production were not stimulated in the absence of ZnCl 2 (data not shown), indicating that the signals for the induction of PAI-1 and fibronectin were rescued by the exogenous receptors.
To evaluate whether T␤R-I/JD1(⌬150 -181) is able to restore TGF-␤ antiproliferative effect, DNA synthesis assay was performed by measuring the incorporation of [ 3 H]thymidine into the DNA (Fig. 4, A and B). Upon treatment with TGF-␤, [ 3 H]thymidine incorporation into the DNA of Mv1Lu cells was inhibited dose-dependently up to 97% (Fig. 4A), whereas TGF-␤ had no effect on the [ 3 H]thymidine incorporation in the R4 -2 cells transfected with the vector alone. When R4 -2 cells transfected with the wild-type T␤R-I were treated with TGF-␤ in the presence of ZnCl 2 , [ 3 H]thymidine incorporation into the DNA was inhibited by 65-75%, whereas only a marginal inhibition was observed in the absence of ZnCl 2 . In contrast, R4 -2 cells transfected with T␤R-I/JD1(⌬150 -181) were refractory to TGF-␤ growth inhibition in the presence or the absence of ZnCl 2 (Fig. 4, A and B). These results suggested that the N-terminal half of the cytoplasmic juxtamembrane domain of T␤R-I was not required for signaling a PAI-1 response, whereas it was essential for signaling growth inhibitory activity.
Serine 172 and Threonine 176 Are Essential for Signaling Growth Inhibitory Activity-The inability of T␤R-I/JD1(⌬150 -181) to mediate a growth inhibitory signal raised the possibility that the N-terminal half of the cytoplasmic juxtamembrane domain of T␤R-I contains a site for interaction with downstream component that transduces a signal specific for growth inhibition. Alternatively, such a deletion might change the structural conformation, yielding a receptor that is unable to transduce signals even if the substrate interaction sites were preserved. To address these questions, missense mutations instead of deletion were introduced into certain serine and threonine residues in the T␤R-I juxtamembrane region that was deleted in T␤R-I/JD1(⌬150 -181). As an initial attempt, serine 165, serine 172, and threonine 176 were chosen because these serine and threonine residues were rather conserved among the type I receptors for the TGF-␤ superfamily (Fig. 1), especially in ActR-IB, which transduces growth inhibition and PAI-1 signals by activin A (28). Serine and threonine residues were mutated simultaneously or individually to alanine and valine residues, respectively, resulting in four different expression constructs including T␤R-I/JM123(S165A/S172A/T176V), T␤R-I/JM1(S165A), T␤R-I/JM2(S172A), and T␤R-I/ JM3(T176V). These constructs were stably transfected into R4 -2 cells, and their expression, TGF-␤ binding, and physical association with T␤R-II were examined by affinity crosslinking with 125 I-TGF-␤1 followed by immunoprecipitation using anti-T␤R-II antiserum (Fig. 2). All the different receptor mutants were expressed on the cell surface and bound TGF-␤ in complex with T␤R-II in a Zn 2ϩ -dependent manner.
Kinase Activity of T␤R-I and Its Mutant Derivatives in Vitro-The differences among T␤R-I and its mutant derivatives in their ability to restore responsiveness to TGF-␤ might FIG. 2. Binding of 125 I-TGF-␤1 to wild-type T␤R-I and its mutant derivatives. Parental Mv1Lu cells or R4 -2 cells transfected with wild-type T␤R-I and its mutant derivatives were pretreated with or without 100 M ZnCl 2 for 6 h, followed by affinity cross-linking with 125 I-TGF-␤1 using disuccinimidyl suberate. Cross-linked complexes were immunoprecipitated with an antiserum against T␤R-II. Immune complexes were analyzed by SDS-gel electrophoresis under reducing conditions and Bio-Imaging Analyzer. Cross-linked complexes of T␤R-II and T␤R-I/JD1(⌬150 -181) are indicated by arrows. Cross-linked complexes of wild-type T␤R-I, T␤R-I/JM123(S165A/S172A/T176V), T␤R-I/ JM1(S165A), T␤R-I/JM2(S172A), and T␤R-I/JM3(T176V) are indicated as T␤R-I with an arrow. WT, wild-type T␤R-I. be due to altered catalytic activity of their receptor kinase. To address this issue, kinase activity was determined by expressing the cytoplasmic regions of T␤R-I and its mutants as GST fusion proteins in Escherichia coli and testing their kinase activities in vitro. The protein products of wild-type T␤R-I (GST-WT) and all the mutant constructs including GST-JD1(⌬150 -181), GST-JM123(S165A/S172A/T176V), GST-JM1(S165A), GST-JM2(S172A), and GST-JM3(T176V) became phosphorylated (Fig. 5). These observations indicate that all the mutant constructs of T␤R-I used in these experiments were active as kinases at least in vitro.

DISCUSSION
Recent studies on transmembrane serine/threonine kinases have disclosed that certain members of TGF-␤ superfamily exert their multiple effects through binding to unique sets of heteromeric complexes between type I and type II receptors. In the case of TGF-␤, T␤R-II is a constitutively active kinase and capable of binding TGF-␤ in the absence of T␤R-I (22), whereas T␤R-I requires T␤R-II for the ligand binding. The T␤R-I kinase appears to be activated by formation of a hetero-oligomeric complex composed of TGF-␤, T␤R-II, and T␤R-I. In the complex, several serine and threonine residues in the GS domain of T␤R-I become phosphorylated by T␤R-II, and the phosphorylation of GS domain is essential for TGF-␤ signaling (22,26,27); however, the functional role of phosphorylated serine and threonine residues in the GS domain as well as the mechanism of signaling after the phosphorylation are largely unknown. In addition, functional importance of the T␤R-I cytoplasmic region other than the GS domain remains to be elucidated.
In the present communication, we studied the role of the N-terminally flanking region of the T␤R-I GS domain by mutating this region and testing its ability to restore the signaling activity in T␤R-I-defective R4 -2 cells. When expressed in R4 -2 cells, like wild-type T␤R-I, the deletion mutant T␤R-I/ JD1(⌬150 -181) and other missense mutants including T␤R-I/ JM123(S165A/S172A/T176V), T␤R-I/JM1(S165A), T␤R-I/ JM2(S172A), and T␤R-I/JM3(T176V) were all cross-linked with radioiodinated TGF-␤ and co-immunoprecipitated with T␤R-II (Fig. 2), indicating that this region in T␤R-I is dispensable at least for its expression and binding to TGF-␤ on the cell surface and forming a complex with T␤R-II.
The signaling activities of these mutant T␤R-I constructs were tested for some of the most characteristic responses to TGF-␤; i.e. PAI-1 and fibronectin induction and growth inhibition. Wild-type T␤R-I and all the missense mutants restored PAI-1 and fibronectin responses in R4 -2 cells (Fig. 3, A and B), indicating that serine 165, serine 172, and threonine 176 of T␤R-I are not needed to transduce a signal for PAI-1 and fibronectin induction.
Antiproliferative response was also restored by the wild-type T␤R-I and one of the receptor mutants, T␤R-I/JM1(S165A); however, the other mutants including T␤R-I/JD1(⌬150 -181), T␤R-I/JM123(S165A/S172A/T176V), T␤R-I/JM2(S172A), and T␤R-I/JM3(T176V) were unable to rescue this response (Fig. 4,  A and B). Because the wild-type T␤R-I and all the mutant T␤R-I were similar in their activities to bind TGF-␤, form a complex with T␤R-II, and phosphorylate themselves in vitro, the differences in their ability to restore the antiproliferative response does not seem to be at the level of ligand-receptor complex formation or basal kinase activity. Rather, T␤R-I/ JM123(S165A/S172A/T176V), T␤R-I/JM2(S172A), and T␤R-I/ JM3(T176V) are likely to be impaired in interacting with a specific substrate that transduces antiproliferative response but not PAI-1 and fibronectin responses.
From our present data, it is not easy to deduce the mechanistic significance of serine 172 and threonine 176 of T␤R-I in TGF-␤ signaling. Although it was reported that TGF-␤-induced phosphorylation of these residues was not detected in vivo (22), it is still possible that T␤R-II may phosphorylate these residues as minor phosphorylation site(s). Alternatively, these residues might be constitutively phosphorylated even in the absence of TGF-␤, which would not be detected as the ligand-induced phosphorylation sites. It is also possible that serine 172 and threonine 176 in T␤R-I may not be themselves phosphorylated, but their integrity is essential to maintain the proper confor-mation of T␤R-I to interact with its substrates.
It was reported that whereas Mv1Lu cells expressing SV40 T-antigen were refractory to the antiproliferative effect of TGF-␤, TGF-␤ induced the expressions of junB mRNA and extracellular matrix proteins including PAI-1, fibronectin, and thrombospondin in these cells (34,35). In addition, we have previously shown that growth inhibition and extracellular matrix production by TGF-␤ are sensitive and insensitive, respectively, to phorbol 12-myristate 13-acetate in prostatic carcinoma cells (36). These observations have suggested that the signals induced by TGF-␤ that lead to growth inhibition and to extracellular matrix production should differ at a certain step within the signaling cascade from the receptor to the nucleus. In this regard, the present study is of particular importance. T␤R-I mutants including T␤R-I/JM123(S165A/S172A/T176V), T␤R-I/JM2(S172A), and T␤R-I/JM3(T176V) had signaling activity for extracellular matrix protein responses but not growth inhibition. Although other responses including expressions of junB and thrombospondin should be determined, identification of such mutant forms of T␤R-I strongly suggests that the sig- B, inhibition of [ 3 H]thymidine incorporation in R4 -2 cells expressing wild-type T␤R-I and its mutant derivatives. Cells were pretreated with 100 M ZnCl 2 followed by incubation with 15 ng/ml of TGF-␤1 and processed as described above. The data are plotted as the average percentage of inhibition Ϯ standard deviation. WT, wild-type T␤R-I.

FIG. 5. Kinase activity of wild-type T␤R-I and its mutant derivatives in vitro.
Glutathione-Sepharose beads that attached the indicated GST fusion proteins were incubated with kinase buffer containing 1 Ci of [␥-32 P]ATP for 15 min at 4°C. Proteins were resolved on an SDS-polyacrylamide gel under reducing conditions and analyzed by Bio-Imaging Analyzer. WT, wild-type T␤R-I. nals for growth inhibition and extracellular matrix production are diverged closely at the receptor level. In conclusion, serine 172 and threonine 176 within the T␤R-I juxtamembrane region preceding the GS domain are essential for signaling the TGF-␤ antiproliferative response and might be involved in the interaction with the downstream substrate responsible for growth inhibition.