©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperative Binding of Transforming Growth Factor (TGF)-2 to the Types I and II TGF- Receptors (*)

Carlos Rodriguez (1)(§), Feng Chen (1) (2), Robert A. Weinberg (1) (2)(¶), Harvey F. Lodish (1) (2)(**)

From the (1)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and the (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

TGF-1 binds with high affinity (K = 25-50 pM) directly to the TGF- type II receptor serine-threonine kinase (T-RII) in the absence of expression of the TGF- type I or III receptors (T-RI and T-RIII). The serine-threonine kinase T-RI is essential for TGF-1 signaling but not for binding to T-RII. TGF-2, in contrast, does not bind directly to T-RII, although coexpression of T-RIII does allow binding and cross-linking of TGF-2 to T-RII. Here we show that in transfected COS cells binding and cross-linking of I-TGF-2 to T-RI or T-RII requires expression of both receptors. In cells transfected with the c-myc-tagged human T-RII cDNA, only low amounts of I-TGF-2 cross-linked to T-RI and T-RII were detected even with high concentrations (700 pM) of ligand. Cotransfection of the influenza-hemagglutinin-tagged human T-RI cDNA dramatically increased the binding of TGF-2 to T-RII; the concentration of I-TGF-2 required for half-maximal binding and cross-linking to T-RI and T-RII was 40 pM. Coimmunoprecipitation studies showed that the high affinity receptor for TGF-2 is composed of a hetero-oligomer of T-RI and T-RII. Thus TGF-1 and -2 bind to TGF- receptors in different ways; TGF-1 binds directly to T-RII, while binding of TGF-2 to T-RII requires coexpression of T-RI or T-RIII.


INTRODUCTION

The TGF- (transforming growth factor )()family of cytokines has important functions in growth, development, and differentiation(1) . The three mammalian TGF- isoforms, TGF-1, -2, and -3, share 71-76% amino acid identities. Although disulfide-linked homodimers of 25 kDa are the predominant species, the heterodimers TGF-1.2 and TGF-2.3 have been described(2) . The sequences of the mature, proteolytically processed forms of each TGF- family member are almost entirely conserved across species, and thus there has been evolutionary pressure to retain both the similarities and differences in these isoforms. In most cell types, all three forms share very similar biological activities(3) .

Most mammalian cells express three abundant high affinity receptors, which can bind and be cross-linked to TGF-: the type I (53 kDa), type II (65 kDa), and type III (100-280 kDa) receptors, based upon the molecular mass of the cross-linked products analyzed by gel electrophoresis(4) . T-RI and T-RII, the type I and II receptors, are type I transmembrane proteins with cytosolic domains containing a serine-threonine kinase(5, 6, 7, 8, 9) . Both receptors are essential for intracellular signaling. The TGF- type III receptor, or betaglycan, is a membrane-bound proteoglycan with a short cytoplasmic tail that has no apparent signaling motif(10, 11) . It binds TGF-2 (apparent K 100 pM) with slightly greater affinity than TGF-1 or TGF-3 (apparent K 300 pM)(12, 13) . The main role of betaglycan seems to be in binding and then presenting TGF- ligand to the signaling receptors T-RI and T-RII(14) ; overexpression of T-RIII in L6 myoblasts leads to a dramatic increase in TGF-2 binding to T-RI and T-RII(10, 15) .

Studies in chemically mutagenized cell lines showed that TGF-1 binds T-RII with high affinity (mass 25-50 pM) in the absence of T-RI and that binding of TGF-1 to T-RI requires the presence of T-RII(16, 17) . TGF-1 binds with relatively high affinity to the soluble secreted exoplasmic domain of T-RII(18) . Heteromeric complexes of the type II and type I receptors are found on the surface of many cells after ligand binding, and are important for signal transduction(15, 19, 20) . Addition of TGF-1 allows the cytosolic domains of T-RI and T-RII to interact such that the cytoplasmic domain of T-RI is transphosphorylated by the constitutively active T-RII kinase. Phosphorylation of the cytoplasmic domain of T-RI is believed to activate its kinase activity and to allow its phosphorylation of and/or its association with (unknown) downstream substrates(21) .

Oligomerization of TGF- receptors plays important roles both in ligand binding and signal transduction. Homo-oligomers, probably homodimers, of the types II (22, 23) and III (22) receptors exist on the cell surface in the absence or presence of TGF-1 or TGF-2. Hetero-oligomers of the types II and III receptors are probably transient species, most likely intermediates in the transfer of a TGF- ligand from a type III to a type II receptor(15) . Heteromeric complexes of T-RI and T-RII, and of T-RII and T-RIII, have been detected after addition of TGF-1 (15, 19, 20) but it is not known whether the T-RIT-RII complexes exist in the absence of TGF-1 ligand.

If the type II and type I receptors indeed mediate TGF- -induced signals, then it is puzzling that TGF-2 is as potent as TGF-1 and TGF-3 in its ability to arrest the growth of cells (ED 5-20 pM)(24) , since TGF-2 does not bind well to either the type I or the type II receptors. TGF-2 does interact with the type II receptor, since reintroduction of the cloned type II receptor into mink lung cell mutants lacking the type II receptor restored the ability of these cells to be growth-inhibited by TGF-2(19) . Depending on the cell line TGF-2 binds to T-RII and T-RI with an affinity 10- to 100-fold lower than does TGF-1(24, 25, 26, 27) . TGF-2 does not bind at all to the soluble secreted exoplasmic domain of T-RII, and in transfected COS cells, binding of TGF-2 to T-RII requires coexpression of T-RIII (18).


MATERIALS AND METHODS

Epitope-tagged Receptors

The expression vectors for the epitope-tagged TGF- receptors have been described previously(22, 29) .

Transient Transfection of COS-7 Cells

COS-7 cells (American Type Culture Collection CRL 1651) were grown in Dulbecco's modified Eagle's medium supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum (Life Technologies, Inc.). Approximately 30% confluent cells in a 10-cm diameter dish were transfected with different combinations of the plasmids pcDNA-1 containing the N-terminal Myc-tagged human T-RII receptor (22) and CMV7 containing the C-terminal HA-tagged T-RI receptor(28, 29) , using the DEAE-dextran/chloroquine method described previously(5) .

Receptor Cross-linking and Immunoprecipitation

TGF-1 and TGF-2 (R& Systems, Minneapolis, MN) were iodinated by the chloramine-T method as described(30) . COS cells were used in binding studies 2 days after transfection, as detailed in Ref. 22. Affinity labeling of transfected cells was done in the presence of I-TGF-1 or I-TGF-2 and the cross-linking agent disuccinimidyl suberate (DSS; Pierce) as described previously(31) . After binding and cross-linking, portions of the cell lysates were analyzed directly by electrophoresis through a 10% SDS-polyacrylamide gel, exposed overnight to a Fuji BAS III screen, and quantified with the BAS-2000 bio-imaging analyzer (Fuji). Subsequently the gel was exposed for 3 weeks to a preflashed Kodak XAR-5 film. The remainder of the lysate was immunoprecipitated with either the anti-HA monoclonal antibody 12CA5 (32) (Harvard Monoclonals, Boston, MA) or the monoclonal antibody 9E10 that recognizes a human c-myc epitope (33) (BAbCO, Berkeley, CA) as described previously(22) . The immunoprecipitates were also analyzed by SDS-PAGE, autoradiography, and bio-imaging quantification.


RESULTS

To study the binding properties of TGF-2 to T-RI and T-RII, we transfected the full-length human receptors into COS cells. TGF-1 binds efficiently to T-RII expressed in COS cells, and binding is not enhanced by coexpression of T-RI (6).()The cotransfection experiment in Fig. 1confirms this point, and serves as an essential control for the experiments with TGF-2. Almost undetectable binding of I-TGF-1 to either T-RI or endogenous T-RII occurred when cells were transfected with T-RI alone (Fig. 1, lane1). In contrast, substantial binding of I-TGF-1 to T-RII but not to endogenous T-TI occurred after transfection of T-RII alone (lane2). This confirms that binding of TGF-1 to T-RII does not require expression of T-RI(19) . In addition, and as previously reported(6) , after cotransfection of T-RI and T-RII I-TGF-1 is bound and cross-linked to both receptors (Fig. 1, lane3). We do not know why less I-TGF-1 was cross-linked to T-II when T-RI was coexpressed; one possibility is that cotransfection of both receptors decreased surface expression of T-RII.


Figure 1: Binding and cross-linking of I-TGF-1 to transfected COS cells expressing T-RI or/and T-RII. COS cells were transfected with 1 µg of T-RI-HA (lane1), 1 µg of T-RII-Myc cDNA (lane2), or 1 µg of each cDNA (lane3). Forty-eight hours after transfection, the cells were incubated 2 h at 4 °C with 50 pMI-TGF-1. The cells were then incubated for 10 min at 4 °C in the presence of 50 mM of the cross-linking reagent DSS. The cell lysates were resolved by electrophoresis through a 10% SDS-polyacrylamide gel and autoradiographed using Kodak XAR film.



Consistent with our previous results(18) , Fig. 2A (lanes 1-4) show that, in COS cells transfected with T-RII cDNA, little I-TGF-2 is bound and cross-linked to T-RII or endogenous T-RI or T-RIII, even at high concentrations (700 pM) of TGF-2. Importantly, cotransfection of T-RI dramatically increased the amount of I-TGF-2 cross-linked to T-RII and T-RI (lanes 5-8). Quantification (Fig. 2B) showed that, at concentrations of TGF-2 less than 300 pM, over 10 times more I-TGF-2 was cross-linked to T-RII when T-RI was coexpressed. After coexpression of both T-RII and T-RI, the concentration of I-TGF-2 required for half-maximal binding and cross-linking to cell surface T-RI and T-RII was 40 pM. In the cotransfection experiments, the amount of I-TGF-2 cross-linked to T-RI was higher than that to T-RII. This may reflect a real difference in the proportion of both receptors in the binding complex or differences in the efficiency of ligand-receptor cross-linking.


Figure 2: Binding and cross-linking of I-TGF-2 to transfected COS cells expressing T-RI and T-RII. COS cells were transfected using the dextran-chloroquine technique with 1 µg of TB-RII-Myc cDNA without (lanes 1-4) or together with (lanes 5-8) 1 µg of T-RI-HA cDNA. Forty-eight hours after transfection, the cells were incubated at 4 °C with increasing concentrations of I-TGF-2: 6 pM (lanes1 and 5), 30 pM (lanes2 and 6), 150 pM (lanes3 and 7), or 750 pM (lanes4 and 8). After cross-linking with DSS, as detailed in the legend to Fig. 1, the cell lysates were resolved by electrophoresis through a 10% SDS-polyacrylamide gel and autoradiographed using Kodak XAR film. PanelA shows the autoradiogram. In PanelB, the gel was exposed for a short period of time to a Fuji BAS III screen; the individual protein bands were quantified using a Fuji BAS-2000 Bio-imaging analyzer. The amount of radioactivity in each protein species is expressed in arbitrary units. Closedsquares, I-TGF-2 cross-linked to T-RI; closedtriangles, I-TGF-2 cross-linked to T-RII; solidlines, cells transfected with both T-RI-HA and T-RII-Myc; dashedlines, cells transfected only with T-RII-Myc.



To confirm that the cross-linked receptor species correspond to the transfected TGF- receptors and not to endogenous COS cell receptors, the cell lysates from the experiment in Fig. 2were immunoprecipitated with either anti-HA or anti-Myc antibodies (Fig. 3). When the cells were transfected only with T-RII-Myc and cross-linked to I-TGF-2, the anti-Myc antibody precipitated no I-TGF-2 cross-linked to T-RII (Fig. 3, lanes 11-14). Similarly, when cells were transfected only with T-RI-HA and cross-linked to I-TGF-2, only trace amounts of labeled receptors could be detected (Fig. 4, lanes 1-4) and the anti-HA antibody precipitated no I-TGF-2 cross-linked to T-RI (data not shown). Thus, ITGF-2 cannot bind either to T-RII-Myc or T-RI-HA when expressed alone in COS cells; the low level of binding of I-TGF-2 to type I and type II receptors singly expressed in COS cells (Fig. 2A, lanes 1-4) most likely involves endogenous type I and II COS cell receptors since these are not precipitated by the anti-epitope antibodies. However, when COS cells were transfected both with T-RII-Myc and T-RI-HA and cross-linked to I-TGF-2, both anti-Myc and anti-HA antibodies immunoprecipitated I-TGF-2 cross-linked to both T-RII-Myc and T-RI-HA (Fig. 3, lanes 5-8 and 15-18). Thus, both T-RII and T-RI are associated in a tight complex after ligand binding. The total amount of both receptors immunoprecipitated with anti-Myc antibodies is lower than that with anti-HA, most likely reflecting a lower efficiency of immunoprecipitation by the anti-Myc antibody.()


Figure 3: Binding and cross-linking of I-TGF-2 to transfected COS cells expressing T-RI and T-RII: analysis by immunoprecipitation. The cell lysates from the experiment in Fig. 2 were immunoprecipitated overnight in the presence of 1% Triton X-100 using 10 mg/ml anti-HA or anti-Myc (9E10) antibody. Lanes 1-8 represent the samples in lanes 1-8 of Fig. 1A immunoprecipitated with the anti-HA antibody, respectively, and lanes 11-18 represent the samples in lanes 1-8 of Fig. 1A immunoprecipitated with the anti-Myc antibody. The immunoprecipitates were resolved by electrophoresis through a 10% SDS-polyacrylamide gel and autoradiographed using Kodak XAR film.




Figure 4: Binding and cross-linking of I-TGF-2 to transfected COS cells expressing increasing amounts of T-RI in the presence or absence of T-RII. COS cells were transfected with 0 µg (lanes1 and 5), 0.1 µg (lanes2 and 6), 0.3 µg (lanes3 and 7), or 1 µg (lanes4 and 8) of T-RI-HA cDNA without (lanes 1-4) or together with (lanes 5-8) 1 µg of T-RII-Myc cDNA. Two days after transfection, the cells were incubated at 4 °C for 2 h with 150 pMI-TGF-2, then for 10 min at 4 °C in the presence of 50 µM DSS. The cell lysates were analyzed as in Fig. 1.



The increased amount of type II receptor cross-linked to I-TGF-2 after T-RI overexpression indicates that T-RI increases the affinity of TGF-2 for T-RII. On the other hand, the amount of type I receptor cross-linked to I-TGF-2 dramatically increases when this receptor is coexpressed with T-RII, suggesting that T-RII increases the affinity of T-RI for TGF-2. Fig. 4demonstrates directly that T-RII increases the affinity of TGF-2 for T-RI. In this study, different concentrations of T-RI-HA cDNA were transfected alone or cotransfected with a fixed amount of T-RII-Myc cDNA. Transfected cells were incubated at 4 °C for 2 h with a fixed amount of TGF-2 (150 pM) and then subjected to cross-linking with DSS. Transfection of T-RI-HA in the absence of transfected T-RII does allow a small but significant binding of I-TGF-2 to T-RI-HA (Fig. 4, lanes 1-4); presumably this is due to endogenous T-RII in the COS cell clone used in this study interacting with the transfected T-RI. Binding of I-TGF-2 to T-RI-HA was dramatically increased when T-RII was cotransfected (Fig. 4, lanes 5-8).


DISCUSSION

The secreted exoplasmic domain of the human TGF- type II receptor binds to and can be cross-linked to I-TGF-1 with an apparent affinity of 200 pM; in contrast, TGF-2 does not bind to this receptor domain (K > 10 nM)(18) . The ability of I-TGF-1 to bind directly to the extracellular domain of the human TGF- type II receptor suggests that the type II receptor may be the primary binding subunit for TGF-1. Several facts support this notion. First, mutant cell lines that lack cell-surface type II receptors and are resistant to growth inhibition by TGF-1, such as DR mink lung cells and Hep 3B-TR cells, also lack type I receptors that are able to bind ligand, and expression of the type II receptor in these cells restores binding and cross-linking of TGF-1 to cell surface type I receptors(19) . Second, several type I receptors for TGF- and activin have been cloned. When expressed alone in transfected cells none are able to bind TGF-1 or any other ligand tested. When coexpressed in COS cells with the type II TGF- receptor all of these species are able to bind TGF-1, and when the type II activin receptor is coexpressed all are able to bind and be cross-linked to activin. Thus, the nature of the type II receptor determines the nature of the ligand that is bound to the type I receptor, even though only certain combinations of type II and I receptors are apparently able to transduce TGF-1 or activin signals(9, 34) .

TGF-2 does not bind at all to the soluble secreted extracellular domain of T-RII (18) or to T-RII expressed in transfected COS cells (Ref. 18 and this work), yet T-RII is essential for TGF-2 signaling(16) . Thus, binding of TGF-2 to T-RII might require other receptor subunits on the cell surface. One of these could be T-RIII, since expression of T-RIII enhances binding of TGF-2 to T-RII in transfected COS cells and also in L6 myoblasts(15, 18) . Furthermore, the affinity of TGF-2 for T-RI and T-RII is higher in cells that endogenously express high numbers of T-RIII than in cells that do not(24) .

However, growth inhibition by TGF-2 does not require expression of T-RIII(15) . The data presented here strongly indicate the existence of a high affinity TGF-2 receptor that is a hetero-oligomer of the two serine-threonine kinase receptors, T-RI and T-RII. In COS cells overexpressing T-RI or T-RII alone, the amount of receptors cross-linked to iodinated TGF-2 was very low, even at high concentrations of ligand. Cotransfection of T-RI with T-RII dramatically increased the ability of both T-RI and T-RII to be cross-linked to I-TGF-2.

The current model for binding of members of the TGF- family of cytokines to cell surface receptors is based mainly on studies using TGF-1. High affinity binding of TGF-1 to T-RII does not require the presence of T-RI since mutant cells lacking functional T-RI display a normal pattern of binding of TGF-1 to T-RII (16, 19). Binding and cross-linking of TGF-1 to T-RI is dependent on prior binding of TGF-1 to T-RII(19) . This interaction presumably involves the extracellular and/or transmembrane domains of both receptors since a mutant truncated T-RII, lacking a complete cytoplasmic domain, restores binding of TGF-1 to T-RI in a cell line that lacks a functional T-RII(35) .

Our data indicate that interaction of TGF-2 with TGF- receptors follows a different sequence of events. Since expression of both T-RI and T-RII are required for TGF-2 to bind with high affinity to either receptor, cell surface T-RI and T-RII probably interact in the absence of ligand. A weak ligand-independent interaction between T-RI and T-RII has been observed when the proteins are overexpressed in insect (36) and COS cells(29) . Since most of the cell surface T-RII is in homodimers(22) , heteromers of T-RI and T-RII could represent a small proportion of the total receptors on the cell surface. We cannot say whether these presumed heteromers would contain one or two subunits of T-RI or T-RII. We hypothesize that the small number of high affinity receptors for TGF-2 in Mv1Lu epithelial cells are preformed T-RI-T-RII complexes. In the Mv1Lu cell line, TGF-2 apparently binds only to a small subset of the T-RI and T-RII receptors that can bind TGF-1(24) . However, the affinity of binding of TGF-2 to these high affinity receptors is the same (20 pM TGF-2 required for half-saturation) as for binding of TGF-1 to a larger number of cell surface receptors, and the concentrations of TGF-1 and TGF-2 required for growth inhibition are also the same (24). Thus, these presumably preformed T-RI and T-RII heteromers could be the preferred receptors for signaling by TGF-2.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant R01 CA-63260 (to H. F. L.) and National Institutes of Health/NCI Outstanding Investigator Grant 5R35 CA-39826 (to R. A. W.). 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.

§
Recipient of Fellowship FE-41295 from the Medical Research Council of Canada.

Research Professor of the American Cancer Society.

**
To whom correspondence should be addressed. Tel.: 617-258-5216; Fax: 617-258-9872; E-mail: lodish@wi.mit.edu.

The abbreviations used are: TGF-, transforming growth factor ; T-RI, T-RII, and T-RIII, type I, II, and III TGF- receptors; PAGE, polyacrylamide gel electrophoresis; DSS, disuccinimidyl suberate.

C. Rodriguez and H. F. Lodish, unpublished observations.

R. G. Wells and H. F. Lodish, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Petra Knaus, Kunxin Luo, Riki Perlman, and Rebecca Wells for helpful discussions of the manuscript.


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