©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation-dependent Interaction of the Cytoplasmic Domains of the Type I and Type II Transforming Growth Factor- Receptors (*)

Ruey-Hwa Chen , Harold L. Moses (1), E. Miko Maruoka , Rik Derynck (§) , Masahiro Kawabata (1)

From the (1) Departments of Growth and Development and Anatomy and Programs of Cell Biology and Developmental Biology, University of California, San Francisco, California 94143-0640 and the Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor- (TGF-) transduces signals through its type I and type II receptors. Both receptor types have previously been shown to interact in a heteromeric complex in the presence of TGF-. We have now characterized these interactions between both receptor types using a combination of yeast two-hybrid interaction assays and coimmunoprecipitation analyses. Our results indicate a direct association between the cytoplasmic domains of the two receptor types. Mutation analysis of these cytoplasmic domains reveals that this direct interaction requires kinase activity and, thus, depends on phosphorylation, probably via a transphosphorylation mechanism. Furthermore, the two receptor types already have an inherent affinity for each other in the absence of TGF-, and the heteromeric complex can be detected in coimmunoprecipitations under these conditions. Taken together, our results reveal a novel mechanism of receptor complex formation, whereby two different cytoplasmic domains directly associate with each other. This interaction may play a major role in activation of serine/threonine kinase receptors.


INTRODUCTION

Transforming growth factor (TGF-)() belongs to a family of multifunctional proteins which regulate cell proliferation, differentiation, and formation of the extracellular matrix (1, 2) . TGF- exerts its biological activities through binding to specific cell surface receptors. Most cells express three classes of TGF- receptors, defined by their sizes and ability to bind and cross-link I-TGF- (3, 4) . The type III TGF- receptor is a proteoglycan (5, 6) and may be involved in presenting ligand to type I and type II receptors, but does not directly mediate TGF- signaling (7, 8) . In contrast, both type II and type I receptors are required for TGF--induced signaling (9, 10) . Dominant negative inhibition of the type II receptor signaling suggests the existence of two receptor-mediated signaling pathways, one of which requires a functional type II receptor, presumably in combination with the type I receptor, and leads to the antiproliferative effect of TGF-. The other pathway is associated with the type I receptor without the necessity of type II receptor signaling and directs TGF--induced expression of several genes (11) .

The type II receptor is a transmembrane serine/threonine kinase, the cytoplasmic domain of which can be autophosphorylated when expressed in Escherichia coli(12) . This receptor is constitutively autophosphorylated when transfected into mammalian cells (13) , which is consistent with its homodimeric complex formation (14, 15, 16) . The type I TGF- receptor was identified using chemical cross-linking techniques as a 53-kDa glycoprotein (17) , and several closely related type I TGF- receptors have been cloned (18, 19, 20, 21, 22) . They contain an intracellular serine/threonine kinase domain, but do not bind ligand unless coexpressed with the type II receptor. The specificity of ligand binding to the type I receptor is determined by the nature of the type II receptors (19, 22, 23) . Although only one of them, ALK-5/R4, restores TGF- responsiveness in a mutant cell line lacking type I receptors (20, 21) , another type I receptor, Tsk 7L, is involved in TGF--induced transdifferentiation of epithelial cells (24) . The physiological importance of the different type I receptors in the various biological activities of TGF- still remains to be determined.

Previous studies suggest that heteromeric complex formation between type II and type I receptors is required for ligand binding to the type I receptor and may play a role in TGF- signaling. A physical interaction of the two receptor types has been shown by coimmunoprecipitation experiments, in which type II or type I receptor-specific antibodies precipitated both ligand-bound type II and type I receptors. This interaction with the type II TGF- receptor was apparent with all type I receptors (8, 19, 22, 23, 25, 26) . Furthermore, it has been suggested that ligand cooccupation is required for the physical association of the two receptor types (13) . In the present studies, we characterized the interaction of both receptor types. As a model, we studied the association of type II receptor with the type I receptor Tsk 7L. The cytoplasmic domains of the two receptors can undergo a direct physical association with each other as determined by coimmunoprecipitations and yeast two-hybrid assays. This direct association depends on the kinase activity of the receptors. In addition, coimmunoprecipitation studies demonstrated that both receptor types interact in the absence of TGF- and thus have an inherent affinity for each other. The phosphorylation-dependent interaction of two cytoplasmic domains represents a novel mechanism for dimerization and/or oligomerization of receptors, which may be required for signaling of TGF--related factors through their transmembrane serine/threonine kinase receptors.


MATERIALS AND METHODS

Yeast Expression Plasmids

To construct the plasmid pEGcRII encoding the entire cytoplasmic domain of type II receptor inserted in frame to the DNA-binding domain of LexA, a 3.5-kilobase pair HpaI/XhoI fragment containing the cytoplasmic domain of the type II receptor (amino acids 192-567) was removed from pH2-3FF (12) and ligated into pEG202 (28) that was digested by BamHI, filled in with Klenow DNA polymerase I, and digested with XhoI. pJGcRI allows the expression of a fusion protein between the B42 acidic activation domain and the cytoplasmic domain of type I receptor Tsk 7L. To make this plasmid, the entire cytoplasmic sequences (amino acids 147-509) of the Tsk 7L was PCR-amplified and cloned into pJG4-5 (28) . pJGcRII, encoding the B42-type II cytoplasmic domain fusion, was constructed by excising the EcoRI fragment from pEGcRII and then cloned into pJG4-5. pEGcRI, containing the LexA DNA binding domain-type I receptor cytoplasmic domain fusion, was made by excising the EcoRI/XhoI fragment from pJG4-5 and then cloned into pEG202. To construct the plasmid pAScRII containing the cytoplasmic domain of the type II receptor fused to the 3` end of the GAL4 DNA binding domain, a 2.3-kilobase pair SmaI/BamHI fragment was excised from pEGcRII and cloned into the SmaI/BglII sites of the pAS1-CYH2 (29) . Plasmid pACTcRI allows expression of the fusion protein of the GAL4 activation domain and the type I receptor Tsk 7L cytoplasmic domain. To construct this, the entire cytoplasmic domain (amino acids 147-509) of the type I receptor Tsk 7L was generated by PCR and cloned into pACTII (29) .

To make the deletion mutants of the type II receptor cytoplasmic domain and the kinase-defective mutants of the type I and type II receptor cytoplasmic domains, the two cytoplasmic domains were cloned into M13mp19 vector. Deletions or point mutations were generated using the Sculptor in vitro mutagenesis system (Amersham Corp.) and oligonucleotide primers to introduce the desired mutations. The mutated type II cytoplasmic domains containing the different deletions were subcloned into pJG4-5 (28) . The point-mutated kinase-defective cytoplasmic domains were subcloned into pAS1-CYH2 (22) for the type II mutant and into pACTII (29) for the type I mutant.

The deletion mutants of type I receptor cytoplasmic domain were constructed as follows. Plasmids pJGcRI-(147-235) and pJGcRI-(147-414) were generated by removing the EcoRI/XmnI and EcoRI/MscI fragments from the type I receptor cytoplasmic domain in pJGcRI and then inserting the mutated cDNA into pJG4-53, respectively. pJG4-53 was constructed by inserting two annealed oligonucleotides (5`-AATTCATCCCGGGTACTCGAGTAAGTAAGTAA and 5`-TCGATTACTTACTTACTCGAGTACCCGGGATG) between the EcoRI and XhoI sites of plasmid pJG4-5, thereby inserting stop codons in all three frames. To construct pJGcRI-(147-360), an EcoRI/NsiI fragment derived from pJGcRI was first inserted into pSP72 (Promega) to be converted to EcoRI/XhoI fragment and then cloned into pJG4-53. To generate pJGcRI-(237-509), an XmnI/XhoI fragment was removed from pJGcRI, converted to EcoRI/XhoI fragment by inserting into and subsequently excising from pSP72, and cloned into pJG4-53. Plasmids pJGcRI-(361-509) and pJGcRI-(416-509) were constructed by removing the NsiI/XhoI and MscI/XhoI fragments from pJGcRI, converting them into EcoRI/XhoI fragments using strategy described above, and cloning them into pJG4-53.

Two-hybrid Assays

Yeast transformation was performed as described (32) . Y190 transformants were plated on Sc media lacking Trp, Leu, His, but including 25 mM 3-aminotriazole (Sigma). EGY48 transformants were isolated on glucose-Ura His Trp plates and assayed as described (28). Interaction was determined by screening for -galactosidase activity using a filter lift assay (33) . Quantitation of -galactosidase activity in yeast was performed using o-nitrophenyl--D-galactoside as substrate (33) .

Mammalian Expression Plasmids

Plasmid pIR-HA drives expression of the full-length type I receptor Tsk 7L linked to an HA epitope tag (GLYDVPDYASLG) at its C terminus. The entire coding region of Tsk 7L (18) was amplified by PCR and cloned into the expression plasmid, pRK5 (27) , together with a synthetic adaptor encoding the HA epitope tag. pIRC-myc is an expression plasmid for the entire cytoplasmic domain (amino acids 153-509) of the type I receptor Tsk 7L with an N-terminal myc epitope tag. An EcoRI fragment for the cytoplasmic domain of the Tsk 7L was generated by PCR and ligated to the 3` of a single-stranded synthetic adaptor encoding the myc epitope tag (GEQKLISEEDLN) preceded by an initiator methionine. The ligated product was subcloned into the expression plasmid pRK7 (27) . The construction of pIIRC-Flag, which allows expression of the cytoplasmic domain of the type II receptor with an N-terminal Flag tag, has been described before (16) .

Transient Transfections, Cell Surface Biotinylations, and Double Immunoprecipitations

293 cells were transfected using the calcium-phosphate precipitation method (30) . The conditions for treatment with TGF- or its neutralizing antibody have been described previously (16) . Metabolic labeling of cells was initiated at 36-48 h after transfection using S-protein-labeling mix (DuPont NEN). Labeled cells were lysed and subjected to double immunoprecipitations as described (16) . To detect the receptors located at cell surface, cells were labeled using sulfosuccinimdyl 6-(biotinamino) hexanoate (Pierce) at 48 h after transfection using procedures described (31) and then lysed for immunoprecipitation. The biotinylated proteins were detected with the enhanced chemiluminescence system (Amersham Corp.) using streptavidin-conjugated horseradish peroxidase as probe.

RESULTS

Direct Interaction of the Cytoplasmic Domains of the Type I and Type II Receptors

Since the type I and type II receptors have the ability to interact in a heteromeric complex, we evaluated the involvement of different receptor domains in this interaction. In this context, we examined whether the cytoplasmic domains of the two receptor types undergo a direct physical interaction. For this purpose, we used the yeast two-hybrid system, which lacks endogenous type I and type II receptors and scores direct physical interactions between proteins, based on transcriptional activation of reporter genes (34) . Two expression plasmids were constructed by fusion of the cytoplasmic domains of type II and type I receptors to the LexA DNA-binding domain and the nuclear localized B42 acidic activation domain, respectively. Cotransformation of the two plasmids in yeast strain EGY48 (28) resulted in transactivation of the Leu and lacZ reporter genes, as detected by its ability to grow in the absence of leucine and its blue color when stained for -galactosidase activity (). No transactivation was found when yeast was transformed with only one of the plasmids. Similar transactivation was observed when yeast was cotransformed with a pair of the reciprocal plasmids, in which the type I cytoplasmic domain was fused to the LexA DNA-binding domain, and the type II cytoplasmic domain was made as a hybrid with the B42 activation domain (). The specificity of this interaction was further confirmed using a different two-hybrid system (29) , in which the coexpression of a hybrid GAL4 DNA-binding domain-type II cytoplasmic domain and a fusion protein of the GAL4 activation domain and the type I cytoplasmic domain resulted in transactivation of the His and lacZ reporter genes in the yeast strain Y190. Taken together, these results indicate a direct and specific association between the cytoplasmic domains of the type I and type II receptors. In contrast, no direct interaction was detected in the same two-hybrid assays between the type II receptor cytoplasmic domains, the type I receptor cytoplasmic domains, and between the type II receptor cytoplasmic domain () and the cytoplasmic domain of TGF- as a control protein.

We next examined whether this association of the two types of cytoplasmic domains also occurs in vivo. Cells were cotransfected with the cytoplasmic portions of type I and type II receptors, preceded by an initiator methionine and C-terminally tagged with myc and Flag epitopes, respectively. The lysates of metabolically labeled, cotransfected cells were subjected to immunoprecipitations using tag-specific antibodies. As shown in Fig. 1, the anti-Flag antibody precipitated not only the type II receptor, but also a band with the same mobility as the type I receptor (lane 2). Such coimmunoprecipitation was not detected from cells transfected with only the type II cytoplasmic domain (Fig. 1, lane 1). Similarly, the anti-myc antibody precipitated not only the myc-tagged type I cytoplasmic domain but also a band with a mobility identical to the Flag-tagged type II receptor cytoplasmic domain (lane 3). To confirm the identity of the coprecipitated band, we performed a double immunoprecipitation analysis, in which low stringency immunoprecipitates using an anti-myc antibody were solubilized and reprecipitated under high stringency conditions using an anti-Flag antibody. Such double immunoprecipitations revealed that the coprecipitated band was recognized by the anti-Flag antibody and thus corresponds to the type II receptor cytoplasmic domain (Fig. 1, lane 4), thus indicating the association of the two cytoplasmic domains. As expected, the two sequential immunoprecipitation steps resulted in a considerably lower intensity of the two receptor bands. Taken together, these results indicate that the two cytoplasmic domains can interact independent of the extracellular/transmembrane domains and can be involved in the formation of the heteromeric complex between the type I and type II receptors.


Figure 1: The cytoplasmic domains of type I and type II receptors can interact with each other. 293 cells transfected with pIIRC-Flag, an expression plasmid encoding the Flag-tagged type II cytoplasmic domain (20) (II) or cotransfected with pIIRC-Flag and pIRC-myc, which expresses a myc-tagged cytoplasmic domain of the Tsk 7L type I receptor (D), were metabolically labeled. Cell lysates were subjected to single (lane 1) or double (lanes 2-4) immunoprecipitations using antibodies as indicated (M, anti-myc; F, anti-Flag). cRI and cRII indicate the cytoplasmic domains of the type I and type II receptors, respectively. Molecular weight markers are shown to the right. The cytoplasmic domain of the type I receptor coimmunoprecipitates with the cytoplasmic domain of the type II receptor (compare lane 2 with lane 1) and vice versa (lane 3). Double immunoprecipitations show coprecipitation of type II receptor cytoplasmic domain in type I receptor immunoprecipitates as well as associated type I receptor cytoplasmic domain (lane 4).



The Interaction of the Two Cytoplasmic Domains Depends on the Kinase Activity

Because the interaction between the two cytoplasmic domains represents a novel mechanism of receptor interactions that may be a feature unique to the TGF- receptor family, we investigated whether specific regions in these domains are involved in their association. A panel of deletion mutants that span the length of the type II cytoplasmic region was constructed and individually fused with the B42 activation domain (Fig. 2A). Their capacity to directly and specifically interact with the cytoplasmic domain of the type I receptor was tested again using the two-hybrid system. As shown in Fig. 2A, deletion of the spacer region upstream from the kinase domain or the C-terminal tail region immediately downstream from the kinase sequences did not affect association with the type I cytoplasmic portion. However, deletion of any part of the type II receptor kinase domain tested abolished its ability to interact with the type I receptor cytoplasmic portion. Using a similar approach, the subregion in the type I cytoplasmic domain involved in the interaction with the type II cytoplasmic domain was also determined. A panel of deletion mutants of the type I cytoplasmic domain, each lacking a portion of the kinase domain, was constructed and individually fused to the B42 transactivation domain (Fig. 2B). None of these mutants were able to interact with the type II cytoplasmic portion in the same yeast two-hybrid assays. Thus, our data indicate that an intact kinase domain, but not the flanking sequences, is required for the association between the cytoplasmic domains of the type I and type II receptors.


Figure 2: Kinase domains are required for the association between the cytoplasmic portions of type I and type II receptors. Deletion mutants of the cytoplasmic domains of the type I (A) and type II (B) receptors were used to define the interacting domains and are shown schematically. Transmembrane domains (TM) of the two receptors are indicated. The numbers in parentheses indicate the amino acid number of the cytoplasmic sequence present in the fusion proteins; when preceded by a ``d,'' the numbers indicate the deleted segments. The deletion mutants of the cytoplasmic domains of the type II and type I receptors were expressed as fusion proteins with the B42 activation domain and these plasmids were named JG-cRII and JG-cRI, respectively. EGY48 yeast cells (21) were cotransformed with the indicated panel of plasmids for type II deletion mutants together with the expression plasmid pEG-cRI which fuses the LexA domain and the cytoplasmic domain of type I receptor (A) or with plasmids for the panel of pJG-cRI deletion mutants and pEG-cRII containing the type II receptor cytoplasmic domain (B). Binding was determined by the dark blue staining following a filter lift assay for -galactosidase activity (25).



The results obtained with the deletion mutants suggest that the overall structure of the kinase domains per se may be critical for association between the two cytoplasmic domains and that deletion of any portion of the kinase domains may alter such structure, thus disrupting the association. However, it was equally possible that the kinase activities of the two receptors play a role in the association, since any deletion within the kinase domains tested is likely to inactivate the kinase activity. To test the latter possibility in yeast that lacks endogenous TGF- receptor kinase activity, kinase-defective type I and type II receptor cytoplasmic domains were constructed by replacing the lysine in the ATP-binding site with an arginine, and the mutated cytoplasmic domains were then fused to the GAL4 activation and DNA-binding domains, respectively. The strengths of the interactions between the wild type and mutant as well as between the two mutants were tested in the yeast two-hybrid system. As shown in , inactivation of the kinase activity in both receptors abolished the direct association as assessed by the lack of -galactosidase activity. Combination of the type II wild type and type I kinase-defective mutant showed a somewhat weaker interaction compared with the kinase-active pair. Furthermore, the type II kinase-negative mutant showed only a very weak interaction with the wild type type I receptor. These data indicated that the kinase activity of the two receptors, especially the type II receptor, is required for the direct association of both cytoplasmic domains, probably via a transphosphorylation mechanism. To examine the role of the kinase activity in the heteromerization of these two domains in mammalian cells, we generated expression plasmids for the cytoplasmic domains identical to the ones used in Fig. 1, except that, as in the yeast two-hybrid expression plasmids, we replaced the lysine in the ATP-binding site of each cytoplasmic domain with an arginine. Unfortunately, the kinase-inactive versions of the cytoplasmic domains had a much lower intracellular stability than the kinase-active versions, thereby precluding an evaluation of their association in transfected cells using coimmunoprecipitation analysis (data not shown).

Interaction of Full-size Type I and Type II Receptors

The experiments outlined above demonstrated that the kinase-active cytoplasmic domains of the type I and type II receptors have an inherent affinity for each other which results in their association. Furthermore, they indicate that the kinase activity of the type II receptor plays a major role in this association, which could be due to an autophosphorylation of the cytoplasmic domains. In mammalian cells, the type II receptor forms a constitutive homodimer (14, 15, 16) which is autophosphorylated both in the presence and absence of TGF- (13) . Based on these results, we evaluated whether the intact, full-size receptors also had an inherent affinity for each other, which could result in an association in the absence of TGF-.

Expression plasmids for HA-epitope-tagged type I Tsk 7L and myc-tagged type II receptors (16) were constructed and cotransfected into 293 cells. The lysates of metabolically labeled, cotransfected cells were subjected to immunoprecipitations using tag-specific antibodies. These analyses demonstrated a diffuse band of the size of the type I receptor in the type II receptor immunoprecipitates which was absent in cells expressing only the type II receptor (Fig. 3, lanes 2-4). Unfortunately, the expression level of the type II receptor was consistently much higher than that of the type I receptor. However, in reciprocal experiments, bands of the size of the type II receptor were clearly apparent in type I receptor immunoprecipitations when both receptor types were coexpressed, but not when only the type I receptor was expressed (Fig. 3, lanes 7-9). These data thus suggest association of both receptor types. To further evaluate the identity of the coprecipitated bands, we performed double immunoprecipitations using first anti-myc and then anti-HA antibodies. These analyses revealed that the HA-tagged type I receptor coprecipitated and thus was associated with the myc-tagged type II receptor (Fig. 3, lanes 12 and 13). In control experiments, no coprecipitation could be detected in untransfected cells, nor in 293 cells transfected with only the myc-tagged type II receptor or the HA-tagged type I receptor (Fig. 3, lanes 5, 10, 14, and 15). The reciprocal immunoprecipitation using first the HA- and then the myc-specific antibodies further documented this association (Fig. 3, lane 16). This double immunoprecipitation revealed a high level of myc-tagged type II receptor coprecipitated with the HA-tagged type I receptor using the HA antibody, since a comparable level of type I receptor was recovered from two sequential immunoprecipitations with HA antibody (Fig. 3, compare lanes 16 and 17). Furthermore, the association of the two receptors was also detected when lysates of cotransfected cells were first immunoprecipitated using the myc or HA antibody and then subjected to Western blotting using the HA or myc antibody, respectively (data not shown). Interestingly, the amounts of coprecipitated HA-tagged type I receptor did not differ when the cotransfected cells were treated with TGF- or a neutralizing antibody that interferes with TGF- binding to its receptors (35) (Fig. 3, compare lanes 13 and 14).


Figure 3: Heteromeric complex formation between the type I and type II receptors in metabolically labeled, transiently transfected cells. 293 cells cotransfected with equal amounts of expression plasmids for the HA-epitope-tagged Tsk 7L type I receptor, pIR-HA, and the myc-tagged type II receptor, pIIR-myc (20) (D), or transfected with pIIR-myc alone (II), pIR-HA alone (I), or untransfected (-) were metabolically labeled and treated with TGF- (+) or the neutralizing antibody (-) as indicated. In each transfection, 25 µg of plasmid DNA was used. Cell lysates were subjected to single (lanes 1-11) or double (lanes 12-17) immunoprecipitations using conditions as described (20) and antibodies as indicated (M, anti-myc; H, anti-HA) and analyzed by denaturing gel electrophoresis under reducing conditions. The molecular markers (111, 74, and 45 kDa) are indicated as dots on both sides. The type I (RI) and type II (RII) receptors are indicated. Lane 1 is a shorter exposure of a pIIR-myc transfection, similar to lane 2, albeit from different experiments (hence the differences in the degradation products of the type II receptor). In contrast to lane 2 (transfection with pIIR-myc only), the type I receptor was coprecipitated in type II receptor immunoprecipitates, when pIR-HA was cotransfected with pIIR-myc (lanes 3 and 4). In immunoprecipitations of the type I receptor, the type II receptor coprecipitated with the type I receptor when pIIR-myc was cotransfected with pIR-HA (lanes 8 and 9), but not in the absence of pIIR-myc (lane 7). Double immunoprecipitation using first the myc and then the HA antibody showed the presence of the HA-tagged type I receptor (most intense band) and some myc-tagged type II receptor (lanes 12 and 13). Furthermore, the myc-tagged type II receptor was detected in double immunoprecipitations using first the HA and then the myc antibody (lane 16).



In principle, the coprecipitation of both receptor types could have been due to overcrowding of these receptors at the surface of cotransfected cells, thereby leading to an increased and, to some extent, nonspecific level of oligomerization or aggregation. However, no coimmunoprecipitation was detected in cells overexpressing the myc-tagged type II receptor and a control transmembrane protein, TGF-, using the same cotransfection and immunoprecipitation methods (16). In addition, to address whether this constitutive interaction occurs under physiological conditions, i.e. at receptor expression levels comparable with receptor levels in untransfected cells, the epitope-tagged type II and type I receptors were expressed in 293 cells at low levels, which based on cell surface cross-linking with I-TGF-, were comparable with the endogenous receptor levels of C2C12 cells (data not shown). The ligand-independent association of the two receptor types could be demonstrated by double immunoprecipitation analysis under these conditions as well (Fig. 4). Finally, we performed coimmunoprecipitation analysis of the receptors at the cell surface, that were labeled using surface biotinylation, thus excluding the intracellular receptor pool (Fig. 5). Also under these conditions was the type II receptor expression considerably higher than the type I receptor levels, thereby requiring sequential immunoprecipitation analysis, as in Fig. 3and Fig. 4. As is apparent especially from lanes 9-12, both receptor types associate with each other at the cell surface in the absence of ligand binding, consistent with the results from the S-labeled receptors in total cell lysates.


Figure 4: Formation of the heteromeric type I-type II receptor complex in cells expressing a low level of transfected receptors, comparable to endogenous receptor levels. 293 cells were cotransfected with 2 µg of pIR-HA and 2 µg of pIIR-myc in the presence of 21 µg of carrier DNA. Cells were treated with TGF- (+) or neutralizing antibody (-), metabolically labeled, and subjected to single (lanes 1 and 2) or double immunoprecipitations (lanes 3 and 4) using antibodies as indicated. The molecular weight markers and the type I and II receptors are indicated. The double immunoprecipitations using first the myc and then the HA antibody showed the presence of the HA-tagged type I receptor (most intense band) and some myc-tagged type II receptor (lanes 3 and 4).




Figure 5: Formation of the heteromeric receptor complex at the surface of cotransfected cells. 293 cells were cotransfected with pIR-HA and pIIR-myc (D) or transfected with pIIR-myc alone (II), pIR-HA alone (I), or untransfected (-). This experiment was carried out in the absence of TGF- and the presence of a neutralizing antibody (16), and immunoprecipitations were carried out using antibodies as indicated. The molecular markers (111, 74, and 45 kDa) are indicated as dots to the right. The type I (RI) and type II (RII) receptors and a frequently observed degradation product of the type II receptor (asterisk) are indicated. Single immunoprecipitations show the type II receptor as a double band (because of the degradation product) and the type I receptor as a single band. Double immunoprecipitations of cells expressing both receptor types show the presence of the HA-tagged type I receptor in type II receptor immunoprecipitates (lane 9) and myc-tagged type II receptor in type I receptor immunoprecipitates (lane 11). Lanes 10 and 12 show control double immunoprecipitations in which the type II receptor and type I receptor were subjected to two sequential immunoprecipitations.



Taken together, these results indicate that both receptor types have already an affinity for each other in the absence of TGF-. Under our immunoprecipitation conditions, a similar degree of receptor association was apparent in both the presence and absence of TGF-, implicating the presence of a pre-existing heteromeric receptor complex independent of ligand occupation.

We next examined whether the ability of both types of full-size receptors to associate with each other was solely based on the possible direct interaction of the cytoplasmic domains. We therefore generated expression plasmids for a Flag-tagged truncated type I (23) and a myc-tagged truncated type II receptor (16) lacking most of their cytoplasmic domains. Double immunoprecipitation analyses showed that the Flag-tagged truncated type I receptor was coimmunoprecipitated with the myc-tagged truncated type II receptor using the anti-myc antibody (Fig. 6, lanes 11 and 12) and vice versa (Fig. 6, lanes 9 and 10). No coprecipitated truncated type I or type II receptor was detected in cells transfected with only the truncated type II or type I receptor respectively (Fig. 6, lanes 13 and 14). Treatment of cells with TGF- did not stimulate their association (Fig. 6, compare lanes 9 and 10; 11 and 12), similar to the results obtained using the two full-length receptors. Thus, as in the case of the type I and type II receptors, the extracellular and transmembrane domains display already an affinity toward each other. This affinity is high enough to allow the detection of the associated heteromeric complex by coimmunoprecipitation analysis.


Figure 6: Interaction between the truncated type I and type II receptors lacking most of their cytoplasmic domains. 293 cells cotransfected with pIRDN-Flag, an expression plasmid for the Flag-epitope tagged, cytoplasmically truncated Tsk 7L type I receptor (15) and pIIRDN-myc, which expresses the myc-tagged, cytoplasmically truncated type II receptor (20) (D), with pIIRDN-myc alone (II), or with pIRDN-Flag alone (I), or untransfected (-) were metabolically labeled and treated with TGF- (+) or with a neutralizing antibody (-). Single (lanes 1-8) or double (lanes 9-14) immunoprecipitations were performed using antibodies as indicated (M, anti-myc; F, anti-Flag). tRII and tRI indicate the truncated type II and type I receptors, respectively. Molecular weight markers are shown to the right. Both the truncated type II and I receptors migrated as double bands reflecting differences in glycosylation. The highest band of the type I receptor (lanes 1-3) comigrates with a band of the type II receptor (lanes 5-7). The lowest band in the type II receptor lanes, which is most apparent in lanes 9-12, corresponds to a frequently observed degradation product of the receptor. Double immunoprecipitations using first the Flag and then the myc antibody showed the presence of the myc-tagged type II receptor in the type I receptor immunoprecipitates (lanes 9 and 10). Double immunoprecipitations using antibodies in a reversed order showed the presence of the Flag-tagged type I receptor in the type II receptor immunoprecipitates (lanes 11 and 12).



DISCUSSION

Receptor dimerization is essential for kinase activation of growth factor receptors that are themselves transmembrane tyrosine kinases or are associated with cytoplasmic tyrosine kinases (36, 37) . Typically, occupation of the receptors by ligands induces dimerization of adjacent extracellular domains and leads to interactions between the cytoplasmic domains and their rapid transphosphorylation. As a result, a cascade of signaling events is activated and leads to the various growth factor-stimulated biological responses. In the case of TGF- receptors, which could be considered as prototypes for the recently discovered transmembrane serine/threonine kinase receptors, formation of heteromeric complexes between type I and type II receptors in the presence of TGF- has been demonstrated following cross-linking with I-TGF- (8, 19, 22, 23, 25, 26) . In addition, Wrana et al.(13) proposed, based on coimmunoprecipitation analysis of cotransfected cells, that ligand induces the interaction of the type II and type I receptors. Analyses of the ligand-bound heteromeric complex (8, 13, 19, 22, 23, 25, 26) and the constitutive homodimerization of the type II receptor independent of ligand binding (14-16) strongly suggest that the heteromeric TGF- receptor complex may be a tetramer which combines two type II and two type I receptors. In the current study we further characterized the structural basis of the heteromeric interaction between the type I and type II receptors using a combination of double immunoprecipitation analyses and yeast two-hybrid assays.

We have shown that the cytoplasmic domains of the type I and type II receptors are able to interact directly with each other, as is apparent from yeast two-hybrid assays and double immunoprecipitation analyses. If the expression of -galactosidase in the yeast two-hybrid system is a measure of the strength of the interaction, we conclude that the association between both types of cytoplasmic domains is comparable with the reported interactions between pRB and phosphatase 12 (33) or cdk2 and the cdk-inhibitor p21 (29) . This specific association between two different cytoplasmic domains represents a novel mechanism of receptor interactions and may contribute to the stability of the heteromeric receptor complex. Analyses of cytoplasmic domain mutants in the two-hybrid system indicate that the kinase activity is essential for direct association. Furthermore, the two kinase-active cytoplasmic domains can undergo a heteromeric interaction in vivo. Whether this physical interaction of the cytoplasmic domains is mediated through phosphorylated amino acids or results from conformational changes induced by cytoplasmic domain phosphorylation remains to be determined. In this context, it is possible that specific sequences in the cytoplasmic domains bind to phosphorylated serines and/or threonines in the opposing receptor domain, similar to the interactions of SH2 domains with phosphotyrosine residues (36) . Our results also suggest the existence of transphosphorylation of adjacent cytoplasmic kinase domains in the heteromeric TGF- receptor complex. Thus, transphosphorylation of both receptors may induce the interaction between the cytoplasmic domains and play a major role in the stabilization of the receptor complex. The observation that elimination of the type II receptor kinase activity diminishes the association more dramatically than inactivation of the type I receptor kinase suggests that transphosphorylation of the type I receptor by the type II receptor kinase is more important for this interaction than the reciprocal phosphorylation event. This is consistent with the recent conclusion that the type II receptor phosphorylates the type I receptor in the heteromeric complex (13) . Thus, in a tetrameric receptor complex of two type II and two type I receptors (15) , the phosphorylation of the type II receptor cytoplasmic domains may result from ligand-independent autophosphorylation (13) and mediate the direct interaction with the type I receptor cytoplasmic domain.

The constitutive autophosphorylation of the type II receptor cytoplasmic domain (13) and the direct association of the kinase-active cytoplasmic domain of the type II receptor with the cytoplasmic domain of the type I receptor suggest that both receptor types may have an inherent affinity for each other. This possibility was evaluated by double immunoprecipitation analyses using metabolically labeled cells cotransfected with differentially tagged type I and type II receptors. Using our experimental conditions, we demonstrated that both receptor types can already associate with each other in the absence of TGF- and in the presence of a neutralizing anti-TGF- antibody that interferes with receptor binding to endogenous TGF-. Thus, our results indicate the presence of a pre-existing heteromeric type I-type II receptor complex and show that ligand binding is not required for formation of this complex. Furthermore, we did not detect a difference in the amount of heteromeric complex in cells treated with or without TGF-. The association of both receptor types in the absence of TGF- was not a result of overexpression of these receptors at the cell surface, since heteromerization was also apparent at physiological expression levels and no association with an unrelated transmembrane protein, e.g. transmembrane TGF-, was detected using overexpression conditions. The ability of both receptor types to associate in the absence of TGF- may seem inconsistent with the previously reported conclusion that ligand binding induces formation of the heteromeric complex (13) . However, our results are not necessarily in contradiction. In the case of tyrosine kinase receptors, two receptors can undergo a low affinity dimerization in the absence of ligand, and ligand binding stabilizes this interaction thus resulting in a dimeric complex which can be immunoprecipitated as such (38) . Similarly, ligand occupation may not be required for low affinity heteromerization of type I and type II receptors as suggested (13) , but may increase the stability of this complex. However, in contrast with receptor tyrosine kinases, the interaction between both receptor types in the absence of ligand occupation may be of sufficiently high affinity to allow their coimmunoprecipitation. Thus, whether or not ligand induces complex formation may be a semantic issue and a reflection of the sensitivity of the experimental conditions.

Whereas the direct association of the cytoplasmic domains is likely to play a role in the formation and stabilization of the heteromeric complex, our results also indicate that this cytoplasmic association is not absolutely required for the heteromeric interaction. Indeed, truncated receptors lacking the cytoplasmic domains still have the ability to associate. This is reminiscent of tyrosine kinase receptors, which dimerize through interaction of their extracellular/transmembrane segments (36, 37, 38) . Thus, multiple contact points are involved in the formation and stabilization of the heteromeric complex of the type I and type II receptors.

In the case of tyrosine kinase receptors, overexpression of truncated receptors lacking most of their cytoplasmic kinase domains abrogates receptor function (39, 40, 41) . Similar dominant-negative inhibition of receptor functions by overexpressing cytoplasmically truncated receptors has also been demonstrated in the case of TGF- receptor family, such as activin (42) and TGF- type II receptors (11, 43, 44), suggesting formation of receptor complexes via interactions between their extracellular/transmembrane domains. In accordance, we have now provided direct evidence for the involvement of this domain in the formation of the receptor complex.

The characteristics of the heteromeric TGF- receptor complex, determined in the present study, are somewhat different from typical tyrosine kinase receptors, in which the extracellular/transmembrane domains mediate ligand-induced dimerization (36, 37, 38) . However, this complex may resemble to some extent the receptors for insulin and insulin growth factor-1, which are more stable homodimers in the absence of ligand (45) . Similarly, transmembrane guanylyl cyclase receptors exist as homomers and/or heteromers and in fact may form a tetrameric protein complex in the absence of ligand (46) . How ligand binding induces receptor activation is largely unknown for any of these receptors, but is likely a consequence of ligand-induced conformational changes. In the case of the TGF- receptor family, ligand binding may stabilize a pre-existing heteromeric complex, thereby inducing a conformational alteration and, as a consequence, exposing critical amino acids in the cytoplasmic domains. These substrate sites could then undergo transphosphorylation by the adjacent cytoplasmic kinase domain in the receptor complex, as documented for the type I receptor phosphorylation by the type II receptor (13) . Phosphorylation of these critical residues presumably plays a key role in the recruitment of downstream signaling proteins and/or receptor activation. Future studies will be conducted to evaluate this model and to determine the contributions of the homomeric and heteromeric receptor complexes in TGF--mediated signal transduction.

  
Table: Interaction between the cytoplasmic domains of type II receptor and type I receptor Tsk 7L


  
Table: Effect of kinase activity of the type I and type II receptors on the association between their cytoplasmic domains

Y190 yeast was transformed with various plasmids as indicated, and -galactosidase activity was determined by the colony lift method and quantitated using o-nitrophenyl--D-galactoside as substrate (33). The expression levels of the type II and type I cytoplasmic domain fusion proteins were verified using Western blot analyses and were very similar in the yeast transformed with the different sets of plasmids (data not shown).



FOOTNOTES

*
This work was supported by grants from the National Institutes of Health (to R. D. and H. L. M.) and the American Cancer Society (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.: 415-476-7322; Fax: 415-476-1499.

The abbreviations used are: TGF-, transforming growth factor-; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Drs. R. Brent and S. Elledge for providing the yeast two-hybrid reagents and Drs E. Filvaroff and S. Lawler for stimulating discussions.

Note Added in Proof-Chen and Weinberg (Chen, R.-H., and Weinberg, R. A.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1565-1569) recently reported the ligand-independent association of the type I and type II TGF- receptors in COS-1 cells, which is consistent with our results.


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