©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
GH3 Pituitary Tumor Cells Contain Heteromeric Type I and Type II Receptor Complexes for Transforming Growth Factor and Activin-A (*)

(Received for publication, August 15, 1994; and in revised form, November 1, 1994)

Aristidis Moustakas (1)(§) Toru Takumi (1)(¶) Herbert Y. Lin (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

Transforming growth factors beta (TGF-betas) and activins induce and inhibins block secretion of follicle-stimulating hormone by rat GH3 pituitary tumor cells. Cheifetz et al. (Cheifetz, S., Ling, N., Guillemin, R., and Massagué, J.(1988) J. Biol. Chem. 263, 17225-17228) reported that GH3 cells express a 50-kDa surface protein, termed the type IV TGF-beta receptor, that directly binds all of these peptide hormones. Here we show that GH3 cells express the previously identified type I and type II receptors for TGF-beta and activin-A. Immunoprecipitation of affinity-labeled surface binding proteins with antisera specific to known receptors demonstrated independent heteromeric complexes of TGF-beta types I and II receptors and of activin types I and II receptors. As judged by ligand-binding and cross-linking analysis, TGF-beta binding to the TGF-beta receptors is not inhibited by activin-A and activin-A binding to its receptors is not inhibited by TGF-beta. Screening of a cDNA library from GH3 cells for potential receptor serine-threonine kinases yielded the known types I and II TGF-beta and activin receptors. The presumed common intracellular signaling pathway for TGF-beta and activin in GH3 cells appears to be mediated by distinct cell-surface receptors.


INTRODUCTION

The transforming growth factors-beta (TGF-betas), (^1)activins and inhibins are structurally similar dimeric polypeptides that regulate cell growth, differentiation, and development (reviewed in (1, 2, 3) ). These polypeptides belong to a large superfamily of growth factors which also includes mammalian bone morphogenetic proteins and Müllerian inhibiting substance(4) . Inhibins are heterodimers of alpha and beta chains while activins are dimers of beta chains(1) . Mammals have three homodimeric isoforms of TGF-beta, termed TGF-beta1, -beta2, and -beta3, and at least two heterodimeric isoforms, TGF-beta1.2 and TGF-beta2.3(2, 3) . Activins induce synthesis and release of follicle-stimulating hormone by pituitary cells(5, 6) , stimulate steroidogenesis in granulosa cells (7, 8) , stimulate erythroid differentiation(9) , and induce mesodermal tissues during amphibian development(10, 11) . In general, inhibins have opposing effects to those of activins(1, 12) . TGF-betas generally mimic the function of activins in the above-mentioned systems(12, 13) and exhibit a number of distinct regulatory functions, including stimulation of extracellular matrix deposition (3) and suppression of immune cell growth(14) . Both activins/inhibins and TGF-betas inhibit growth of many cells(15, 16, 17) .

Specific cell-surface receptors mediate the physiological effects of activins and TGF-betas (reviewed in (18, 19, 20, 21) ). Three distinct receptors for the TGF-betas and activin-A have been identified through their ability to bind and be chemically cross-linked to radioiodinated ligands: types I (TGF-betaRI and ActRI), II (TGF-betaRII and ActRII), and III receptors (TGF-betaRIII and ActRIII), of molecular mass 55, 80, and >100 kDa, respectively. The types I and II receptors for TGF-betas and activin-A are transmembrane serine-threonine kinases(16, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) which are essential for signal transduction, while the type III receptors present ligands to the types I and II receptors. The type II receptors require their corresponding type I receptors for signaling, while binding of TGF-betas or activin-A to the respective type I receptors requires coexpression of the corresponding type II receptor(16, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46) .

In most cells, activins/inhibins and TGF-betas bind to distinct sets of receptors. It has been reported that GH3 rat pituitary tumor cells express a single high affinity receptor for both activins/inhibins and TGF-betas(47) , reflecting the similar physiological effects of these hormones on pituitary cells. This 55-60-kDa surface protein could be affinity-labeled by I-TGF-beta1; labeling was inhibited by TGF-beta1, TGF-beta2, activin-AB, and inhibin-B at concentrations in the high picomolar to low nanomolar range(47) . Thus, GH3 cells apparently express a novel type of cell-surface TGF-beta receptor (type IV) capable of recognizing several members of the TGF-beta superfamily.

Here we show that GH3 cells express heteromeric type I-type II receptor complexes for the TGF-betas and for activin-A that have been reported in other cultured cell systems. We find no evidence for a type IV receptor. We hypothesize that in GH3 cells the TGF-betas and activins use different receptor complexes to induce similar physiological effects, presumably by activating common downstream intracellular effectors.


MATERIALS AND METHODS

Cell Culture

GH3 cells (CCL 81.2: American Type Culture Collection, Rockville, MD) were grown as monolayers in Dulbecco's modified Eagle's medium supplemented with penicillin, streptomycin, L-glutamine, 10% horse serum, and 5% calf serum (Life Technologies, Inc.).

Generation of Anti-peptide Antibodies

We have described a polyclonal rabbit antiserum, called alpha(TGF-betaRII), raised against a peptide corresponding to a C-terminal sequence of the human TGF-betaRII (49) . Similarly, a polyclonal antiserum, alpha(ALK-5), specific for a juxtamembrane cytoplasmic epitope (Val-158 to Asp-179, VPNEEDPSLDRPFISEGTTLKD) of ALK-5, the human TGF-betaRI(31) , and one, alpha(ALK-4), for the corresponding epitope (Gln-160 to Asp-181, QRLDMEDPSCEMCLSKDKTLQD) of ALK-4, human ActRI-B(16, 35) , were raised by Research Genetics (Huntsville, AL) according to standard protocols (50) . Antiserum alpha(ALK-2), against the homologous juxtamembrane epitope of the human type I receptor ALK-2 (35) for TGF-beta or activin (29, 30, 35) , was a gift from Dr. K. Miyazono (Uppsala, Sweden). For immunoprecipitations using alpha(TGF-betaRII), immunoglobulins (IgGs) were prepared from the crude rabbit serum by ammonium sulfate precipitation followed by DEAE-cellulose chromatography(50) . Crude antisera were used for all other immunoprecipitations.

Receptor Cross-linking

TGF-beta1, -beta2, and -beta3 were supplied by R & D Systems (Minneapolis, MN), and activin-A was a gift of Dr. Y. Eto (Ajinomoto Inc., Kawasaki, Japan). These were iodinated as described(49) . We have described (37) procedures for binding and cross-linking of 0.05 and 0.5 nMI-TGF-beta1, -beta2, or activin-A to cells grown on six-well trays or 100-mm dishes (Corning Inc., Corning, NY). Cross-linked proteins were resolved by 5-10% linear gradient SDS-PAGE under reducing conditions and autoradiographed on XAR film (Kodak, Rochester, NY) at -70 °C or on a phosphorimager (Fujix, BAS 2000, Fuji Photo Film Co. Ltd., Japan). To test the sensitivity to the reductant dithiothreitol (DTT) of ligand binding to type I receptors, cells were preincubated for 5 min at 37 °C with 1 mM DTT and washed to remove the reducing agent prior to addition of radioligands(49) .

Receptor Immunoprecipitation

Cell-surface receptors were cross-linked to 0.2 or 0.6 nMI-TGF-beta1, -beta2, or activin-A as described above. Detergent extracts were diluted to 1 ml (final volume) of ice-cold immunoprecipitation buffer (phosphate-buffered saline with 1% Triton X-100, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride). Extracts were precleared by incubation with preimmune serum followed by incubation with protein A-Sepharose (Sigma). Then, 10 µg/ml IgG fraction or 10 µl/ml crude sera were added in the absence or presence of an equimolar amount of the respective immunizing peptide. After precipitation with protein A-Sepharose the immunocomplexes were thoroughly washed in immunoprecipitation buffer supplemented with 0.2% SDS, dissolved in Laemmli loading buffer, and subjected to 5-10% linear gradient SDS-PAGE, followed by autoradiography or phosphorimager analysis. All antibodies specifically precipitated metabolically labeled receptors and affinity-labeled receptors from transfected cells expressing the appropriate recombinant receptor cDNA and from cell lines expressing endogenous receptors(^2)(35, 37, 49) .

Reverse Transcription Polymerase Chain Reaction

cDNA synthesized from GH3 cell mRNA was used as a DNA template for PCR amplification. Primers corresponding to the most conserved amino acid sequences in the kinase domain of the known members of the TGF-beta receptor family were used; the sequences of the clones and the details of the reverse transcription-PCR procedures are described in full detail elsewhere(48) . Amplified cDNA products were purified from acrylamide gels and ligated to a pCRII vector (Invitrogen, San Diego, CA). Random clones were isolated and sequenced using Sequenase (U. S. Biochemical Corp.).

Rapid cDNA Cloning by PCR Screening

A cDNA library was constructed from GH3 cells as described(25) . The GH3 library was screened as described elsewhere(48) , and single positive clones were obtained. The cDNA sequences were determined as described above.


RESULTS

Cross-linking of I-TGF-beta1 to GH3 cells resulted in a single predominant protein species of 70 kDa (Fig. 1, lane2), confirming the result in reference(47) , where the type IV receptor was described. This protein species is equivalent to 57 kDa, the approximate molecular mass of the type I TGF-beta and activin receptors, if the molecular mass of the cross-linked monomeric TGF-beta1 is subtracted; it has the same size as the cross-linked type I receptors in many other cell lines that express endogenous receptors or transfected recombinant type I receptors. Fig. 1(lane 1) shows that prior treatment of the cells with DTT resulted in partial inhibition of labeling of the 70-kDa species, suggesting that this species might consist of a heterogeneous population of receptors, some of which exhibit the typical DTT sensitivity of TGF-beta type I receptors. Importantly, cross-linking of 0.05 nMI-TGF-beta1 to the 57-kDa receptor was efficiently competed by a 100-fold excess of unlabeled TGF-beta1 (Fig. 1, lane6) and by a 500-fold excess of TGF-beta3 (lane 12) but not by a 500-fold excess of TGF-beta2 (lane 9) or activin-A (lane15). Thus, this 70-kDa species resembled in its binding properties TGF-beta receptors characterized in other cell lines(35, 37, 49) . In contrast to results in (47) , up to 25 nM activin-A could not inhibit binding of TGF-beta1 (lanes 13-15) or TGF-beta2 (data not shown) to this surface receptor.


Figure 1: Chemical cross-linking of I-TGF-beta1 to GH3 cells. Confluent GH3 monolayers were incubated with 0.05 nMTGF-beta1 alone (lanes 1 and 2) or together with increasing concentrations of unlabeled TGF-beta1 (0.5, 1, 5, and 25 nM in lanes 3-6, respectively), TGF-beta2 (1, 5, and 25 nM in lanes 7-9, respectively), TGF-beta3 (1, 5, and 25 nM in lanes10-12, respectively) or activin-A (1, 5, and 25 nM in lanes13-15, respectively). Cells in lane1 were treated with 1 mM DTT for 5 min at 37 °C prior to ligand binding. After cross-linking with DSS, detergent extracts were analyzed by 5-10% SDS-PAGE. The positions of molecular mass markers (in kDa) are indicated on the left margin and that of the type I receptor on the right margin of the panel. Only the relevant portion of the autoradiogram is shown.



Binding of the TGF-betas to the type I receptor requires co-expression of the type II TGF-beta receptor(20, 21) , but in GH3 cells affinity-labeling experiments revealed the presence only of type I-like receptors; type II receptors were undetectable ( Fig. 1and (47) ). Type II receptors were visualized by immunoprecipitation analyses of GH3 proteins affinity-labeled with TGF-beta1 using specific anti-receptor antisera (Fig. 2). Antibody alpha(ALK-5), raised against the type I TGF-beta receptor, specifically immunoprecipitated both the 70-kDa TGF-beta1 and the TGF-beta2-affinity-labeled type I receptors together with small amounts of an affinity-labeled protein of the expected size of the type II receptor (Fig. 2, lanes 8 and 14). Importantly, antibody alpha(TGF-betaRII), specific for the type II TGF-beta receptor, specifically immunoprecipitated the 70-kDa TGF-beta1 and the TGF-beta2-affinity-labeled type I receptors together with large amounts of the type II receptor (lanes 9 and 16). Immunoprecipitation was inhibited by incubation of the antibodies with their corresponding immunogenic peptide (lanes13 and 15). Neither antibody precipitated receptors affinity-labeled with I-activin-A (lanes4 and 5; see below). Antibodies alpha(ALK-4) and alpha(ALK-2) also immunoprecipitated a small amount of the 70-kDa TGF-beta1 and TGF-beta2-affinity-labeled type I receptors (lanes 6, 7, 10, and 12); this was surprising since ALK-4 and ALK-2 are thought to be type I receptors specific for activin-A. However, TGF-beta will bind to these receptors if co-expressed in COS cells with large amounts of the type II TGF-beta receptor (^3)(35) or, in the case of ALK-2, when overexpressed in murine epithelial cells(28) . Thus, GH3 cells express the conventional type I (ALK-5, ALK-4, and ALK-2) and type II TGF-beta receptors.


Figure 2: Immunoprecipitation with anti-receptor antibodies of GH3 proteins cross-linked to I-TGF-beta1, -beta2, and activin-A. GH3 cells were chemically cross-linked to 0.6 nMI-labeled activin-A (lanes 1-5), TGF-beta2 (lanes 6-9), or TGF-beta1 (lanes 10-16). Detergent extracts were immunoprecipitated with antibodies alpha(ALK-2) (lanes 1, 6, and 10), alpha(ALK-4) (lanes 2, 3, 7, 11, and 12), alpha(ALK-5) (lanes 4, 8, 13, and 14), or alpha(TGF-betaRII) (lanes5, 9, 15, and 16) alone or in the presence of equimolar amounts of the respective immunogenic peptides (lanes 2 and 11 for alpha(ALK-4), lane13 for alpha(ALK-5), and lane15 for alpha(TGF-betaRII)). The resulting immunocomplexes were analyzed by 5-10% SDS-PAGE. The positions of molecular mass markers (in kDa) are indicated on the leftmargin and of the type I and II receptors on the rightmargin of the panel. Only the relevant portion of the autoradiogram is shown.



Fig. 3shows that cross-linking of I-activin-A to GH3 cells resulted in a single predominant protein species of 74 kDa (lane 2), equivalent to 61 kDa if the molecular mass of the cross-linked monomeric activin-A is subtracted. This species migrates slower than the TGF-beta1 affinity-labeled type I receptor of 70 kDa (compare lanes 3, 9, and 14 of Fig. 2), and has the molecular size expected for type I activin receptors(16, 35) . Fig. 3(lane 1) shows that labeling of this receptor with I-activin-A was somewhat affected by prior treatment of cells with DTT, similar to results obtained with the TGF-beta type I receptor (Fig. 1). Importantly, binding of 0.5 nMI-activin-A to this GH3 cell receptor was inhibited by as little as a 10-fold excess of unlabeled activin-A (lanes 3-6) but not at all by as much as 250 nM (500-fold) of any TGF-beta isoform (lanes 7-15). Thus, in contrast to the results in (47) , GH3 cells express distinct type I receptors for TGF-beta and for activin-A. We find no evidence for a receptor that can directly bind to both ligands.


Figure 3: Chemical cross-linking of I-activin-A to GH3 cells. Confluent GH3 monolayers were incubated with 0.5 nMI-activin-A alone (lanes 1 and 2) or together with increasing concentrations of unlabeled activin-A (5, 10, 50, and 250 nM in lanes 3-6, respectively), TGF-beta1 (10, 50, and 250 nM in lanes 7-9, respectively), TGF-beta2 (10, 50, and 250 nM in lanes 10-12, respectively), and TGF-beta3 (10, 50, and 250 nM in lanes 13-15, respectively). Cells in lane 1 were treated with 1 mM DTT for 5 min at 37 °C prior to ligand binding (marked as +), whereas cells in lane2 were not (marked as -). After cross-linking with DSS detergent, extracts were analyzed by 5-10% SDS-PAGE. The positions of molecular mass markers (in kDa) are indicated on the left margin and of the type I receptor on the right margin of the panel. Only the relevant portion of the autoradiogram is shown.



The GH3 receptor affinity-labeled by activin can be immunoprecipitated by alpha(ALK-4), specific for the type I activin receptor, together with small amounts of an affinity-labeled protein of the expected size of the type II activin receptor (Fig. 2, lane 3). Since we do not have antisera specific for the type II activin receptor, we are unable to confirm the identity of this species by specific immunoprecipitation. Neither GH3 receptor affinity-labeled by activin-A can be immunoprecipitated by alpha(ALK-5), specific for the type I TGF-beta receptor, or by alpha(ALK-2) (Fig. 2, lanes 4 and 1, respectively).

We extended our analysis of type I and type II receptors expressed in GH3 cells by screening mRNA and a cDNA library made from these cells by means of the polymerase chain reaction(48) . We used sets of degenerate primers corresponding to the most conserved amino acid sequences in the kinase domain of the known members of the TGF-beta receptor family.^3 Of a total of 27 PCR clones analyzed, one was 99% identical to the corresponding region of the human type II TGF-beta receptor and encoded its rat counterpart(26) . Three other types of cDNA clones were isolated; nine corresponded to ALK-2, also called ActRI or tsk-7L(28, 29, 30, 35) . Twelve corresponded to ALK-4(16, 35) and one to ALK-5, the type I TGF-beta receptor(31) . The immunoprecipitation studies described here showed that the protein products of all these clones were expressed in functional form on the plasma membrane of GH3 cells. Taken together, our PCR cloning and affinity-labeling/immunoprecipitation studies show that GH3 cells express ``conventional'' types I and II receptors for activin and TGF-beta and provide no evidence for a novel type IV TGF-beta receptor.


DISCUSSION

Here we report that GH3 pituitary tumor cells express conventional heteromeric type I and type II receptor complexes for TGF-betas and activin-A, rather than a type IV receptor (47) that binds all of these growth factors. Since TGF-betas and activins bind to distinct GH3 cell-surface receptors yet induce common physiological responses, certain intracellular signal transduction molecules may interact with receptors for both hormones. Since virtually nothing is known of intracellular proteins that mediate signaling by the TGF-beta and activin receptors, it is difficult to speculate what these common molecules might be.

Our affinity-labeling experiments ( Fig. 1and Fig. 3) demonstrate two distinct type I receptors on the surface of GH3 cells, one specific for the TGF-betas and one for activins. Whether inhibins might utilize the same receptor complex as activins remains an open question and was not addressed here. The type I TGF-beta receptor migrates slightly faster (70 kDa) than the activin receptor (74 kDa) upon SDS-PAGE. Using the same SDS-PAGE system, we have consistently observed this difference in mobility when we compared the affinity-labeled recombinant type I TGF-beta receptor ALK-5 and the type I activin receptor ALK-4 expressed in transfected COS cells together with the corresponding type II receptors.^3

Our immunoprecipitation experiments established that in GH3 cells at least some of these type I receptors are in heteromeric complexes with type II receptors. The principal TGF-beta receptor is a heteromer of ALK-5 and TGF-betaRII, while the principal activin receptor is a heteromer of ALK-4 and the type II activin receptor. Taken together, our results argue that the major type I TGF-beta and activin receptors expressed in GH3 cells are ALK-5 and ALK-4, respectively, the predominant type I receptors found in other cells(16, 31, 35) . Our PCR cloning studies are consistent with this conclusion. However, the possibility remains that GH3 cells may also express additional type I-like receptor proteins, which may have escaped detection by our experimental approaches. Importantly, the competition experiments in Fig. 1and Fig. 3indicate that any GH3 protein that directly binds TGF-beta (i.e. in the absence of any other receptor protein) cannot bind activin with measurable affinity, nor can any activin-binding protein also bind TGF-beta.

Our immunoprecipitation experiments (Fig. 2) suggest the presence of type II receptors for both TGF-beta and activin-A on the surface of GH3 cells, although the identity of the activin-A type II receptor was not confirmed by direct antibody precipitation. As we reported previously (37) , the use of anti-receptor antibodies increases the sensitivity of detection of minor affinity-labeled receptor components. The type II receptors determine the ligand recognized by the type I receptors(36, 37) . Thus, it is interesting that in GH3 cells both activin-A and TGF-beta ligands can be cross-linked to the type I receptors with higher efficiency than to the type II receptors ( Fig. 1and Fig. 3). This phenomenon may reflect initial binding of activin and TGF-beta ligands to their respective type II receptors, followed by efficient shuttling, or presentation, of the bound ligand to type I receptors. Thus, the cell surface may contain solitary, affinity-labeled type I receptors as well as type I receptors in complexes with type II receptors. An alternative hypothesis is that, upon ligand binding to the type II receptor, TGF-betas or activins undergo a conformational change that makes them more accessible to the cross-linking agent and/or more able to bind to the type I receptor.

An important unanswered question is the exact stoichiometry of the heteromeric type I-type II receptor complex for TGF-beta and activin, both in GH3 cells, where more type I receptor is detected than type II by affinity labeling, and in Hep3B, Rat-1, and Mv1Lu cells, in which approximately equal amounts of types I and II receptors are detected by affinity-labeling. We have shown that the type II receptor forms homo-oligomers, probably homodimers, both in the absence and presence of TGF-beta ligands(51) . We hypothesize that the heteromeric complex contains two copies of the type I and two of the type II receptors, but the exact composition may differ in different types of cells.

Interestingly, the antibody specific for ALK-4 (activin type I receptor-B) immunoprecipitated not only activin affinitylabeled heteromeric type I-type II receptor complexes, but also TGF-beta affinity-labeled heteromeric receptor complexes. This is in disagreement with the assignment of activin-A as the sole ligand that can specifically signal through the ALK-4 type I receptor (also called ActRI-B; (16) and (35) ). Our results do agree with the ligand-binding studies performed in COS cells co-expressing type I and II receptors^3(35) ; when we expressed TGF-betaRII together with ALK-4, the ligand binding specificity of ALK-4 (and TGF-betaRII) for all three TGF-beta isoforms was indistinguishable from that of the known functional type I TGF-beta receptor ALK-5^3. Despite the fact that ALK-5 but not ALK-4 was able to reconstitute functional TGF-beta responses in mutant mink lung epithelial cells defective in type I receptors(16, 33, 35) , it is plausible that ALK-4 serves as a type I TGF-beta receptor in other cells, such as pituitary cells. Possibly ALK-4 mediates cell responses different from the growth inhibition and induction of the plasminogen activator inhibitor type I gene that have been studied mainly in Mv1Lu cells.

ALK-2, like ALK-4, is able to bind TGF-beta when co-expressed in COS and epithelial cells with the type II TGF-beta receptor(28, 29, 30, 32, 35) . ALK-2 was also thought to transduce signals by activin, but not by TGF-beta, but the ability of ALK-2 to mediate biological responses to TGF-beta has been uncovered recently. (^4)We found that a small amount of TGF-beta can be affinity-cross-linked to ALK-2 on GH3 cells (Fig. 2, lanes 6 and 10). These results cannot be attributed to receptor overexpression (such as occurs in COS cells), since we studied endogenous receptors expressed in very low numbers per cell.

Thus GH3 cells express type I receptors that are capable of binding both TGF-beta and activin. It is difficult to compare the properties of the type I receptors of GH3 cells we described here with those of the GH3 cell type IV receptor(47) . We do not detect inhibition of binding of radiolabeled TGF-beta1 by activins, and it is possible that the competition detected in (47) was due to impurities in the activin and inhibin preparations available then. The electrophoretic mobility of the type IV receptor (47) resembles that of the type I receptors. However, binding of either TGF-beta or activin to a type I receptor requires the presence of the appropriate type II receptor; ligands apparently bind first to a type II receptor and then are shuttled to a type I receptor. Thus, binding of TGF-beta to any type I receptor is not inhibited by the presence of activin, although it is by other TGF-beta isoforms, since activin cannot bind to the type II TGF-beta receptor. Similarly, TGF-beta does not inhibit binding of activin to its type I receptor. These results indicate the necessity for careful analysis of specific receptors in various cell types before the ligand specificity and function of the members of this complex growth factor receptor family can be assigned conclusively.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA-63260 and HL 41484 (to H. F. L.). 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.

§
Postdoctoral fellow of the Anna Fuller Fund (Grant 719).

Fellow of the Human Frontier Science Program. Present address: Dept. of Pharmacology II, Faculty of Medicine, Osaka University, Suita, Osaka 565, Japan.

**
Present address: Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Boston, MA 02114 and Dept. of Medicine, Harvard Medical School, Boston, MA 02115.

§§
To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-9872; lodish{at}wi.mit.edu.

(^1)
The abbreviations used are, TGF-beta, transforming growth factor beta; TGF-betaRI, TGF-betaRII, and TGF-betaRIII, TGF-beta receptor types I, II, and III, respectively; ActRI and ActRII, activin receptor types I and II, respectively; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; PCR, polymerase chain reaction; DSS, disuccinimidyl suberate.

(^2)
A. Moustakas, unpublished results.

(^3)
T. Takumi, A. Moustakas, H. Y. Lin and H. F. Lodish, submitted for publication.

(^4)
R. Derynck, personal communication.


ACKNOWLEDGEMENTS

We thank P. I. Knaus and Y. I. Henis for providing anti-peptide antibodies and for helpful discussions, and U. Klingmüller, K. Luo, P. Scherer, and R. C. Wells for helpful conversations and for reviewing this manuscript. We especially thank K. Luo for assistance with preparation of the figures. We also thank Celtrix Laboratories, Inc., Palo Alto, CA, and R& Systems, Minneapolis, MN for their generous gifts of TGF-beta1 and -beta2; Y. Eto (Ajinomoto Inc., Kawasaki, Japan) for activin-A; and K. Miyazono (Ludwig Institute for Cancer Research, Uppsala, Sweden) for the alpha(ALK-2) antibody.


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