Distinct Roles of the Intracellular Domains of Transforming Growth Factor+ Type I and Type I1 Receptors in Signal Transduction*

Transforming growth factor+ (TGF-p) transduces sig- nals through binding to type I (TPR-I) and type I1 (TPR-11) serinekhreonine kinase receptors. TPR-I requires TPR-I1 for ligand binding, whereas TPR-I1 requires TPR-I for signaling. We generated two different chimeric TGF-P receptors, Le. TPR-1.2 containing the extracellu- lar domain of TpR-I and the intracellular domain of TPR-11, and TPR-2.1 containing the extracellular domain of TpR-I1 and the intracellular domain of TPR-I. TPR-2.1 bound '2sI-TGF-P1 alone, whereas TPR-1.2 bound the ligand only in the presence of TPR-I1 or TPR-2.1. When transfected into a mutant mink lung epithelial cell line that lacks functional TPR-11, TPR-I1 cDNA, but not TPR- 2.1 cDNA, restored the responsiveness to TGF-61 with regard to transcriptional activation of plasminogen activator inhibitor-1 gene promoter and 12-0-tetradecano-ylphorbol-13-acetate-responsive elements. In a mutant mink lung epithelial cell line lacking TPR-I, TPR-I cDNA stimulated promoter activity, but the TPR-1.2 to immunoprecipitation using the antiserum to TPR-11. The gels were fixed, dried, and subjected to autoradiography. Luciferase Assay-The p3TP-Lux promoter-reporter construct, containing a region of the human plasminogen activator inhibitor-1 gene promoter and three sets of 12-0-tetradecanoylphorbol-13-acetate-re- sponsive elements, and which is suitable for quantification of transcriptional activation by TGF-P1 (6, 13), was obtained from J. Massague. p3TP-Lux was co-transfected into mutant MvlLu cells together with the plasmids containing TGF-P receptor cDNAs. Luciferase activities in the cell lysates after stimulation by various concentrations of TGF-P1 were measured using the luciferase assay system (Promega) and a luminometer (model 1250; LKB) (6).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
TGF-Ps exert their effects through binding to various cell surface receptors and binding proteins (2,3). Among these, type I (TPR-I) and type I1 (TPR-11) receptors are most important for signal transduction. After ligand binding, TPR-I and TOR-I1 form a heteromeric receptor complex, and transduce signals (4-7). Thus, TPR-I1 can bind ligands in the absence of TPR-I but requires TPR-I for signaling. TPR-I, on the other hand, cannot bind ligands in the absence of TPR-11. Recent data suggest that ligand binding induces formation of a hetero-oligomeric receptor complex, most likely a heterotetramer composed of two molecules each of TPR-I and TOR-I1 (8). Moreover, it was shown that TPR-I1 is present as a homo-oligomer, presumably a homodimer, in the absence of the ligand (9, 10).
Both TpR-I and TPR-I1 are serine/threonine kinase receptors (11)(12)(13)(14). The overall structures of TPR-I and TPR-I1 are similar and consist of relatively short extracellular domains with cysteine-rich regions, followed by single transmembrane domains, and intracellular regions containing serinekhreonine kinase domains. The serinehhreonine kinase domains of TPR-I and TPR-I1 have 41% amino acid sequence similarity, but the sequence similarities are less in other parts of the cytoplasmic domains. Two kinase inserts are observed at analogous positions in the kinase domains of TPR-I and TPR-11. In the region preceding the kinase domain of TPR-I, there is a glycine-and serine-rich sequence, termed GS domain, which is conserved in other type I receptors for proteins in the TGF-P superfamily (2, 3), but is not present in TPR-11. After the C termini of the kinase domains, TPR-I1 has a tail of 24 amino acid residues, whereas that of TPR-I is composed of only 5 amino acid residues.
The functional roles of the intracellular regions of TpR-I and TPR-I1 are not fully elucidated. The kinase activities of type I1 and type I receptors are essential for signaling activity at least for the growth inhibition signal (6, 13, 15, 16). However, the C-terminal tail and the kinase inserts of TPR-I1 are not required for signal transduction (17). The serinekhreonine kinase of TPR-I1 is constitutively active. Recent data suggest that after ligand binding and formation of a heteromeric receptor complex, TPR-I1 transphosphorylates the GS domain of TpR-I, which may then activate the TPR-I kinase (15). In order to investigate whether the intracellular domains of TPR-I and TPR-I1 have distinct functional roles in the heterooligomeric complex, or whether they are interchangeable, we generated two different chimeric receptors in which the intracellular domains of TPR-I and TPR-I1 were swapped. In the present study, we showed that the intracellular regions of both TPR-I and TPR-I1 are necessary for signaling. EXPERIMENTAL PROCEDURES Construction of TGF-P Receptor Chimeras-cDNAs for human TPR-I1 (11) and TOR-I (12) were used to create the chimeric TGF-p receptor constructs, TPR-1.2 and TpR-2.1. A polymerase chain reaction (PCR) product encompassing the extracellular and transmembrane domains of TpR-I (amino acids l-153), flanked by an EcoRI site in the N terminus and an HpaI site in the C terminus, was obtained using a Perkin-Elmer DNA Thermal Cycler with Pyrococcus furiosus DNA polymerase (Stratagene) and with linearized human TOR-I cDNA as a template. The primers encompassed nucleotides -5 to 15 for the sense primer and 458-448 for the antisense primer of the TpR-I cDNA sequence. The numbering of nucleotides starts from the first nucleotide of the open reading frames of human TpR-I and TOR-11. Deletion of nu- cleotides 8-64 of the human TPR-I cDNA sequence was observed in the resulting PCR product; therefore, an EcoRI-XmaI fragment of the human TPR-I cDNA (nucleotides -92 to 84) was ligated to the PCR product, which was then ligated to the intracellular domain ofTPR-I1 (amino acids 192-567) at the HpaI site. The resulting TPR-1.2 cDNA was cloned into the pcDNA 3 expression vector (Invitrogen).
A PCR product encompassing the extracellular and transmembrane domains of TPR-I1 (amino acids 1-191) and a part of the intracellular domain of human TPR-I (amino acids 151-238), flanked by an EcoRI site in the N terminus and anXbaI site in the C terminus, was prepared as follows: the primers encompassing nucleotides -11 to 8 of TPR-I1 (sense primer), and nucleotides 467-451 of TpR-I followed by 573-559 of TPR-I1 (antisense primer), and those encompassing nucleotides 559-573 of TPR-I1 followed by 451-467 of TPR-I (sense primer), and nucleotides 714-695 of TPR-I (antisense primer), were amplified using human TPR-I1 and TPR-I cDNAs as templates, respectively. Aliquots of the resulting products were mixed and reamplified using the sense primer (nucleotides -11 to 8 of TPR-11) and the antisense primer (nucleotides 714-695 of TPR-I). The PCR product was then ligated to the intracellular domain of TPR-I (amino acids 239-503) in the pSV7d expression vector at the XhaI site (12). A catalytically inactive TPR-11, termed TPR-II(K277S), was generated by mutation of the ATP-binding Lys-277 to Ser using the altered sites in vitro mutagenesis system (Promega).
Cells and Dunsfection-COS-1 cells were obtained from American Q p e Culture Collection. Chemically mutagenized mink lung epithelial (MvlLu) cells (R mutant clone 4-2 and DR mutant clone 27a; Ref. 4) were gifts from M. Laiho and J. Massague. The cells were cultured in a 5% CO? atmosphere at 37 "C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 unitdml penicillin, and 50 pg/ml streptomycin. Transfection of plasmids into the cells was performed by the calcium-phosphate precipitation method using a mammalian transfection kit (Stratagene) (8). The cells were used for affinity labeling and cross-linking experiments 2 days after transfection or subjected to luciferase assay.
Binding, Affinity Cross-linking, and Immunoprecipitation-TGF-Pl (obtained from H. Ohashi) was iodinated by the chloramine T method (18). Binding and affinity cross-linking using disuccinimidyl suberate (Pierce) were performed as described (8). Cell lysates obtained by affinity cross-linking were directly analyzed by SDS-gel electrophoresis using gradient gels consisting of 5-12s polyacrylamide, or first immunoprecipitated using antisera against TPR-I or TPR-11, as described Formation of a hetero-oligomeric complex between TPR-I1 and TPR-2.1 in COS cells. cDNAs for TPR-I1 and TPR-2.1 were co-transfected into COS cells. After affinity labeling with ' "' 1-TGF-P1 and cross-linking, cross-linked complexes were first immunoprecipitated by preimmune serum or immune serum to TPR-I (anti-R-I ) , followed by immunoprecipitation using preimmune serum or immune serum to TpR-I1 (anti-R-ZZ). Samples were analyzed by SDSgel electrophoresis and autoradiography. i, immune serum; p , preimmune serum.
previously (8), and subjected to SDS-gel electrophoresis. For sequential immunoprecipitation, cross-linked complexes were first immunoprecipitated by the TPR-I antiserum. Immune complexes were eluted with 150 p1 of 20 mM HEPES, pH 7.5, 10 mM MnCl,, 1 mM dithiothreitol, 100 mM NaCI, 0.05% Nonidet P-40, and 10 pg of peptide used for immunization for 2 h a t room temperature (19). After centrifugation, 300 pl of the solubilization buffer (8) was added to the supernatants, which were then subjected to immunoprecipitation using the antiserum to TPR-11. The gels were fixed, dried, and subjected to autoradiography.
Luciferase Assay-The p3TP-Lux promoter-reporter construct, containing a region of the human plasminogen activator inhibitor-1 gene promoter and three sets of 12-0-tetradecanoylphorbol-13-acetate-responsive elements, and which is suitable for quantification of transcriptional activation by TGF-P1 (6, 13), was obtained from J. Massague. p3TP-Lux was co-transfected into mutant MvlLu cells together with the plasmids containing TGF-P receptor cDNAs. Luciferase activities in the cell lysates after stimulation by various concentrations of TGF-P1 were measured using the luciferase assay system (Promega) and a luminometer (model 1250; LKB) (6).

RESULTS
Ligand Binding Properties of Chimeric TGF-P Receptors-In order to investigate the ligand binding properties of the chimeric receptors TPR-1.2, which has the TOR-I extracellular domain and the TPR-I1 intracellular domain, and TOR-2.1, which has the TpR-I1 extracellular domain and the TpR-I intracellular domain, cDNAs for chimeric and wild-type receptors were transfected into COS-1 cells, and the binding of I2'I-TGF-P1 was investigated by affinity cross-linking (Fig. 1, lanes  1-9). TPR-I and TPR-1.2 did not bind the ligand when transfected singly, whereas TPR-I1 and TPR-2.1 bound TGF-P1. Cotransfection of TPR-I1 or TPR-2.1 cDNAs with TPR-I or TPR-1.2 cDNAs allowed TPR-I and TPR-1.2 to bind the ligand. After co-transfection of TPR-I1 and TPR-I cDNAs, cross-linking of '251-TGF-P1 to TPR-I1 became less efficient, as reported previously (14, 20). Immunoprecipitation of the cross-linked complexes using the TOR-I1 or TPR-I antisera (Fig. 1, lanes 10-18), supported the results obtained by cross-linking only. These results indicate that the chimeric TGF-p receptors are efficiently transported to the cell surface and bind ligand; TPR-2.1 can bind TGF-P1 by itself and enables TPR-I and TPR-1.2 to bind ligand after co-transfection. TPR-1.2 can bind TGF-P1 only in the presence of TPR-I1 or TPR-2.1.

Formation of a Hetero-oligomeric Complex between TpR-II
and TpR-8.1-TPR-I1 was shown to form an oligomeric complex on the cell surface (8)(9)(10). In order to investigate whether TPR-I1 and TPR-2.1 form a n oligomer, the cDNAs were transfected into COS cells, and cross-linked complexes were first immunoprecipitated by the antiserum to TPR-I, which recognizes TPR-2.1, and then immunoprecipitated by the TPR-I1 antiserum (Fig. 2). The TPR-I1 cross-linked complex could be observed after the sequential immunoprecipitation (Fig. 2, lane  3). In contrast, cross-linked complexes were not seen, when preimmune sera were used in the first or second precipitation (Fig. 2, lunes 1 and 2). These results indicate that TPR-2.1 forms a heteromeric complex with TPR-I1 on the cell surface in the presence of TGF-P.
Signaling Properties of Chimeric TGF-P Receptors-The signaling activities of the chimeric TGF-P receptors were investigated using the p3TP-Lux transcriptional activation assay. When the TPR-I1 cDNA was transfected into the DR mutant of MvlLu cells, which lacks functional TPR-11, the response to TGF-P was restored in these cells (Fig. 3A). On the other hand, significant transcriptional response was not observed by either TPR-2.1, a catalytically inactive mutant of TPR-II(K277S), or When the cDNAs were transfected into the R mutant cells, which lack functional TPR-I, the responsiveness to TGF-/3 was restored by TPR-I, but not by TPR-1.2 (Fig. 3B ). TPR-2.1, which formed a hetero-oligomer with TPR-I1 in the transfected COS cells, did not induce the transcriptional response by TGF-P1 after transfection into the R mutant (Fig. 3 0 . We then investigated whether co-transfection of the TPR-1.2 cDNA with the TGF-P receptor cDNAs containing the extracellular domain of TPR-11, transduced TGF-P signals using the R mutant MvlLu cells. As shown in Fig. 3B, TPR-2.1, but not TPR-11, restored the responsiveness to TGF-P1 in the presence of TPR-1.2. However, the degree of transcriptional stimulation was less than that induced by the transfection of TPR-I cDNA in the same cells.
These results indicate that heteromeric combinations of TPR-I and TPR-I1 intracellular domains, i.e. TPR-I and TPR-11, or TPR-1.2 and TPR-2.1, transduce intracellular signals upon stimulation by TGF-P1 with regard to the p3TP-Lux transcriptional response, whereas homomeric combinations of the intracellular domains induced by TPR-I and TPR-2.1, or by TPR-I1 and TPR-1.2, are ineffective.

DISCUSSION
TGF-/3 induces the formation of a heteromeric receptor complex of TPR-I1 and TPR-I, which is critical for signal transduction. However, the functional roles of the intracellular domains of TpR-I1 and TPR-I, both of which have serinehhreonine kinase activity, have not been fully elucidated. The present data show that homo-oligomerization of TPR-I intracellular domains, achieved by the transfection of the TPR-2.1 cDNA into the DR mutant cells containing endogenous wild-type TPR-I, or homo-oligomerization of the TPR-I1 intracellular domains, achieved by the transfection of TPR-1.2 cDNA into the R mutant cells containing endogenous wild-type TPR-11, do not efficiently transduce intracellular signals (Table I and Fig. 4). In contrast, hetero-oligomerization of the intracellular domains of TPR-I1 and TPR-I, achieved by simultaneous expression of TPR-I and TpR-11, or TPR-1.2 and TPR-2.1, transduces signals. These results suggest that the intracellular domains of both TPR-I and TPR-I1 have distinct functions and both are required for signal transduction.
We have recently shown that TPR-I1 and TPR-I form a hetero-oligomer, most likely a heterotetramer, after ligand binding (8). Moreover, TPR-I1 has been shown to form a homo-oligomer in the presence and absence of the ligand (9, 10). However, the homo-oligomerization of TPR-I1 observed in the R mutant cells are not enough for signal transduction (8). We show here that TPR-2.1, which can form a hetero-oligomer with TPR-11, did not induce any transcriptional response by TGF-P1 after transfection into the R mutant (Fig. 3C). Since TPR-I1 forms a homooligomer in the absence of the ligand, TPR-2.1 may form a heteromeric complex with TPR-I1 without the ligand. We did not observe any significant increase in the luciferase activity without ligand stimulation (data not shown); however, the possibility that certain signals other than the p3TP-Lux signal are transduced by the TPR-I1 and TPR-2.1 complex in the absence of the ligand has not been ruled out. These results suggest that ligand-induced association of the intracellular domains of TPR-I and TPR-I1 to higher orders of oligomers may be important for efficient signal transduction.
The mechanism of activation of the kinase domains of the TGF-P receptor complex is not fully determined. The ligand initially binds to TPR-11, which then recruits TPR-I to the ligand-TPR-I1 complex. Recent data revealed that TPR-I1 transphosphorylates the GS domain of TPR-I, which then triggers the activation of TPR-I kinase (15). Type I receptors appear to act downstream of the type I1 receptors and specify the properties of intracellular signals (21). Type I receptors, but not type I1 receptors, were shown to interact with FKBP-12, a binding protein of the macrolyde FK506 and rapamycin (22); thus, FKBP-12 may act downstream of type I receptors in signal transduction.
The present data show that hetero-oligomerization of the intracellular domains of TPR-I1 and TpR-I are required for signal transduction with regard to the p3TP-Lux transcrip-  tional activation. The possibility still remains, however, that homo-oligomerization of intracellular domains of TPR-I or TpR-I1 may transduce certain signals, other than the p3TP-Lux transcriptional activation. Our future studies will be aimed at exploring the functional roles of TPR-I1 and TPR-I intracellular domains, e.g. whether the distinct roles of the intracellular domains of TPR-I and TPR-I1 is due to a difference in substrate specificities of the two receptor kinases, or whether the difference is due to regions outside the kinase domains, such as the GS domain. It will also be important to determine the interactions of TPR-I and/or TPR-I1 with downstream signal transduction molecules.