Biosynthesis of the Type I and Type II TGF-beta Receptors
IMPLICATIONS FOR COMPLEX FORMATION*

(Received for publication, September 12, 1996, and in revised form, January 21, 1997)

Rebecca G. Wells Dagger §, Haya Yankelev Dagger , Herbert Y. Lin Dagger and Harvey F. Lodish Dagger par **

From the Dagger  Whitehead Institute, Cambridge, Massachusetts 02142, the § Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, and the par  Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
Note Added in Proof
REFERENCES


ABSTRACT

The TGF-beta type I and type II receptors (Tbeta RI and Tbeta RII) are signaling receptors that form heteromeric cell surface complexes with the TGF-beta s as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-beta -responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-beta 1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-beta 1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for Tbeta RI and Tbeta RII. Our results, which suggest that stable complexes between type I and II TGF-beta receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.


INTRODUCTION

The transforming growth factor-beta s (TGF-beta 1,1 -beta 2, and -beta 3) are the prototypical members of a growing superfamily of peptide growth factors that includes the activins and inhibins, bone morphogenetic proteins, Müllerian inhibiting substance, and the product of the Drosophila decapentaplegic (dpp) gene. Members of the TGF-beta subfamily are multifunctional, with wide-ranging effects on growth, differentiation, immune response, and extracellular matrix organization and biogenesis (1-3). They are among the most important and versatile growth factors yet described.

The three major cell surface receptors for TGF-beta are termed types I, II, and III (Tbeta RI, II, and III, respectively) (4-7). Tbeta RI and Tbeta RII are the signaling receptors, whereas Tbeta RIII appears to promote ligand binding to Tbeta RI and Tbeta RII (8, 9). The type I and II receptors are serine/threonine kinases with approximately 40% identity in their kinase domains (7). Like their ligands, they are members of a large superfamily (10-13); receptors with related serine/threonine kinase domains include activin receptors, bone morphogenetic protein receptors, the Drosophila saxophone, thick veins, and punt gene products, and the Caenorhabditis elegans Daf-1 and Daf-4 proteins (14-26). Within this superfamily, type I- and type II-like kinases fall into distinct subcategories. Tbeta RI-like receptors have a highly conserved juxtamembrane region rich in glycine and serine residues, termed the GS domain, whereas Tbeta RII and similar receptors have a serine- and threonine-rich C-terminal extension.

When overexpressed in COS cells, all three types of TGF-beta receptors form homo-oligomers (likely dimers) on the cell surface even in the absence of TGF-beta (27, 28).2 It is not known, however, whether they form homo- or heterodimers in the absence of ligand when expressed at lower, more physiologic concentrations on the surface of nontransfected TGF-beta -responsive cells. Interactions between the cytoplasmic domains of Tbeta RI and Tbeta RII have been detected in both the yeast two-hybrid system and in COS cells overexpressing the cytoplasmic domains of both receptors (29, 30).3 Studies using chimeric receptors with the extracellular domain of the erythropoietin receptor and the cytoplasmic domains of Tbeta RI or Tbeta RII showed that both homodimerization of the cytoplasmic domain of the type I TGF-beta receptor and heterodimerization with the cytoplasmic domain of the type II receptor is required for intracellular signal transduction leading to growth inhibition (31).

The type II receptor is able to bind TGF-beta 1 and -beta 3 independent of the presence of the type I receptor; Tbeta RI requires the presence of Tbeta RII to bind these ligands (32-34). Both receptors are required for high affinity binding of TGF-beta 2, which appears to bind to a preformed complex of Tbeta RI and Tbeta RII (35). Coimmunoprecipitation studies with ligand-bound receptors demonstrated that Tbeta RI, Tbeta RII, and TGF-beta ligand form a ternary complex on the cell surface (9, 34). The association between Tbeta RI and Tbeta RII expressed at physiologic concentrations appears to be ligand-dependent, at least when tested with TGF-beta 1 (29, 36). Several lines of evidence suggest that this ligand-induced complex may be a heterotetramer, containing at least two copies of each signaling receptor. Weis-Garcia and Massague (37) showed that kinase-deficient and activation-deficient type I receptors can functionally complement each other, implying that the activated complex contains two molecules of Tbeta RI. Additionally, dimers of the Tbeta RI cytoplasmic domain are required for ligand response (31). Henis et al. (27) showed that the type II receptor is a homo-oligomer with or without TGF-beta . This, in combination with the results of two-dimensional gel analyses (38), makes a heterotetramer the most likely minimum cell surface receptor complex.

Once a complex has formed, signaling is initiated by the transphosphorylation of Tbeta RI by Tbeta RII, a constitutively active kinase, on serine and threonine residues in the GS domain (29, 30, 36). This phosphorylation and a functional Tbeta RI kinase are required for downstream signaling (36, 37, 39-41). MADR2, one member of a newly identified class of proteins required for serine/threonine kinase receptor signaling, is a substrate of the TGF-beta receptor complex; it becomes phosphorylated and transmits signals directly to the nucleus (42, 43). Several other cytoplasmic proteins that interact with Tbeta RI or Tbeta RII have been isolated, although their role in signaling has yet to be determined (44-49).3

As part of our ongoing studies into early events in the TGF-beta response, we have studied the biosynthesis of the type I and II TGF-beta receptors in nontransfected, TGF-beta -responsive, mink lung epithelial cells (Mv1Lu). We show here that the rates and efficiencies of Golgi processing of newly made Tbeta RI and Tbeta RII are very different, as are the apparent half-lives of the Golgi-processed, mature receptors. We also show that although TGF-beta 1 has no effect on Golgi processing of either receptor, it markedly reduces the stability of the mature forms of both. This suggests that the two receptors are synthesized independently and neither arrive on the cell surface as a preformed complex nor, in the presence of ligand, remain on the cell surface as a stable complex. In addition, the rapid turnover of the type II receptor has potential implications for regulation, especially since the Endo H-resistant (and therefore presumably cell surface) form of Tbeta RII is even more unstable in the presence of ligand.


MATERIALS AND METHODS

Cell Lines and Cell Culture

Mv1Lu mink lung epithelial cells were obtained from the American Type Culture Collection and were grown in modified Eagle's medium (Life Technologies, Inc.) with 10% decomplemented fetal calf serum (Life Technologies, Inc.) supplemented with 2 mM glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and nonessential amino acids (JRH Bioscience) in a 5% humidified CO2 atmosphere. COS7 cells (American Type Culture Collection) and HYB2 cells (a gift of Dr. A. Geiser (National Institutes of Health) (50)) were grown in Dulbecco's modified Eagle's medium with the same additives except no nonessential amino acids.

Binding and Cross-linking of Iodinated TGF-beta

TGF-beta 1 (a gift of R & D Systems) was iodinated by the chloramine T method as described (51), with the following modifications. TGF-beta 1 (1 µg) in 10 µl of 1 M sodium phosphate, pH 7.2, was mixed with 2 µl of Na125I (0.2 µCi). The reaction was initiated by the sequential addition of three 2-µl portions of chloramine T (0.1 mg/ml). The reaction was stopped by the sequential addition of 10 µl of acetyl tyrosine (100 mM), 100 µl of potassium iodide (100 mM), and 100 µl of urea-saturated acetic acid. For the binding and cross-linking experiment shown in Fig. 5, a modification of the method of Wang et al. (4) was used. Subconfluent Mv1Lu cells in standard medium were incubated at 37 °C in tunicamycin (Boehringer Mannheim; stock of 3 mg/ml in DMSO) at a concentration of 2 µg/ml for times ranging from 0 (no tunicamycin added; duplicate plates) to 11 h. Cells were rinsed once with KRH binding buffer (50 mM Hepes, pH 7.5, 128 mM NaCl, 1.3 mM CaCl2, 5 mM MgSO4, 5 mM KCl) with 0.5% fatty acid-free bovine serum albumin (Sigma) and then incubated at 37 °C in KRH binding buffer with either 2 µg/ml tunicamycin or an equivalent volume of DMSO (0 time points). After 30 min, this material was aspirated and 3 ml of ice-cold KRH binding buffer containing 50 pM 125I-TGF-beta 1 was added. Plates were incubated at 4 °C with rotation for 3 h and then rinsed 3 times with ice cold KRH. The ligand was then cross-linked to the receptor with the addition of 0.5 mg/ml disuccinimidyl suberate (Pierce) from a 100-fold concentrated stock in DMSO, followed by incubation at 4 °C for 15 min. Glycine was added to 20 mM, and plates were incubated for 10 min at 4 °C. Plates were rinsed twice with ice-cold phosphate-buffered saline (PBS), and lysed at 4 °C for 20-30 min in 1 ml lysis buffer (PBS with 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, pH 8.0, and 1 mM phenylmethylsulfonyl fluoride). Insoluble material was pelleted by microcentrifugation at 14,000 rpm for 10 min at 4 °C and discarded. Cleared lysates were immunoprecipitated at 4 °C overnight with <FR><NU>1</NU><DE>200</DE></FR> volume of a polyclonal rabbit antiserum (alpha -IIC (9)) raised against the C-terminal 16 amino acids of the human type II receptor; this epitope has 15 of 16 amino acids identical to the mink lung receptor (34). One-twentieth volume of protein A-Sepharose CL-4B beads (Sigma) was added, and the lysates were incubated with rotation for 30 min. Protein A-Sepharose beads were rinsed twice with lysis buffer and once with PBS, and bound protein was eluted by boiling in 0.5% SDS. The eluate from one of the 0 time point plates was treated overnight with N-glycosidase F (3000 units/50 µl; New England Biolabs). Samples were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis. Autoradiographs were quantified with a LaCie Silverscanner II and MacBAS (Fuji) software.


Fig. 5. Binding of radioiodinated TGF-beta to tunicamycin-treated Mv1Lu cells. Mv1Lu cells were incubated with 2 µg/ml tunicamycin at 37 °C for the times noted. Cells were then cooled to 4 °C and allowed to bind and subsequently become cross-linked to 125I-TGF-beta . Labeled cells were lysed and immunoprecipitated with alpha -IIC. The 0 h time point was prepared in duplicate; one half of the combined immunoprecipitated eluant was treated with N-glycosidase F before SDS-polyacrylamide gel electrophoresis (0*) and the other half was untreated (0). II+, II-, I+, and I- indicate the type II and I receptors with (+) or without (-) N-linked sugars.
[View Larger Version of this Image (60K GIF file)]


Metabolic Labeling

Mv1Lu cells were grown to subconfluence in standard medium on 10-cm plates. They were rinsed once with PBS and then starved in serum-free Dulbecco's modified Eagle's medium minus cysteine and methionine (ICN) with glutamine and penicillin/streptomycin for 5-5.5 h at 37 °C. The medium was then replaced with fresh medium supplemented with 0.2 mM oxidized glutathione (Boehringer Mannheim) and 0.5 mCi/ml of a mixture of [35S]methionine and [35S]cysteine (Express; New England Nuclear) and incubated at 37 °C for 10 min (pulse-labeling). Plates were quickly rinsed twice with warmed chase medium (serum-free Dulbecco's modified Eagle's medium with glutamine and penicillin/streptomycin) and then incubated for the designated chase times at 37 °C in the presence of either 100 pM TGF-beta 1 ("+ ligand") or 50 µg/ml of Pan-Specific TGF-beta Neutralizing antibody (R & D Systems; "- ligand"), the IgG fraction of a polyclonal rabbit antiserum that neutralizes TGF-beta 1, beta 1.2, beta 2, beta 3, and beta 5. Plates were then rinsed three times with ice-cold PBS and lysed in 1 ml of lysis buffer. Cleared lysates were precleared overnight with protein A-Sepharose and then immunoprecipitated with <FR><NU>1</NU><DE>100</DE></FR> volume of antibody alpha -IIC or a polyclonal rabbit antiserum raised against the juxtamembrane cytoplasmic domain of the human type I receptor (7) (an epitope with 22 of 22 amino acids identical to the mink lung receptor (37)), at 4 °C for 2 h. After a 30-min incubation with protein A-Sepharose, bound beads were rinsed three times with lysis buffer plus 0.5% SDS and then twice with PBS. Protein was eluted into 0.5% SDS. For type II receptor immunoprecipitates, the eluate was split in two; one half was treated with Endo H (Genzyme, 100 mIU/ml) in the presence of 100 mM sodium citrate, pH 6.0, at 37 °C overnight. For type I receptor immunoprecipitates, one-third of the eluate was treated with Endo H, and another one-third with N-glycosidase F. In cases where the type I and II receptors had identical chase times, plates were labeled and chased in parallel; the pairs of lysates from such a time point were pooled and then split before immunoprecipitation to ensure that comparison between the two receptors at a given time point was valid. Samples were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis; gels were fluorographed with 2,5-diphenyloxazole, dried, and placed on Kodak XAR film, which was scanned as above.

Control immunoprecipitations (not shown) were performed in the presence of an excess of the immunizing peptide to confirm the identity of the type I and II receptors. Also, because the type I receptor antibody immunoprecipitates a number of unrelated but peptide-competable proteins, COS7 cells mock transfected or transfected with the ALK-5 (type I receptor) cDNA (7) (a gift of Dr. K. Miyazono, Japanese Foundation for Cancer Research, Tokyo, Japan) were pulse-labeled and analyzed on each gel to confirm the proper size of the type I receptor.


RESULTS

Both the ER and Golgi-processed Forms of the Type II Receptor Have Relatively Short Metabolic Half-lives

To determine the half-life of the native type II receptor, we metabolically labeled TGF-beta -responsive mink lung epithelial cells, chased them from 0 to 4 h, and immunoprecipitated detergent extracts with an anti-type II receptor antibody. The newly synthesized receptor (Fig. 1A, left panel, 0 h time point) migrates as a doublet that shifts from 62 and 65 kDa to 58 and 61 kDa after treatment with Endo H (Fig. 1B, left panel), indicating that it is present in the endoplasmic reticulum. This Endo H-sensitive material disappears with a half-life of 30-35 min (Fig. 2A, control). A smear from 67 to 77 kDa, representing the Endo H-resistant receptor, appears at 0.5 h of chase (Fig. 1, A and B, left panels). This Golgi-processed receptor has a heterogeneous population of complex N-linked sugars. More than 50% of the steady state population of cellular receptors, including the majority of the cell surface form, is also O-glycosylated (52).4 During the chase, approximately 90% of pulse-labeled Endo H-sensitive receptor is converted to the Endo H-resistant, Golgi-processed material (Fig. 2B), demonstrating efficient conversion of the ER to the mature form, with minimal ER degradation.


Fig. 1. The ER and mature, Golgi-processed, forms of the type II receptor have relatively short half-lives. Mink lung cells were pulse-labeled for 10 min at 37 °C in 0.5 mCi/ml [35S]methionine/cysteine and then chased at 37 °C for the times indicated in nonradioactive medium containing either 100 pM TGF-beta 1 or 50 µg/ml of a pan-specific TGF-beta neutralizing antibody (- ligand). Cells were lysed and immunoprecipitated with alpha -IIC, an anti-peptide antibody specific for the C terminus of the type II receptor. Eluted material was split in two; one half was treated with Endo H (B), and the other half was left untreated (A). Brackets indicate the Endo H-sensitive (ER) and mature (M) forms of the receptor. The asterisks indicate non-peptide-competable, contaminant bands.
[View Larger Version of this Image (56K GIF file)]



Fig. 2. Quantitation of the Endo H-sensitive and -resistant forms of newly made type II receptor. Bands representing Endo H-sensitive (A) and -resistant (B) forms of the type II receptor (from Fig. 1A) were scanned and quantified with MacBAS software and plotted as a percentage of the total amount of labeled protein present at the end of the pulse (time point 0). Control samples (- ligand) represent cells chased in the presence of a pan-specific TGF-beta neutralizing antibody.
[View Larger Version of this Image (17K GIF file)]


The Golgi-processed form of Tbeta RII, like the Endo H-sensitive form, disappears rapidly, with a half-life of approximately 2.5 h (Fig. 2B). The smear of Endo H-resistant material represents both intracellular and cell surface receptors. Over half of the total population of Endo-H resistant Tbeta RII, however, is sensitive to digestion of intact cells with proteinase K (data not shown), indicating that approximately half of the total Endo-H resistant Tbeta RII is on the cell surface at steady state. Thus, our data suggest that the half-life of cell surface Tbeta RII is also approximately 2.5 h.

Similar experiments (data not shown) with the HYB2 line of TGF-beta -responsive human cells (50) also show rapid metabolism of the type II receptor, with a half-life of the Endo H-sensitive Tbeta RII of 25-30 min and a half-life of the Endo H-resistant form of approximately 3 h.

Golgi-processed Type II Receptor Is Degraded More Rapidly in the Presence of TGF-beta

To assess the effect of TGF-beta on the stability of the Endo H-sensitive and -resistant forms of Tbeta RII, the above experiments were performed in parallel in the presence of 100 pM TGF-beta 1 (Fig. 1, A and B, right panels). The Endo H-sensitive receptor disappeared with a t1/2 of 30-35 min, identical to the rate in control cells (Fig. 2A). This indicates that ER stability, processing efficiency, and the rate of ER to Golgi transport of newly made Tbeta RII are unaffected by the presence of TGF-beta . Endo H-resistant Tbeta RII, however, is less stable in the presence of TGF-beta , with a half-life of 1.7 h compared to 2.5 h in control cells (Fig. 2B). Importantly, all control experiments (- ligand) were performed in the presence of a neutralizing antibody against the TGF-beta s (see "Materials and Methods"); thus, it is unlikely that residual or endogenously synthesized TGF-beta masked an even more marked effect of the ligand.

Performing the same experiment on HYB2 cells, we obtained a similar lack of effect of TGF-beta on the fate of the Endo H-sensitive Tbeta RII, and a significant although less marked effect on the stability of the Golgi-processed receptor (data not shown).

The Type I Receptor Is Metabolized More Slowly than the Type II Receptor

The half-life of the type I receptor was determined by metabolically labeling Mv1Lu cells in parallel with the experiments above (Figs. 1 and 2) and immunoprecipitating the cell lysates with an anti-type I receptor antibody. Lysates from similarly labeled, mock- and Tbeta RI-transfected COS7 cells (not shown) were run alongside the Mv1Lu cell material to confirm identification of the Tbeta RI bands. Pulse-labeled Tbeta RI migrates on SDS-polyacrylamide gel electrophoresis as a single species of 52 kDa (Fig. 3A, left panel). The gel mobility shifts to 49 kDa after treatment with Endo H, indicating that newly synthesized Tbeta RI contains a high mannose, N-linked oligosaccharide characteristic of the ER (Fig. 3B, left panel). The Endo H-sensitive ER form of Tbeta RI disappears with a half-life of 2.8-3 h (Fig. 4A), markedly longer than the half-life of 30-35 min of newly made, Endo-H-sensitive type II receptor. At no time during the chase period could we detect a specific higher molecular weight form of Tbeta RI; we presume that the Golgi-processed, Endo H-resistant form of Tbeta RI migrates within the background "smear" above 52 kDa. By treating the Tbeta RI immunoprecipitates with N-glycosidase F, however, removing all N-linked sugars, we can quantify the total (Endo H-sensitive and -resistant) amount of metabolically labeled Tbeta RI in the cell (Fig. 3C). We calculate the amount of mature (Endo H-resistant) Tbeta RI by subtracting the amount of labeled Endo H-sensitive receptor (Fig. 3B) from the total amount (N-glycosidase F-treated) of Tbeta RI (Fig. 3C). Fig. 4 shows that no more than 50% of pulse-labeled Tbeta RI is converted to an Endo H-resistant form, compared with approximately 90% of Tbeta RII (Fig. 2B).


Fig. 3. The type I receptor exits the ER more slowly than the type II. Mink lung cells were 35S-labeled and chased as in Fig. 1, except that chase times ranged from 0 to 10 h. This experiment was performed in parallel with that in Fig. 1. The chase medium contained either 100 pM TGF-beta 1 or 50 µg/ml of a pan-specific TGF-beta neutralizing antibody (- ligand). Cells were lysed and immunoprecipitated with an anti-peptide antibody specific for the type I receptor. Eluted material was divided into three portions; one third was treated with Endo H (b), one third was treated with N-glycosidase F (c), and one third was left untreated (a). The solid arrowhead shows the location of the deglycosylated receptor at 49 kDa; the open arrowhead shows the location of the 52-kDa receptor bearing Endo H-sensitive oligosaccharides that is seen in A. The asterisk shows a nonspecific contaminant band.
[View Larger Version of this Image (52K GIF file)]



Fig. 4. Quantitation of the Endo H-sensitive and -resistant forms of newly made type I receptor. Labeled Endo H-sensitive (Fig. 3B) and total cellular Tbeta RI (Fig. 3C) were quantified as in Fig. 2 and were plotted as a percentage of the amount of (Endo H-sensitive) labeled Tbeta RI present at the end of the pulse labeling (0 h time point). The difference between the total cellular (B) and Endo H-sensitive (A) curves was assumed to represent Endo H-resistant material and is plotted in C. Control cells (- ligand) represent cells chased in the presence of a pan-specific TGF-beta neutralizing antibody.
[View Larger Version of this Image (24K GIF file)]


Because of the slow and incomplete conversion of pulse-labeled Tbeta RI to an Endo H-resistant form, we cannot use the data in Fig. 4B to calculate a precise half-life of Golgi-processed Tbeta RI. Clearly, however, it is significantly longer than the 2.5-h half-life of the mature, Golgi-processed type II receptor (compare control curves in Figs. 2B and 4C).

The presence of TGF-beta has no effect on the metabolic stability of the Endo H-sensitive form of Tbeta RI (Fig. 4A), nor on the rate by which this material is converted into an Endo H-resistant form (Fig. 4C). The Golgi-processed, Endo H-resistant form of Tbeta RI, however, is less stable in the presence than the absence of TGF-beta 1 (Fig. 4C); this is similar to the decrease in stability of the Golgi-processed type II receptor in the presence of ligand (Fig. 2B).

Ligand-binding and Cross-linking Experiments Confirm Different ER to Cell-surface Processing Times for the Type I and Type II Receptors

We performed ligand-binding and cross-linking experiments to confirm that the ER to Golgi half-lives calculated from pulse-chase experiments (above) correlate with ER to cell surface half-lives. In the experiment depicted in Fig. 5, Mv1Lu cells were incubated in tunicamycin for times ranging from 0 to 11 h. The cells were chilled, and 125I-TGF-beta 1 was bound and then cross-linked to cell surface TGF-beta receptors. Tunicamycin blocks the synthesis of dolichol pyrophosphoryl N-acetyl glucosamine, an essential intermediate in the addition of N-linked sugars to newly synthesized polypeptides. In preliminary experiments, we showed that the concentration of tunicamycin used, 2 µg/ml, completely inhibited N-linked glycosylation of both receptors.

Deglycosylated Tbeta RII appears on the cell surface as early as 1 hour after addition of tunicamycin, indicating that it is transported from the ER to cell surface in less than this time. The half-time for appearance of the maximum amount of deglycosylated type II receptor on the cell surface is approximately 2 h; the actual half-time for its formation is less than this, since the time from addition of tunicamycin to depletion of dolichol pyrophosphoryl N-acetyl glucosamine is unknown. In contrast, deglycosylated type I receptor does not appear on the cell surface until 3 h after addition of tunicamycin; the half-time for appearance of the maximum amount of deglycosylated type I receptor is approximately 4 h. The half-lives of the glycosylated forms of the cell surface receptors cannot be readily determined from this type of experiment, because at the time of addition of tunicamycin there may be substantial intracellular (ER or Golgi) pools of Tbeta RI or Tbeta RII that can subsequently move to and then be removed from the cell surface. Thus, the fact that glycosylated cell surface Tbeta RII is depleted much more rapidly than Tbeta RI (Fig. 5, compare II+ and I+) provides only an indirect indication that their actual half-lives are different. After 4-5 h of treatment with tunicamycin, however, all cell surface Tbeta RII is nonglycosylated, whereas all cell surface Tbeta RI is fully glycosylated. (We see the same results regardless of whether lysates are immunoprecipitated with antibodies against Tbeta RI or Tbeta RII or are not immunoprecipitated; data not shown.) These results are consistent with the different rates of processing from the ER of newly made types I and II receptors depicted in Figs. 2 and 4. They also indicate that complexes between types I and II TGF-beta receptors do not occur until the proteins have reached a post-ER compartment (presumably the cell surface) and have important implications for our understanding of formation of receptor complexes, as discussed below.

The decreased amount of cell surface receptors after long incubations in tunicamycin may represent decreased protein synthesis (a known consequence of tunicamycin treatment, affecting some proteins more than others). Receptors lacking N-linked oligosaccharides bind ligand and are immunoprecipitated as efficiently as fully glycosylated receptors,4 so the lower signal is not the result of inefficient binding and cross-linking or immunoprecipitation. Decreased formation or stability of nonglycosylated type I/II receptor complexes is unlikely, given that our findings are similar for nonimmunoprecipitated lysates.


DISCUSSION

Our principal finding is that the types I and II TGF-beta receptors are metabolized very differently. The ER form of the type II receptor has a half-life of 30-35 min and is efficiently (approximately 90%) processed by Golgi enzymes; the Golgi-processed forms of the type II receptor have a relatively short metabolic half-life, about 2.5 h. In contrast, the Endo H-sensitive, ER form of the type I receptor has a half-life of 2.8-3 h, and less than 50% of pulse-labeled type I receptor is converted to an Endo H-resistant form. The exact half-life of Golgi-processed type I receptor is difficult to determine because of the large ER pool of receptor subunits being slowly processed to the Golgi at the same time that Golgi-processed receptors are being degraded (Fig. 4, A and C). Nonetheless, the half-life is considerably longer than that of the type II receptor. Addition of TGF-beta 1 to cells causes a decrease in the stability of the Endo H-resistant forms of both receptors. These conclusions, derived from experiments in which TGF-beta -responsive mink lung epithelial cells were metabolically pulse-chase labeled, were confirmed by TGF-beta 1 binding and cross-linking experiments on cells treated with tunicamycin for various times. In unpublished studies, similar results were obtained using a TGF-beta -responsive human cell line (HYB2). Because these cell lines express native, rather than transfected, receptors, it ensures that the concentrations and ratios of the types I and II TGF-beta receptors are physiologic.

Our results are consistent with recent studies on the metabolism of the type II receptor in bone-forming cells, which show that after cycloheximide treatment, the amount of cell surface Type II receptor able to bind to iodinated ligand decreased with a half-life of 2 h (53). This is an indirect measure of receptor biosynthesis and stability, however, and provides no information about the type I receptor, which requires cell surface type II receptor for binding to TGF-beta . To the best of our knowledge, biosynthetic studies have not been undertaken for any other members of the receptor superfamily, and there are no published studies of metabolically labeled receptors in nontransfected cell lines.

Our pulse-chase experiments indicate that newly made type II receptor moves quickly and efficiently through the ER after synthesis. This suggests rapid and efficient folding within the ER because only correctly folded and processed proteins are transported to the Golgi apparatus (54). Similarly, the soluble extracellular domain of the human TGF-beta type II receptor is efficiently synthesized in COS-7 cells and binds 125I-TGF-beta with the same specificity and affinity as the normal cell surface Tbeta RII (52).5 Thus, the rapid folding and efficient processing of the type II receptor is a property mainly of its extracellular domain, which contains six potential disulfide bonds and two (three in human) N-linked oligosaccharides, as well as an undetermined number of O-linked sugars.

The Golgi-processed form of Tbeta RII is degraded with a half-life of approximately 2.5 h (Fig. 2B). Because more than half of the total population of Endo-H resistant Tbeta RII is sensitive to digestion of intact cells with proteinase K and thus at steady state is on the cell surface, our data suggest that the half-life of cell surface Tbeta RII also is approximately 2.5 h. This short half-life is similar to the half-lives reported for the mature forms of the nonrecycling interferon receptor (3 h) (55), the IL-2 receptor (1 h) (56), and the erythropoietin receptor (45 min) (57). In contrast, other receptors have longer half-lives: the asialoglycoprotein receptor (20 h) (58), the low density lipoprotein receptor (11 h) (59), and the transferrin receptor (20 h) (60). After exiting the cell surface, type II receptors do not appear to undergo proteolytic cleavage or storage within the cell, because experiments with 125I-TGF-beta -bound receptors show no release of smaller labeled fragments into the medium (data not shown). Furthermore, by analysis of immunoprecipitates of metabolically labeled cells, we have not seen specific smaller proteolytic degradation products of Tbeta RII (data not shown).

We speculate that the short metabolic half-life of the type II receptor has implications for regulation of TGF-beta signaling that go beyond its impact on complex formation. The rapid turnover of the type II receptor potentially allows receptor numbers to change quickly in response to various stimuli, an important characteristic for a growth factor receptor with wide-ranging effects on differentiation and development. The decreased stability of the mature form of Tbeta RII in response to ligand suggests that TGF-beta itself may regulate the numbers of its receptor and that the type II receptor may be a limiting factor in the cellular response to TGF-beta . Phosphorylation of Tbeta RII following ligand binding may be a signal for receptor degradation, in a manner analogous to many receptors spanning seven membranes, which undergo internalization and down-modulation after ligand binding (61).

The newly made ER form of the type I receptor disappears from cells with a half-time of nearly 3 h, 5-6 times as long as for the type II receptor. Less than one-half of the newly made receptors ever acquire Endo-H-resistant oligosaccharides. Processing of the type I receptor thus is similar to that of the erythropoietin receptor, which likely folds inefficiently (57, 62, 63). A mutant erythropoietin receptor is transported from the ER more efficiently than the wild type receptor and is expressed in elevated numbers at the cell surface (63); this is the first example of a mutant polypeptide that is processed in the ER more efficiently than its wild type counterpart. We speculate that inefficient folding and processing of the wild type erythropoietin receptor and potentially the type I TGF-beta receptor in the ER is one mechanism for controlling the number of plasma membrane receptors.

The fact that the types I and II TGF-beta receptors exit the ER at different rates implies that they do so separately. Fig. 5 provides perhaps the most vivid demonstration of the different rates of ER exit of the two receptors: after 4 h of treatment with tunicamycin, all cell surface Tbeta RII was nonglycosylated, whereas all cell surface Tbeta RI remained fully glycosylated. Because the rates and extents of ER to Golgi processing of the two receptors are different and because the mature forms of the two receptors are degraded with different half-lives, our results imply that stable complexes between the types I and II TGF-beta receptors do not occur until the proteins have reached a post-ER compartment, presumably the cell surface. Given our results, it would be possible to conclude the opposite---that hetero-oligomers form in the ER---only if one receptor was synthesized in excess and accumulated in the ER awaiting production of the other receptor. This scenario would imply that the first receptor could not become correctly folded and mature to the Golgi unless complexed to the second receptor. Both the type I and type II receptors, however, mature normally to the cell surface even when expressed alone in COS cells (36).6 Furthermore, the soluble exoplasmic domain of the type I receptor, like that of the type II, is efficiently secreted by transiently transfected COS cells, as well as by stably transfected Chinese hamster ovary cells,5 again implying that folding of the exoplasmic domain of Tbeta RI occurs efficiently in the absence of Tbeta RII. Our results showing different rates of degradation of Golgi-processed Tbeta RI and Tbeta RII are also consistent with the idea that stable complexes between the two receptors do not occur in the ER; indeed, they suggest that even cell surface complexes are short-lived.

Studies with TGF-beta 1 showed that formation of a Tbeta RI/Tbeta RII heteromeric complex is dependent on the presence of TGF-beta (29, 36). TGF-beta 2, however, binds to neither cell surface signaling receptor expressed in the absence of the other, suggesting the existence of a preformed Tbeta RI/Tbeta RII complex (35). Our present experiments imply that if Tbeta RI/Tbeta RII complexes do occur on the cell surface in the absence of TGF-beta , they are relatively unstable and do not form in the ER. Although our preliminary data suggest that the type II receptor forms dimers cotranslationally,6 it is unlikely that heteromeric complexes with the type I receptor also form at this point.

The cytoplasmic domains of Tbeta RI and Tbeta RII may have an intrinsic but weak affinity for each other, as interactions between the two have been detected in both the yeast two-hybrid system and COS cells overexpressing the cytoplasmic domains of both receptors (29, 30).3 Such interactions, however, have not been detected in TGF-beta -responsive cells, and we do not know whether they affect receptor trans-activation and signal transduction. Although weak interactions between the cytoplasmic domains of Tbeta RI and Tbeta RII expressed at physiologic concentrations are not, in themselves, sufficient for generating growth inhibitory signals, they could, if they exist, serve to initiate formation of heterodimers of Tbeta RI and Tbeta RII that become stabilized by the binding of a TGF-beta ligand.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA63260 (to H. F. L.) and DK02290 (to R. G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Present address: Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Boston, MA 02114.
**   To whom correspondence should be addressed: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-9872; E-mail: lodish{at}wi.mit.edu.
1   The abbreviations used are: TGF-beta , transforming growth factor-beta ; Tbeta RI, type I TGF-beta receptor; Tbeta RII, type II TGF-beta receptor; ER, endoplasmic reticulum, Endo H, endoglycosidase H; PBS, phosphate-buffered saline.
2   L. Gilboa and Y. I. Henis, personal communication.
3   R. Perlman and R. A. Weinberg, personal communication.
4   R. G. Wells and H. F. Lodish, manuscript in preparation.
5   H. Yankelev, R. G. Wells, and H. F. Lodish, unpublished results.
6   R. G. Wells and H. F. Lodish, unpublished results.

ACKNOWLEDGEMENTS

We thank Kohei Miyazono (Japanese Institute for Cancer Research, Tokyo, Japan) for Tbeta RI cDNA, Andrew Geiser (National Institutes of Health) for HYB2 cells, and James Weatherbee (R & D Systems) for TGF-beta 1. We are grateful to Stephanie Watowich for helpful discussions and to Ralph Lin and Riki Perlman for critically reviewing the manuscript.


Note Added in Proof

Koli and Arteaga (Koli, M., and Arteaga, C. L. (1997) J. Biol. Chem. 272, 6423-6427) recently published similar data. Their data agree with ours in showing a reduction in the half-life of Tbeta RII in the presence of TGF-beta . They see no effect of ligand, however, on the half life of the type I receptor. This difference may be due to different assumptions about the identity of a 55-kDa form of the type I receptor. We have identified it as an Endo H-sensitive, ER form, whereas Koli and Arteaga identify it as the mature, Endo H-resistant receptor.


REFERENCES

  1. Massague, J. (1990) Ann. Rev. Cell Biol. 6, 597-641 [CrossRef]
  2. Roberts, A. B., and Sporn, M. B. (1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds), Vol. 95/I, pp. 419-472, Springer-Verlag, Berlin
  3. Kingsley, D. M. (1994) Genes Dev. 8, 133-146 [CrossRef][Medline] [Order article via Infotrieve]
  4. Wang, X. F., Lin, H. Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and Weinberg, R. A. (1991) Cell 67, 797-805 [Medline] [Order article via Infotrieve]
  5. Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massague, J. (1991) Cell 67, 785-795 [Medline] [Order article via Infotrieve]
  6. Lin, H. Y., and Wang, X. F. (1992) Mol. Reprod. Dev. 32, 105-110 [Medline] [Order article via Infotrieve]
  7. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C. H., and Miyazono, K. (1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  8. Lopez-Casillas, F., Wrana, J. L., and Massague, J. (1993) Cell 73, 1435-1444 [Medline] [Order article via Infotrieve]
  9. Moustakas, A., Lin, H. Y., Henis, Y. I., Plamondon, J., O'Connor-McCourt, M. D., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22215-22218 [Abstract/Free Full Text]
  10. Massague, J. (1992) Cell 69, 1067-1070 [Medline] [Order article via Infotrieve]
  11. Massague, J., Attisano, L., and Wrana, J. L. (1994) Trends Cell Biol. 4, 172-178 [CrossRef]
  12. Lin, H. Y., and Lodish, H. F. (1993) Trends Cell Biol. 3, 14-19 [CrossRef]
  13. Derynck, R. (1994) Trends Biochem. Sci. 19, 548-553 [CrossRef][Medline] [Order article via Infotrieve]
  14. Georgi, L. L., Albert, P. S., and Riddle, D. L. (1990) Cell 61, 635-645 [Medline] [Order article via Infotrieve]
  15. Mathews, L. S., and Vale, W. W. (1991) Cell 65, 973-982 [Medline] [Order article via Infotrieve]
  16. Attisano, L., Carcamo, J., Ventura, F., Weis, F. M., Massague, J., and Wrana, J. L. (1993) Cell 75, 671-680 [Medline] [Order article via Infotrieve]
  17. Ebner, R., Chen, R. H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993) Science 260, 1344-1348 [Medline] [Order article via Infotrieve]
  18. Estevez, M., Attisano, L., Wrana, J. L., Albert, P. S., Massague, J., and Riddle, D. L. (1993) Nature 365, 644-649 [CrossRef][Medline] [Order article via Infotrieve]
  19. ten Dijke, P., Ichijo, H., Franzen, P., Schulz, P., Saras, J., Toyoshima, H., Heldin, C. H., and Miyazono, K. (1993) Oncogene 8, 2879-2887 [Medline] [Order article via Infotrieve]
  20. Brummel, T. J., Twombly, V., Marques, G., Wrana, J. L., Newfeld, S. J., Attisano, L., Massague, J., O'Connor, M. B., and Gelbart, W. M. (1994) Cell 78, 251-261 [Medline] [Order article via Infotrieve]
  21. Nellen, D., Affolter, M., and Basler, K. (1994) Cell 78, 225-237 [Medline] [Order article via Infotrieve]
  22. Xie, T., Finelli, A. L., and Padgett, R. W. (1994) Science 263, 1756-1759 [Medline] [Order article via Infotrieve]
  23. Liu, F., Ventura, F., Doody, J., and Massague, J. (1995) Mol. Cell. Biol. 15, 3479-3486 [Abstract]
  24. Letsou, A., Arora, K., Wrana, J. L., Simin, K., Twombly, V., Jamal, J., Staehling-Hampton, K., Hoffmann, F. M., Gelbart, W. M., Massague, J., and O'Connor, M. B. (1995) Cell 80, 899-908 [Medline] [Order article via Infotrieve]
  25. Ruberte, E., Marty, T., Nellen, D., Affolter, M., and Basler, K. (1995) Cell 80, 889-897 [Medline] [Order article via Infotrieve]
  26. Takumi, T., Moustakas, A., Lin, H. Y., and Lodish, H. F. (1995) Exp. Cell Res. 216, 208-214 [CrossRef][Medline] [Order article via Infotrieve]
  27. Henis, Y. I., Moustakas, A., Lin, H. Y., and Lodish, H. F. (1994) J. Cell. Biol. 126, 139-154 [Abstract]
  28. Chen, R.-H., and Derynck, R. (1994) J. Biol. Chem. 269, 22868-22874 [Abstract/Free Full Text]
  29. Ventura, F., Doody, J., Liu, F., Wrana, J. L., and Massague, J. (1994) EMBO J. 13, 5581-5589 [Abstract]
  30. Chen, F., and Weinberg, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1565-1569 [Abstract]
  31. Luo, K., and Lodish, H. F. (1996) EMBO J. 15, 4485-4496 [Abstract]
  32. Laiho, M., Weis, F. M. B., and Massague, J. (1990) J. Biol. Chem. 265, 18518-18524 [Abstract/Free Full Text]
  33. Laiho, M., Weis, F. M. B., Boyd, F. T., Ignotz, R. A., and Massague, J. (1991) J. Biol. Chem. 266, 9108-9112 [Abstract/Free Full Text]
  34. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X. F., and Massague, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  35. Rodriguez, C., Chen, F., Weinberg, R. A., and Lodish, H. F. (1995) J. Biol. Chem. 270, 15919-15922 [Abstract/Free Full Text]
  36. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  37. Weis-Garcia, F., and Massague, J. (1996) EMBO J. 15, 276-289 [Abstract]
  38. Yamashita, H., ten Dijke, P., Franzen, P., Miyazono, K., and Heldin, C.-H. (1994) J. Biol. Chem. 269, 20172-20178 [Abstract/Free Full Text]
  39. Brand, T., and Schneider, M. D. (1995) J. Biol. Chem. 270, 8274-8284 [Abstract/Free Full Text]
  40. Carcamo, J., Zentella, A., and Massague, J. (1995) Mol. Cell. Biol. 15, 1573-1581 [Abstract]
  41. Chen, R.-H., Moses, H. L., Maruoka, E. M., Derynck, R., and Kawabata, M. (1995) J. Biol. Chem. 270, 12235-12241 [Abstract/Free Full Text]
  42. Massague, J. (1996) Cell 85, 947-950 [Medline] [Order article via Infotrieve]
  43. Macias-Silva, M., Abdollah, S., Hoodless, P., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224 [Medline] [Order article via Infotrieve]
  44. Wang, T., Donahoe, P. K., and Zervos, A. S. (1994) Science 265, 674-676 [Medline] [Order article via Infotrieve]
  45. Chen, R. H., Miettinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995) Nature 377, 548-552 [CrossRef][Medline] [Order article via Infotrieve]
  46. Kawabata, M., Imamura, T., Miyazono, K., Engel, M. E., and Moses, H. L. (1995) J. Biol. Chem. 270, 29628-29631 [Abstract/Free Full Text]
  47. Wang, T., Li, B.-Y., Danielson, P. D., Shah, P. C., Rockwell, S., Lechleider, R. J., Martin, J., Manganaro, T., and Donahoe, P. K. (1996) Cell 86, 435-444 [Medline] [Order article via Infotrieve]
  48. Wang, T., Danielson, P. D., Li, B. Y., Shah, P. C., Kim, S. D., and Donahoe, P. K. (1996) Science 271, 1120-1122 [Abstract]
  49. Reddy, K. B., Karode, M. C., Harmony, A. K., and Howe, P. H. (1996) Biochemistry 35, 309-314 [CrossRef][Medline] [Order article via Infotrieve]
  50. Geiser, A. G., Burmester, J. K., Webbink, R., Roberts, A. B., and Sporn, M. B. (1992) J. Biol. Chem. 267, 2588-2593 [Abstract/Free Full Text]
  51. Frolik, C. A., Wakefield, L. M., Smith, D. M., and Sporn, M. B. (1984) J. Biol. Chem. 259, 10995-11000 [Abstract/Free Full Text]
  52. Lin, H. Y., Moustakas, A., Knaus, P., Wells, R. G., Henis, Y. I., and Lodish, H. F. (1995) J. Biol. Chem. 270, 2747-2754 [Abstract/Free Full Text]
  53. Centrella, M., Ji, C., Casinghino, S., and McCarthy, T. L. (1996) J. Biol. Chem. 271, 18616-18622 [Abstract/Free Full Text]
  54. Pelham, H. R. (1989) Ann. Rev. Cell Biol. 5, 1-23 [CrossRef]
  55. Aguet, M., and Blanchard, B. (1981) Virology 115, 249-261 [Medline] [Order article via Infotrieve]
  56. Duprez, V., and Dautry-Varsat, A. (1986) J. Biol. Chem. 261, 15450-15454 [Abstract/Free Full Text]
  57. Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143 [Abstract]
  58. Schwartz, A. L., Fridovich, S. E., and Lodish, H. F. (1982) J. Biol. Chem. 257, 4230-4237 [Free Full Text]
  59. Casciola, L. A., van der Westhuyzen, D. R., Gevers, W., and Coetzee, G. A. (1988) J. Lipid Res. 29, 1481-1489 [Abstract]
  60. Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A. N., Dean, A., and Bridges, K. R. (1983) J. Biol. Chem. 258, 4715-4724 [Abstract/Free Full Text]
  61. Benovic, J. L., Strasser, R. H., Caron, M. G., and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801 [Abstract]
  62. Neumann, D., Wikstrom, L., Watowich, S. S., and Lodish, H. F. (1993) J. Biol. Chem. 268, 13639-13649 [Abstract/Free Full Text]
  63. Hilton, D. J., Watowich, S. S., Murray, P. J., and Lodish, H. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 190-194 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.