Transforming growth factor- (TGF-) ()is a secreted protein that induces a range of cell responses which affect growth and differentiation (for recent reviews see (1) and (2) ). TGF- treatment of epithelial cells results in cell cycle arrest in late G coincident with an inhibition of synthesis and/or activity of some cyclins and cyclin-dependent kinases (3, 4, 5, 6) . In mink lung epithelial cells, TGF- decreases the activities of two G cyclin-dependent kinases, cdk2 and cdk4(4, 6, 7) . TGF- down-regulates the synthesis of cdk4 and constitutive expression of cdk4 results in resistance to TGF--induced growth arrest, suggesting that cdk4 is a major downstream target in TGF--induced growth inhibition(4) . The inhibition of cdk2 activity by TGF- may involve the cdk inhibitor p27, which prevents complex formation of cyclin E/cdk2 as well as cyclin D/cdk 4(8, 9, 10) . Another cdk inhibitor p15, that is TGF--inducible, interferes with the cyclin D/cdk 4 complex in keratinocytes(11) .

While down-regulation of cdk4 and activation of the cdk inhibitors correlates with TGF--induced cell cycle arrest, other components of the cell cycle machinery are also inhibited by TGF-. Indeed, late G arrest by TGF- is associated with decreased synthesis of cdc2 (12) and cyclin A(13, 14, 15, 16, 17) . Cyclin A plays a critical role in cell cycle progression by complexing with and regulating the activities of cdc2 and cdk2. Cyclin A/cdc2 and cyclin A/cdk2 complexes also interact with other proteins involved in the regulation of the G to S transition, including pRB, the pRB-like proteins p107 and p130, and the transcription factor E2F(18, 19, 20, 21, 22) . Inhibition of pRB hyperphosphorylation by cyclin A/cdk2 may be involved in control of G progression by TGF-(23) .

In addition to its antiproliferative effect, TGF- induces the expression of many genes, including those for extracellular matrix proteins. In fact, the induction of plasminogen activator inhibitor type 1 (PAI-1) expression is often used as a biochemical marker for TGF- responsiveness. Induction of PAI-1 expression can be easily measured using a reporter plasmid in which luciferase expression is driven from the PAI-1 promoter in a TGF--dependent manner(24, 25) . This assay is often used in combination with transient transfection experiments to measure cellular responsiveness to TGF- and the corresponding receptor requirements. In contrast, no similarly convenient reporter assay has been developed to measure the growth inhibitory response of TGF-, even though considerable evidence suggests that TGF-'s effects on growth inhibition and gene expression result from divergent signaling cascades (for review, see (26) ).

The early signaling events that lead to growth inhibition or extracellular matrix production by TGF- are largely unknown. TGF- interacts with a set of cell surface receptors (for review, see Refs. 26, 27), including the type I and type II receptors which are essential for signal transduction. Both receptor types belong to an expanding family of transmembrane serine/threonine kinases(26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . Several structural features distinguish type I from type II receptors. The extracellular domain of the type I receptor is shorter than that of the type II receptor. The type I receptor also has a highly conserved GS motif (SGSGSGLP) in the juxtamembrane region preceding the kinase domain, which is absent in the type II receptor (for review, see (26) ). The type II receptor can bind TGF- by itself, but the type I receptors bind TGF- only when coexpressed with the type II receptor, presumably as a result of a physical interaction of these receptors. To date, only one type II receptor for TGF- has been identified(32) , while several type I receptors, i.e. R4/ALK5(34) , Tsk7L (33) , TSR1(31) , have been shown to bind TGF- when coexpressed with the type II TGF- receptor. Despite the structural similarity, certain functional differences among the different type I receptors have been found. For instance, only R4/ALK-5 transduces TGF- signals in Mv1Lu epithelial cells(34, 39) , while Tsk7L mediates TGF- signaling in another epithelial cell line(40) .

The type II receptors(41, 42) , and presumably also the type I receptors, constitutively homodimerize independent of ligand binding. In addition, these two receptor types interact with each other (25, 43, 44) and form a heteromeric (most likely tetrameric) complex which is stabilized by ligand binding(45) . The cytoplasmic domain of the type II receptor is constitutively phosphorylated, independent of ligand binding, due to both autophosphorylation and the activity of cytoplasmic kinases(46, 47) . In the heteromeric complex, the type II receptor kinase phosphorylates the type I receptor (46, 47) and the two types of cytoplasmic domains associate with each other(48) , further stabilizing the heteromeric interaction. This heteromeric complex is likely to be the signaling receptor unit which mediates the biological responses of TGF-.

Epithelial Mv1Lu cells lacking either type I or type II receptor are completely unresponsive to TGF- to induce growth inhibition and gene expression(49, 50) . But the TGF- responsiveness can be restored to these mutant cell lines by transfection with the missing receptor type (25, 39) . These findings support the notion that the two receptors are needed for both types of responses to TGF-. However, separate signaling pathways for the two types of responses have been observed in several studies(51, 52, 53) . For example, cells in which the type II receptor has been functionally inhibited by a truncated type II receptor are resistant to the antiproliferative effect of TGF-, yet still induce the expression of PAI-1 and other gene products(51) . Furthermore, a variety of cell lines, some of which contain defective type II receptors (e.g.(53) ), are resistant to TGF--induced antiproliferation, but remain able to induce various genes including PAI-1 in response to TGF- (see ``Discussion'').

To gain insight into the role of the type I and II receptors in TGF--mediated growth inhibition, we have developed a convenient assay to score the antiproliferative effect of TGF- using an expression plasmid in which a TGF--responsive cyclin A promoter segment drives a luciferase reporter gene. We used this reporter system in transient transfection experiments to examine the function of the TGF- receptors in the growth inhibitory response of Mv1Lu cells. Using chimeric and mutant receptors, we have dissected the role of the two receptors in the growth inhibitory response of TGF- and compared the cyclin A reporter assay with the PAI-1 reporter assay with respect to TGF- responsiveness.

MATERIALS AND METHODS

Construction of the Cyclin A-Luciferase Reporter Plasmid

Construction of Mutant and Chimeric TGF- Receptors

To amplify the RII extracellular/transmembrane (ET) domain: R2E5`, GATAGAATTCACC ATG GGT CGA GGA CTG CT; R2E3`, TCATGGATCC GAC ACG GTA GCA GTA GAA.

To amplify the RII cytoplasmic (C) domain: R2C5`, CTTAGGATCCATG CAC CGA CAG CAG AAG CTG AG; R2C3`, GATGAGGTCGACCTT GGT AGT GTT CAG AGA GC; R2K5`, TATTGGATCCATG CCC ATC GAG CTG GAC AC (for juxtamembrane deletion); R2K3`, ACTTGAGTCGAC GTC CAT ATG CTC CAG CTC AC (for tail deletion).

To amplify the RI ET domain: R4E5`, TAATCGATGAATTC ATG GAG GCA GCA TCG GC; R4E3`, AAAGATCTGGATCC GTT GTG GCA GAT ATA GAC.

To amplify the RI C domain: R4C5`, GATGAATTCATG CGC ACT GTC ATT CAC CAC CG; R4C3`, CAAACTCGAGGTCGAC CAT TTT GAT GCC TTC CTG; R4K5`, GCA GGATCC ATG GTG CTA CAA GAA AGC ATC (for juxtamembrane deletion); R4K3`, GAG GTCGAC CAA TGT CTT CTT AAT TCG CAA (for tail deletion).

To amplify the RI ET domain: R7E5`, AGCC GAATTCACATG GTC GAT GGA GTA AT; R7E3`, GTCTGGATCC CTT CCT GAG AGC AAC TCC.

To amplify the RI C domain: R7C5`, CCACGGATCCATG TTT AAG AGA CGC AAT CAA GA; R7C3`, GGCAGTCGAC ACA GTC AGT CTT CAA TTT GTC.

Amplified receptor cDNA sequences were cloned into the cytomegalovirus promoter-driven mammalian expression vectors pRK5F or pRK5M. These expression vectors are derived from pRK5 (56) but contain the sequences for a myc (57) or FLAG epitope tag (58) between the SalI and HindIII sites, respectively (data not shown). Three truncated receptors lacking the entire extracellular/transmembrane domain (ET) were generated. For example, RII(ET) contains residues 1-191 corresponding to the ET domain of RII, while RI(ET) and RI(ET) include the first 148 residues of RI or RI. Sequences containing ET domains were cloned between the EcoRI and BamHI sites, in frame with the downstream epitope tag, either myc for RI(ET) and RI(ET) or FLAG tag for RII(ET).

In the hybrid receptors, the cytoplasmic (C) domain of one receptor, as a BamHI-SalI fragment (in case of RI, the BglII site substituted for a BamHI site), was placed between the ET domain of a different receptor and the epitope tag. Considering the large number of plasmids, a detailed description of how they were made is not provided in this report; however, further details of the cloning strategies can be obtained upon request. A total of four chimeric receptors between RI and RII were created from the fusion of ET domain of one type to C domain of another. At the junction of these chimeric receptors, three extra amino acid residues (Gly Ser Met) were created corresponding to the restriction sites (BamHI/BglII and the introduced start codon ATG preceding cytoplasmic domain). The nomenclature of the chimeric receptors reflects the origin of the two fused domains. For example, RI(ET)-RII(C) contains ET domain of RI and C domain of RII receptor. All receptor clones were sequenced to ensure that there were no PCR-induced mutations.

Cell Culture

Transfection of COS-1 Cells, Immunoprecipitations, and in Vitro Kinase Reactions

For in vitro kinase assays, immunoprecipitated receptors with specific deletions in the cytoplasmic domains of RI and RII were split into two halves. While half was directly subjected to SDS-PAGE, the other half was used in a kinase autophosphorylation assay. The kinase assay was carried out at room temperature for 30 min in 1 kinase buffer (10 mM HEPES-KOH, pH 7.5, 5 mM MgCl, and 5 mM CaCl) containing 5 µCi of [-P]ATP (5000 µCi/mmol, Amersham) and then stopped by adding equal volume of 2 SDS sample buffer (80 mM Tris, pH 6.8, 3.2% SDS, 16% glycerol, 200 mM dithiothreitol, 0.02% bromphenol blue).

I-TGF- Cross-linking of Receptor Proteins

Functional Assays

R1B-L17 cells, a highly transfectable line of R1B cells were provided by Dr. J. Massagué. Transfection of L17 cells was carried out using the DEAE-dextran method as described (46) at 40% confluence in 6-well plates. For each transfection, 1 µg of receptor expression plasmid or vector control and 3 µg of reporter constructs (1.5 µg of pRKGAL + 1.5 µg of luciferase expression plasmid) were used.

RESULTS

Down-regulation of Cyclin A Expression by TGF- in Mv1Lu Cells

As a first step in developing this assay, we identified the 5` promoter fragment of the cyclin A gene that mediates down-regulation of cyclin A gene expression in response to TGF-. The upstream region of the human cyclin A gene corresponding to nucleotides -516 to +245 contains many potential regulatory elements (54) and was isolated by PCR-based amplification from human genomic DNA. We linked this promoter fragment to the luciferase coding sequence to generate plasmid pCAL2. The luciferase expression driven by this 760-bp fragment of the cyclin A promoter was compared with the luciferase activity from the 7.5-kilobase pair promoter region (nucleotides -7300 to +245) in plasmid CD1(54) . The cyclin A reporter plasmid and a -galactosidase expression plasmid pRKGal were transiently transfected into TGF--responsive Mv1Lu cells, and the luciferase activity was measured 48 h after transfection. Inclusion of pRKGal allowed calibration of the luciferase activity against the -galactosidase activity, providing normalization of transfection efficiency. Both cyclin A plasmids pCAL2 and pCD1 were found to express luciferase, and the luciferase activity was even somewhat higher when using plasmid pCAL2 (see 0 h point in Fig. 1A), indicating that this 760-bp cyclin A promoter fragment is functional in Mv1Lu cells.

Figure 1: A, cyclin A-luciferase reporter assay. Cyclin A-luciferase plasmid pCAL2 or pCD1, together with a -galactosidase expression plasmid pRKGal, were transfected into Mv1Lu cells. Four h after transfection, TGF- was added at 400 pM to the media for the defined times. Luciferase and -galactosidase activity were determined as described under ``Materials and Methods.'' Data are from two experiments with each point determined in triplicates. The vertical bars show the standard deviations. pCAL2 and pCD1 contain the human cyclin A promoter sequence from nucleotide -516 to +245 and from -7300 to +245, respectively. B, regulation of cyclin A-luciferase activity by TGF- in wild-type and mutant Mv1Lu cells. Untransfected cells or DR26 cells transfected with RII or R1B cells transfected with RI were treated (+) or untreated(-) with TGF-. Transfections, TGF- treatment (48 h), and reporter assays were carried out as in panel A. RII, type II TGF- receptor; RI, type I TGF- receptor; Mv1Lu, wild-type Mv1Lu cells; DR26, RII-deficient cells; R1B, RI-deficient cells.

To evaluate the TGF- responsiveness of the cyclin A promoter, both luciferase plasmids were transfected into Mv1Lu cells, and the normalized luciferase activity was calculated at defined time points after initiation of TGF- treatment. In Mv1Lu cells transfected with pCAL2, the luciferase activity was down-regulated by TGF- (Fig. 1A) in a manner similar to that in pCD1-transfected cells. This decreased expression from the cyclin A promoter parallels the inhibition of cyclin A expression by TGF- in Mv1Lu (15, 16, 17) and other epithelial cell lines(13) . The TGF--induced inhibition of luciferase expression was most evident when TGF- treatment was initiated within 6 h after release from contact inhibition (data not shown), and the cyclin A promoter response was maximal when the cells were exposed to TGF- for 48 h (Fig. 1A). These results indicate that the 760-bp cyclin A promoter fragment in pCAL2 contains the TGF--responsive element(s) and that this cyclin A-luciferase assay can be used as transcriptional reporter system to measure the antiproliferative effect of TGF- in transient transfection assays.

Down-regulation of Cyclin A Expression by TGF- Requires Both Type I and Type II TGF- Receptors

The Cytoplasmic Domains of Both Receptors Are Required for TGF- Responsiveness in the Cyclin A-Luciferase Assay

Figure 2: Schematic presentation of mutant TGF- receptors. The construction and nomenclature of the receptor mutants is described under ``Materials and Methods'' and ``Results.'' RI, type I TGF- receptor R4; RI, type I receptor Tsk 7L; RII, type II TGF- receptor. ET, extracellular/transmembrane domain; C, cytoplasmic domain; JM, juxtamembrane domain deletion; T, cytoplasmic tail deletion; JMT, deletion of juxtamembrane domain and cytoplasmic tail.

Figure 3: A, expression of mutant receptors in COS-1 cells. COS-1 cells were transfected with pRK5 (lane 1) or receptor expression plasmids (lanes 2-11). The receptors were immunoprecipitated from S-labeled cell lysates using antibodies specific for the FLAG (F) or myc (M) epitope as indicated. The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. The positions of the receptors and molecular weight markers are shown on the side. Lane 2, RII; lane 3, RI; lane 4, RI; lane 5, RII(ET)-RI(C); lane 6, RII(ET)-RI(C); lane 7, RI(ET)-RII(C); lane 8, RI(ET)-RII(C); lane 9, RII(ET); lane 10, RI(ET); lane 11, RI(ET). B, ligand binding of mutant receptors expressed in COS-1 cells. Binding of I-TGF- to COS-1 cells transfected with plasmids expressing wild-type or mutant receptors was examined. Cross-linked complexes were subjected to SDS-PAGE and autoradiography. Each lane represents an equal amount of protein lysate as determined by BCA assay (Bio-Rad). Migration of the receptors are indicated using the same abreviations for receptors as in Fig. 3A. Lane 1, pRK5; lane 2, RII; lane 3, RI; lane 4, RI and RII; lane 5, RI; lane 6, RIand RII; lane 7, RII(ET)-RI(C); lane 8, RII(ET)-RI(C); lane 9, RI(ET)-RII(C); lane 10, RI(ET)-RII(C) and RII; lane 11, RI(ET)-RII(C); lane 12, RI(ET)-RII(C) and RII; lane 13, RII(ET); lane 14, RI(ET); lane 15, RI(ET) and RII; lane 16, RI(ET); lane 17, RI(ET) and RII.

The ability of the mutant receptors to bind TGF- at the cell surface was determined by binding and cross-linking of I-TGF-. As shown in Fig. 3B, both RII and RII(ET)-RI(C) chimeras with their type II extracellular domain bound I-TGF- (lanes 2, 7, and 8). In contrast, RI and RI(ET)-RII(C) chimeras which have RI extracellular domains did not bind TGF- when expressed alone (lanes 3, 5, 9, and 11). However, these receptors bound ligand when cotransfected with RII (lanes 4, 6, 10, and 12). Similarly, RI(ET) and RI(ET) bound ligand only in the presence of RII (compare lanes 14 and 16 to 15 and 17), whereas RII(ET) bound TGF- by itself (lane 13). Taken together, these results indicate that the mutant receptors were readily expressed at the cell surface and displayed normal ligand binding.

The ability of the mutant TGF- receptor to signal was then examined in the cyclin A-luciferase assay using receptor-deficient Mv1Lu mutant cells. Although wild-type RII restored TGF--dependent down-regulation of cyclin A transcription in RII-deficient DR26 cells, none of the chimeric receptors (RI-RII, RI-RII, RII-RI or RII-RI), when expressed alone, induced transcriptional regulation of cyclin A-luciferase in response to TGF- (Fig. 4, left panel). Similarly, the chimeric receptors did not restore TGF- responsiveness in the RI-deficient R1B cells which contain endogenous RII, even though RI was able to restore the TGF--induced inhibition of cyclin A-luciferase activity to the cells (Fig. 4, right panel). While a single chimeric receptor was unable to confer TGF- responsiveness in the mutant cell lines, coexpression of the two chimeric receptors RII(ET)-RI(C) and RI(ET)-RII(C) resulted in TGF--dependent inhibition of luciferase expression in these cells (Fig. 4). Similarly, coexpression of both chimeric receptors led to TGF--dependent increases of transcription from the PAI-1 promoter in a PAI-1-luciferase assay (data not shown; Table 1). While the coexpression of both chimeras in mutant cells restored the TGF--dependent responses, significant levels of cyclin A inhibition and PAI-1 up-regulation were already observed in the absence of TGF- in these cells. This constitutive signaling in the absence of TGF- resulted in the lower relative level of inhibition of cyclin A expression in the presence of TGF-, when compared with that in the absence of TGF- (Fig. 4). In fact, TGF--independent signaling was also apparent when the two types of cytoplasmic domains (RII and RI) were expressed regardless of what extracellular domains they were fused to (data not shown). These results suggest that the high level expression of both cytoplasmic domains is responsible for ligand-independent signaling and that the two receptor types have an intrinsic heteromeric affinity for each other which is consistent with previous findings(48) . In contrast to the chimeric receptors derived from RI, cotransfection of RII(ET)-RI(C) with RI(ET)-RII(C) did not lead to TGF- responsiveness in the cyclin A-luciferase assay, consistent with the inactivity of the Tsk7L type I receptor in Mv1Lu cells(31, 39) .

Figure 4: Signaling activity of chimeric receptors in mutant Mv1Lu cells using TGF--dependent cyclin A-luciferase reporter assay. Receptor expression plasmids were transfected in DR26 or R1B cells together with the cyclin A-luciferase plasmid pCAL2. Cyclin A-luciferase data are shown as the percentage decrease in luciferase activity relative to cells that did not receive TGF-. In the cases when the cyclin A activity was higher after TGF- treatment, a negative value for the percentage inhibition was observed. The vertical bars show the standard deviations. Receptors are abbreviated as in Fig. 2except that hRII represents wild-type human RII.

Dominant Negative Effect of Chimeric and Truncated Receptors on TGF--dependent Down-regulation of Cyclin A Expression

Figure 5: Dominant negative inhibition of TGF--dependent cyclin A down-regulation by mutant receptors in wild-type Mv1Lu cells. Plasmids used for transfection of Mv1Lu cells are as in Fig. 2except that plasmids RIKR and RIIKR represent the kinase-negative mutants of RI and RII, respectively. Data are presented as in Fig. 4.

Using the TGF--responsive wild-type Mv1Lu cells and the cyclin A-luciferase assay, we also evaluated the effect of expression of truncated receptors lacking the cytoplasmic domain (Fig. 5, right panel). Consistent with the results using a truncated human type II receptor(51, 60) , expression of the mouse RII lacking its cytoplasmic domain, RII(ET), also blocked TGF--dependent inhibition of cyclin A transcription in a dominant negative manner. Overexpression of the truncated RI(ET) type I receptor lacking its cytoplasmic domain, but not RI(ET), in Mv1Lu cells also abolished TGF- responsiveness (Fig. 5, right panel). This effect was also seen in the PAI-1 assay (Table 1). However, the inhibitory effect of RI(ET) was less effective than RII(ET) in the PAI-1 assay (Table 1, data not shown).

Juxtamembrane Domains of Type I and Type II TGF- Receptors Are Required for TGF- Responsiveness

Figure 6: Biological effects of deletion of the cytoplasmic segments of TGF- receptors on TGF--dependent down-regulation of cyclin A-luciferase expression. Cytoplasmic deletion mutants of both RI and RII were as shown in Fig. 2and in legend to Fig. 5. Data are presented as in Fig. 4.

When expression plasmids for these mutant receptors were transfected into COS-1 cells and the kinase activity of the immunoprecipitated receptors was tested in vitro, we observed a correlation between the receptor kinase activity and their biological functions (Fig. 7). While RIT and RIIT maintained their kinase and signaling activities similarly to wild-type receptors, RIJM and RIIJM, like the kinase-negative mutants RIKR and RIIKR, were also kinase inactive and unable to autophosphorylate. Thus, mutant receptors which were inactive in the biological assays were also inactive in the kinase assay.

Figure 7: Expression and in vitro kinase activity of mutant receptors with cytoplasmic deletions. COS-1 cells were transfected with the plasmids indicated and the receptors were immunoprecipitated from S-lysates as described in Fig. 3A. Half of the immunoprecipitated proteins were analyzed by SDS-PAGE (left panels), whereas the other half was subjected to in vitro kinase assay using [gamma]P]ATP prior to gel analysis (right panels). The positions of the receptors and molecular weight markers are shown.

DISCUSSION

Roles of TGF- Receptors in TGF--dependent Down-regulation of Cyclin A Transcription

Based on the TGF- responsiveness of the 760-bp cyclin A promoter element, we have developed a functional assay to monitor cyclin A regulation in response to TGF-. This assay can be used in transient transfection assays to allow a functional evaluation of TGF- receptors and other signaling proteins involved in the antiproliferative effect of TGF-. In spite of the many TGF--induced responses, transcriptional activation of the PAI-1 gene is frequently used as the only indicator for TGF- signaling and receptor function, primarily because of the availability of a highly sensitive and convenient reporter assay that measures luciferase expression from a TGF--responsive promoter element(25) . Before this study, no facile reporter assay has been available to measure the antiproliferative effect of TGF-, even though considerable evidence suggests that the growth inhibition and the induction of gene expression by TGF- result from divergent signaling cascades. The availability of a cyclin A luciferase reporter system allowed us to characterize the role of specific domains of TGF- receptors during TGF--mediated growth inhibition using transient transfection assays.

Our results demonstrate that inhibition of cyclin A transcription by TGF- requires both RII and RI receptors. The cyclin A transcription is inhibited by TGF- in Mv1Lu cells which contain endogenous RI and RII receptors but not in the two mutant cell lines lacking either RI or RII. Introduction of wild-type RI into RI-deficient cells or RII into RII-deficient cells restores TGF--dependent cyclin A transcription. In contrast, various mutant receptors including the chimeric receptors RII(ET)-RI(C), RI(ET)-RII(C), RII(ET)-RI(C), and RI(ET)-RII(C) as well as the kinase-negative point mutants RIKR and RIIKR, and receptors lacking the juxtamembrane domain RIJM and RIIJM, are unable to rescue the ability of mutant cells to respond to TGF-. However, coexpression of RII(ET)-RI(C) and RI(ET)-RII(C), which allows heteromerization of both extracellular and intracellular domains(48) , restores TGF--dependent down-regulation of cyclin A expression in either mutant cell line. In wild-type Mv1Lu cells, individual expression of these mutant receptors inhibits TGF--induced cyclin A down-regulation in a dominant negative fashion. These results suggest that cyclin A expression and, thus, the growth inhibitory effect of TGF- need the cooperative interaction between type I and type II TGF- receptors.

Functional Analysis of TGF- Receptor Domains Using Both Cyclin A and PAI-1 Assays

In our study, we tested in parallel the TGF--dependent cyclin A response and PAI-1 transcriptional response and did not observe an uncoupling of these responses (see Table 1), which is consistent with the proposed requirement of both cytoplasmic domains for full TGF- responsiveness. More specifically, homomerization of cytoplasmic domains, resulting from expression of the RII(ET)-RI(C) chimera in DR26 cells or the RI(ET)-RII(C) chimera in R1B cells, is not sufficient for TGF--induced regulation of cyclin A and PAI-1 expression, suggesting that a functional interaction of the two types of cytoplasmic domains is a prerequisite for TGF- responsiveness. In addition, despite the heteromeric combination of both cytoplasmic domains, homomeric combination of the extracellular/transmembrane domains of type I or type II receptors also fails to restore TGF- responsiveness. In the case of the RI(ET)-RII(C) chimera in DR26 cells which express endogenous RI, this lack of signaling might be due to the inability of the RI extracellular domains to bind ligand in the absence of RII. However, expression of the RII(ET)-RI(C) chimera in R1B cells which have endogenous RII yet lack functional RI also does not activate TGF- responsiveness even though these receptors bind TGF- efficiently. This suggests an active function of the RI extracellular domain in TGF- signaling.

Coexpression of the RII(ET)-RI(C) and RI(ET)-RII(C) chimeras in DR26 or R1B cells results in TGF- responsiveness in both reporter assays, suggesting that the coexistence of both extracellular and cytoplasmic domains in trans provides a functional TGF- receptor unit. TGF- is likely to induce a conformational change in the heteromeric complex of these chimeras in a manner similar to the complex of wild-type RI and RII receptors. The resulting activation promotes the interaction of the receptors with downstream effectors. Our data are in agreement with the recent findings of Okadame et al.(66) , who showed the requirement of both cytoplasmic domains for TGF--induced PAI-1 transcription. We extended their findings by using the cyclin A reporter assay and by examining the effects of chimeric receptors in wild-type Mv1Lu cells.

In wild-type Mv1Lu cells, expression of the RI(ET)-RII(C) or RII(ET)-RI(C) chimera inhibits TGF--induced regulation of cyclin A and PAI-1 expression in a dominant negative manner. This inhibition is likely due to the fact that overexpression of the RI(ET)-RII(C) or RII(ET)-RI(C) chimera sequesters endogenous RII and/or RI away from functional RI-RII receptor complexes. The resulting homomeric complexes of chimeric receptors and the heteromeric complexes between endogenous receptors and the chimeras are then unable to transduce the TGF- signals. This dominant negative interference resembles the inhibitory effect of truncated receptors lacking their cytoplasmic domain and the kinase-negative mutant receptors. The inhibition of TGF- responsiveness by RII(ET) is in accordance with previous results(51, 60, 67) . A dominant negative inhibition, albeit to a lesser extent, was also apparent with overexpression of RI(ET) in Mv1Lu cells, but not with RI(ET) even though the truncated form of Tsk7L inhibited TGF--induced mesenchymal transdifferentiation in NMuMG cells(40) . Thus, overexpression of chimeric, truncated, or kinase-defective receptors derived from RII or RI results in dominant negative interference with TGF- receptor signaling, most likely by interfering with the assembly of functional homo- and heteromeric complexes. After the submission of this manuscript, Vivien et al.(68) and Brand and Schneider (69) reported dominant negative inhibition of TGF- signaling by chimeric and kinase-defective receptors, using assays for TGF--induced gene expression. Their conclusions and our findings based on cyclin A and PAI-1 assays are in agreement; moreover, our cyclin A assay results further extend their observations to TGF--induced growth inhibition.

Finally, we characterized the function of the RI and RII receptor cytoplasmic domains containing defined deletions. Deletion of the C-terminal tail of either receptor did not affect the kinase and signaling activity of RI and RII in both cyclin A and PAI-1 reporter assays. A similar result has been observed in the case of RII using the PAI-1 reporter assay(60) . We also demonstrated that deletion of the juxtamembrane domain inactivates the signaling function of both receptor types. This correlates with a loss of kinase activity as measured by autophosphorylation. In the case of the type I receptor, the juxtamembrane domain contains a conserved SGSGSGLP sequence. This sequence is a major site of phosphorylation by the type II receptor, and its deletion has been shown to inactivate TGF- signaling(46) . Thus, our results indicate the requirement of the juxtamembrane domains for the function and kinase activity of both receptors. These domains may also represent binding sites for cytoplasmic effector proteins.

Coincident Responsiveness in the Cyclin A and PAI-1 Reporter Assays

FOOTNOTES

*

This work was supported by Grant CA63101 from the National Cancer Institute (to R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Growth and Development, University of California at San Francisco, San Francisco, CA 94143-0640. Tel.: 1-415-476-7322; Fax: 1-415-476-1499.

()

The abbreviations used are: TGF-, transforming growth factor-; PAI-1, plasminogen activator inhibitor type 1; PCR, polymerase chain reaction; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

ACKNOWLEDGEMENTS

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