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Endocrinology Vol. 140, No. 4 1760-1766
Copyright © 1999 by The Endocrine Society

ARTICLES

Activin and Inhibin Binding to the Soluble Extracellular Domain of Activin Receptor II1

Cynthia J. Donaldson, Joan M. Vaughan, Anne Z. Corrigan, Wolfgang H. Fischer and Wylie W. Vale2

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
Activins and inhibins belong to the transforming growth factor-ß-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I]activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/ßA or ActRII ECD/ßAßA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/ßA{alpha} was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2–7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
ACTIVINS and inhibins are members of the transforming growth factor-ß (TGFß) superfamily of growth and differentiation factors, which also includes Mullerian inhibiting substance, bone morphogenetic proteins, the Xenopus oocyte vegetal pole-derived growth factor, and the embryonic proteins Drosophilia decapentaplegic, dorsalin, and nodal (1). Activins and inhibins were initially characterized based upon their ability to stimulate and inhibit the release of FSH from anterior pituitary cells, respectively (2, 3). Activins are now known to exert a broad range of biological effects in a great variety of target tissues, and some, but not all, of these effects are functionally antagonized by inhibins (4). Activins are homo/heterodimers of two closely related inhibin/activin ß-subunits, ßA and ßB, and three forms of activin dimers have been characterized to date (activin-A, -B, and -AB) (4). Inhibins, on the other hand, are heterodimers comprising one of the inhibin/activin ß-subunits and the related inhibin {alpha}-subunit (2, 3, 4, 5). Two additional inhibin/activin ß-subunits (ßC and ßD) have been reported, but their biological functions have yet to be determined (6, 7).

Activins, inhibins, and other members of the TGFß superfamily of proteins interact with two classes of structurally related receptors, type I and type II (8, 9, 10, 11, 12). Both receptor types have a single transmembrane domain, an extracellular ligand-binding domain, and a cytoplasmic portion that has serine/threonine protein kinase activity. To date, two type II receptors (ActRII and ActRIIB) and a single type I receptor (ActRIB or ALK-4 = activin receptor-like kinase) (8, 13, 14) have been characterized and shown to be required for activin-specific signal transduction.

Although type II receptors show high affinity binding for their ligand when transiently expressed in COS cells, type I receptors do not appear to bind ligand on their own (8, 10, 15, 16). However, in cells that express both receptor types, cross-linking experiments with either [125I]activin A (10) or [125I]TGFß (17) reveal the presence of two major ligand-receptor complexes with apparent mol wt of 60,000–70,000 and 80,000–90,000, consistent with ligand-bound type I or type II receptors, respectively (18, 19, 20). Several lines of evidence suggest that activin or TGFß binding to the type II receptor is required for the rapid recruitment of the type I receptor into an oligomeric complex with the type II receptor and the ligand (9). Even though the type II receptors are responsible for ligand binding followed by the recruitment and transphosphorylation of the type I receptor, type I receptors are responsible for transduction of the signal to downstream targets that include the recently identified family of proteins, the vertebrate Smads and their Drosophila (Mads) and Xenopus (Xmads) homologs (21, 22, 23, 24, 25, 26).

Deletion of the cytoplasmic protein kinase domain of the type II activin receptor has been shown to inhibit activin signaling in Xenopus (27) and P19 embryonal carcinoma cells (28). This truncation has also been shown to act as a dominant negative mutant that blocks the transcriptional activation of the activin/TGFß-responsive 3TP-lux luciferase reporter in CHO and K562 cells treated with activin (29). These results suggest that deletion of the cytoplasmic domain does not disrupt ligand binding to ActRII. Likewise, a kinase domain truncation in the type II TGFß receptor can act as a dominant negative to decrease the antiproliferative and transcriptional responses of Mv1Lu cells to TGFß (30) or by affecting TGFß control of the developmental regulation of cardiac genes in cardiac myocytes (31). Furthermore, the soluble extracellular domain of the type II TGFß receptor was found to be sufficient for ligand binding (32, 33, 34). To evaluate the nature of the interaction between activin and its type II receptors, we have expressed the extracellular domain of the type II activin receptor (ActRII ECD) as a soluble protein using the baculovirus system (35), as previously presented in abstract form (36). We report here the biochemical characterization of this ECD and show that this soluble protein possesses intrinsic ability to bind ligands with high affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Construction, transfection, and expression of ActRII ECD
A complementary DNA fragment encoding 135 amino acids of the mouse type II activin receptor (10), including the putative signal peptide, the extracellular region, and an engineered stop codon just before the putative transmembrane domain, was generated by PCR using the following primers: 5'-GATCGGATCCATGGGAGCTGCTGCAAAGTTG-3' and 5'-GATCGAATTCTTAGGGTGGCTTCGGTGTAAC-3'. The PCR fragment was subcloned into the BamHI and EcoRI sites of pVL1393 (Invitrogen, Carlsbad, CA) downstream of the viral polyhedron gene promoter, and sequenced for verification.

Recombinant baculovirus was generated with the ActRII ECD-pVL1393 in Trichoplusia ni High Five cells (Invitrogen), using lipofectin (Life Technologies, Grand Island, NY) and BaculoGold (PharMingen, San Diego, CA). Large scale expression of the soluble ActRII ECD was produced in High Five cells in Sf900II-SFM medium for 3 days, with two rounds of amplification of the viral stock (35). The medium containing the soluble ActRII ECD was harvested and clarified by centrifugation. The supernatant was filtered through 0.2-µM pore size filters and stored at -70 C until purification.

Purification of ActRII ECD
Batches of medium (150–180 ml) from High Five cells infected with ActRII ECD baculovirus were dialyzed against lectin equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, 1 mM MgCl2, 1 mM MnCl2, and 1 mM CaCl2, pH 7.5) to remove high levels of glucose. Dialyzed material was gently rotated overnight at 4 C with 5 ml concanavalin A-Sepharose 4B beads (Pharmacia Biotech, Piscataway, NJ). The mixture was packed into a column, and weakly absorbed proteins were removed by washing with 10 vol lectin equilibration buffer. Bound proteins containing sugar moieties were eluted in the same buffer with 0.5 M {alpha}-D-methyl-mannopyranoside and 0.5 M {alpha}-D-methyl-glucopyranoside. The elution product was dialyzed against 20 mM Tris-HCl (pH 8.5), and the retentate was concentrated by lyophilization and reconstituted to about 10 ml with 50 mM Tris-HCl (pH 8.5). The sample was applied to a fast protein liquid chromatography system equipped with a Mono Q HR 5/5 column (1 ml; Pharmacia Biotech) equilibrated in 50 mM Tris-HCl (pH 8.5). Elution was accomplished with a linear gradient of 0–1 M NaCl in the same buffer. The 0.15 M salt zone from anion exchange was further purified, desalted, and concentrated by HPLC chromatography using a Vydac 0.46 x 25-cm C4 silica column. Elution was with a linear gradient of 10–75% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min. At each step of the purification, activity was monitored by immunoblotting, and purity was determined by SDS-PAGE with Coomassie blue or silver staining.

Amino acid analysis and mass spectroscopy
The N-terminal amino acid sequence of purified ActRII ECD was determined using a PE Applied Biosystems 470A protein sequencer (Foster City, CA) equipped with an on-line phenylthiodantoin amino acid analyzer. Mass of the purified ActRII ECD was determined by matrix-assisted laser desorption mass spectrometry as described previously (37).

Deglycosylation
For deglycosylation, purified ActRII ECD was denatured in 0.5% SDS and 5% ß-mercaptoethanol for 10 min at 95 C. After dilution with 20 mM sodium phosphate buffer, pH 7.5, and 1% (vol/vol) Nonidet P-40, the sample was treated with peptide-N-glycosidase F (500 U; New England Biolabs, Inc., Beverly, MA), neuraminidase (10 mU; Genzyme Corp., Cambridge, MA), and/or O-glycanase (2 mU; Genzyme Corp.). Samples were then incubated for 16–20 h at 37 C, denatured with sample buffer, and analyzed by SDS-PAGE and Western blot.

Affinity labeling and chemical cross-linking
Recombinant activin A and inhibin A (provided by J. Mather, Genentech, Inc., South San Francisco, CA) and purified baculovirus-expressed ActRII ECD were iodinated using the chloramine-T oxidation method as previously described (38). Binding experiments were carried out in 100 µl buffer (25 mM HEPES, 135 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.5 mM CaCl2, and 0.1% BSA, pH 7.5) containing either [125I]activin A or [125I]ActRII ECD (1 x 105 cpm/reaction), with or without cold competitor. After incubation at room temperature for 90 min, cross-linking was initiated with 0.5 mM disuccinimydyl suberate (Pierce Chemical Co., Rockford, IL), and samples were incubated for an additional 30 min on ice. The reaction was stopped with the addition of glycine (625 mM, final concentration). The samples were denatured with sample buffer containing 0.5% (vol/vol) 2-mercaptoethanol and analyzed by 10% SDS-PAGE (39) followed by autoradiography at -70 C using Kodak X-Omat AR-5 film (Eastman Kodak, Rochester, NY). The intensity of the protein bands was quantified by computer-assisted transmittance scanning densitometry (Hoefer Scientific, San Francisco, CA).

Antisera, immunoprecipitations, and Western blots
Peptides corresponding to amino acids 1–10 (AILGRSETQE) with the addition of Gly-Lys at the C-terminus or amino acids 102–117 (EVTQPTSNPVTPKPPY) of the processed type II mouse activin receptor (10) were coupled to human {alpha}-globulins via bisdiazotized benzidine and used as immunogens to raise antisera in rabbits. The antiserum to ActRII (amino acids 1–10) was purified by affinity chromatography against the corresponding peptide, as previously described (40). The affinity-purified rabbit antiserum to amino acids 81–113 of porcine inhibin/activin ßA-subunit (anti-ßA) has been previously characterized (40). For immunoprecipitation, cross-linked affinity-labeled complexes were incubated for 4 h on ice with either 20 µg affinity-purified rabbit anti-ßA or 10 µl antiserum directed against ActRII (amino acids 102–117). Samples immunoprecipitated with anti-ActRII (amino acids 102–117) were first pretreated for 15 min with dithiothreitol (7.5 mM, final concentration). For controls, antisera were either blocked with 100 µg of the immunogenic peptide, or equivalent amounts of rabbit {gamma}-globulins were substituted for the specific antisera, respectively. Immunocomplexes were collected after incubation with 25 µl packed protein A-Sepharose (Pharmacia Biotech) at room temperature for 30 min. The beads were pelleted by brief centrifugation and washed three times with RIPA buffer (10 mM NaPO4, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, and 150 mM NaCl, pH 7.5). The samples were denatured with sample buffer plus ß-mercaptoethanol and analyzed on 10% SDS-PAGE, followed by autoradiography. For Western analysis, proteins were separated on 10–20% gradient SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 3% (wt/vol) gelatin in TBS (50 mM Tris-HCl and 150 mM NaCl, pH 7.5) and reacted overnight with anti-ActRII (amino acids 1–10) or anti-ActRII (amino acids 102–117; 1:500 dilution) in TBS with 0.05% Tween-20 and 1% (wt/vol) gelatin. The membranes were then incubated for 2 h at room temperature with alkaline phosphatase-conjugated goat antirabbit IgG (1:3000; Bio-Rad Laboratories, Inc., Hercules, CA), and immunoreactive bands were visualized colorometrically using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrates according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.).

Biological activity in rat anterior pituitary cell cultures
Anterior pituitary cells of male Sprague-Dawley rats were prepared by dispersion with collagenase and plated in 48-well plates (0.15 x 106 cells/well) as previously described (41). Before initiating experiments, the cells were allowed to recover for 72 h in complete medium (ßPJ) supplemented with 2% FBS and appropriate growth factors (41). The cells were washed three times with the same medium and treated for 72 h as indicated. The secretion of FSH was quantified by RIA using kits provided by the National Pituitary and Hormone Distribution Program of the NIDDK.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Construction and expression of the recombinant extracellular domain of the type II activin receptor
Secreted proteins from High Five cells expressing the N-terminal 135 amino acids of the extracellular domain of ActRII (ActRII ECD) were initially analyzed by Western analysis after SDS-PAGE under reducing conditions. Antibodies directed against either the C-terminal ActRII (amino acids 102–117; Fig. 1BGo, lane 1) or the N-terminal ActRII (amino acids 1–10) region (data not shown) revealed the presence of an immunoreactive protein with an apparent mobility of about 25,000. This is significantly greater than that predicted by the sequence of the ActRII ectodomain (mol wt, ~14,000), suggesting that the protein contained carbohydrate moieties. Indeed, two putative Asn-linked glycosylation sites were present in the protein, and subsequent deglycosylation of the purified material confirmed the latter. Moreover, the soluble ActRII ECD was recognized by concanavalin A and could be eluted from a concanavalin A resin with the corresponding sugars, as discussed below.



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  		Figure 1. Purification of ActRII ECD from the medium of baculovirus-transfected High Five cells was monitored by silver staining (A) and Western blot analysis (B). For A and B, lane 1 represents 50 µl expression medium; all other lanes contain the equivalent of 100 µl medium. Lane 2 is after concanavalin A-Sepharose chromatography; lane 3 is after fast protein liquid chromatography on a Mono Q column; lane 4 is after HPLC on a C4 column. The samples were resolved on 10–20% gradient SDS-PAGE, under reducing conditions and were silver stained (A) or transferred to nitrocellulose and probed with an antiserum directed against ActRII (amino acids 102–117; B).

 
Purification and characterization of ActRII ECD
The ActRII ECD was purified by three steps, involving lectin affinity, anion exchange, and HPLC reverse phase chromatography (Fig. 1Go). Concanavalin A-Sepharose 4B, which binds proteins carrying glucose and mannose sugar moieties, was used as a first step in the purification. Bound proteins were eluted with {alpha}-D-methyl-mannopyranoside and {alpha}-D-methyl-glucopyranoside. Analysis of the retained fraction showed a predominant immunoreactive protein with an apparent size of 25,000; a less prominent immunoreactive band of higher mol wt was also observed (Fig. 1Go). The retained material from lectin affinity chromatography was dialyzed to remove salts and sugars and subsequently applied to a Mono Q anion exchange column. The major protein (>80%) eluting in the low salt fraction (0–0.15 M NaCl) migrated with an apparent mol wt of about 25,000 on reducing SDS-PAGE and was recognized by antisera directed against either the C-terminal ActRII (amino acids 102–117; Fig. 1BGo, lane 3) or the N-terminal ActRII (amino acids 1–10; data not shown). For the final purification step, the 0–0.15 M salt zone from Mono Q anion exchange was applied to Vydac C4 silica and eluted using a 0.1% trifluoroacetic acid/acetonitrile buffer system. The fraction that contained the majority of the immunoreactivity showed a single species of about 25,000 mol wt, as visualized by silver staining (Fig. 1Go, lane 4). The final product was estimated to contain ActRII ECD of more than 95% purity.

The calculated mol wt of ActRII ECD was 13,450, whereas the observed weight by mass spectrometry of the purified ActRII ECD was 17,230. This is in good agreement with our estimation of the mass contributed by sugar moieties contained in the recombinantly expressed material. After treatment with N-glycosidase F, the apparent molecular mass of ActRII ECD was reduced, indicating the presence of N-linked carbohydrates (Fig. 2Go). A further reduction in apparent molecular mass was also noted after treatment with neuraminidase to trim possible sialic acids, followed by digestion with O-glycanase to remove O-linked sugars.



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  		Figure 2. Deglycosylation of ActRII ECD was analyzed by Western blot analysis. Baculovirus-expressed and purified ActRII ECD (0.7 µg) was untreated (lane 1) or treated with N-glycosidase F to remove N-linked carbohydrates (lanes 2–4), then further treated with neuraminidase to remove sialic acid residues (lanes 3 and 4) and with O-glycanase to remove O-linked carbohydrates (lane 4). After overnight digestion at 37 C, samples were resolved on 10–20% gradient SDS-PAGE under reducing conditions, blotted onto nitrocellulose, and probed with an antiserum directed against the ActRII (amino acids 102–117).

 
The purified ActRII ECD was also subjected to N-terminal sequence analysis to determine posttranslational processing of the protein expressed by insect cells. Unambiguous identification of phenylthiodantoin amino acids was possible through 28 cycles. Due to a ragged N-terminus, two residues were observed in most cycles. The sequences could be matched to the predicted mature mouse ActRII protein (10) starting at either Ala1 or Arg5. These results show that the putative ActRII signal peptide is proteolytically removed by insect High Five cells to generate the expected N-terminal amino acid sequence Ala1-Ile-Leu-Gly-Arg5-Ser-Glu-Thr-. Based on the estimated ratio of phenylthiodantoin amino acid(s) identified in each cycle, the majority of ActRII ECD was accurately cleaved to yield a fully extended N-terminus. However, the final product contained 10–20% of the slightly shorter protein beginning at Arg5.

Binding characteristics of ActRII ECD
Ligand binding by the purified ActRII ECD was evaluated using either [125I]activin A or [125I]ActRII ECD. When radiolabeled ActRII ECD was bound and cross-linked to activin A, the samples were immunoprecipitated with anti-ßA (amino acids 81–113) or rabbit IgG as a control (Fig. 3Go). Conversely, when radiolabeled activin A was bound and cross-linked to soluble ActRII ECD, immunoprecipitations were performed with the N-terminally directed ECD antiserum. The immunoprecipitated radiolabeled proteins were subsequently resolved by SDS-PAGE under reducing conditions and visualized by autoradiography. With both strategies, immune complexes of 39,000–40,000 and 52,000–55,000 mol wt were present on autoradiograms, providing evidence that these bands contain both inhibin/activin ßA-subunit and ActRII ECD. The 39,000–40,000 mol wt band is consistent with the size of ActRII ECD bound to a monomer of inhibin/activin ßA, whereas the 52,000–55,000 mol wt band would be expected to contain ActRII ECD bound to dimeric activin A (ßAßA).



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  		Figure 3. Chemical cross-linking of [125I]activin A to purified ActRII ECD or of [125I]ActRII ECD to activin A. ActRII ECD (2 ng) was incubated with 100,000 cpm [125I]activin A (A), or conversely, 100,000 cpm [125I]ActRII ECD were incubated with 10 nM activin A (B). After a 90-min incubation at room temperature, the samples were chemically cross-linked for 30 min on ice using disuccinimidyl suberate. The reactions were terminated by the addition of glycine and immunoprecipitated with specific antisera. A, [125I]Activin A/ActRII ECD cross-linked complexes were immunoprecipitated with anti-ActRII (amino acids 102–117); specificity was determined by blocking with 100 µg peptide immunogen. B, [125I]ActRII ECD/activin A cross-linked complexes were immunoprecipitated with either an antiserum to the C-terminus (amino acids 81–113) of the inhibin/activin ßA subunit or with control rabbit {gamma}-globulins. Samples were resolved on 10% SDS-PAGE under reducing conditions and visualized by autoradiography.

 
The ability of the soluble ActRII ECD to bind inhibin A was also determined by cross-linking experiments using [125I]ActRII ECD. The radiolabeled ActRII ECD alone migrated with an apparent size of 25,000 (Fig. 4Go). The expected 39,000–40,000 and 52,000–55,000 mol wt bands were present when activin A was used as the ligand. The same 39,000–40,000 mol wt band was detected in the presence of inhibin A, in agreement with the expected mol wt of ActRII ECD bound to a monomer of inhibin/activin ßA. However, an additional 55,000–57,000 mol wt band was present only with inhibin A as the ligand, consistent with a complex that contains ActRII ECD and dimeric inhibin A ({alpha}ßA; Fig. 4Go).



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  		Figure 4. Chemical cross-linking of [125I]ActRII ECD to activin A or inhibin A. Purified [125I]ActRII ECD (100,000 cpm) was incubated with 3.5 nM activin A or 3 nM inhibin A for 90 min at room temperature and chemically cross-linked using disuccinimidyl suberate. The reaction was stopped by the addition of glycine, and the samples were resolved on 10% SDS-PAGE under reducing conditions and visualized by autoradiography.

 
The affinity of the soluble ActRII ECD for activin A was determined in cross-linking experiments by incubating purified ECD with [125I]activin A in the presence of increasing concentrations of unlabeled activin A (Fig. 5AGo). Activin A inhibited the binding of [125I]activin A to ActRII ECD in a dose-dependent manner (Fig. 5BGo). Densitometric analysis of the cross-linked 39,000–40,000 mol wt complex provided an estimate of the apparent Kd of 2–7 nM. Analysis of the 52,000–55,000 mol wt band gave similar results (data not shown). The intensities of the two cross-linked complexes were similarly diminished in the presence of an equivalent concentration range of unlabeled inhibin A.



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  		Figure 5. Cross-linking of [125I]activin A to soluble ActRII ECD to determine binding affinity. [125I]Activin A (100,000 cpm) was incubated with 2 ng soluble ActRII ECD in the presence of increasing concentrations of cold activin A. After a 90-min incubation at room temperature, the samples were chemically cross-linked with disuccinimidyl suberate for 30 min on ice. The samples were resolved on 10% SDS-PAGE and visualized by autoradiography. Densitometric scanning of affinity-labeled complexes was used to quantify band intensities, and estimates of Kd were calculated using the computer program, LIGAND. A, A representative SDS-PAGE analysis of ActRII ECD/activin A cross-linked complexes; B, the mean ± SEM of densitometric analyses of the intensities of the band representing ActRII ECD/ßA complex from four separate experiments. Equivalent results were obtained when scanning the [125I]ActRII ECD/ßAßA band.

 
Biological activity of ActRII ECD
To determine whether the soluble extracellular portion of ActRII would compete with cellular receptors, the effect of activin A on FSH secretion was evaluated in the presence or absence of ActRII ECD. As previously shown, activin A stimulated FSH secretion from cultured rat anterior pituitary cells (4). The presence of ActRII ECD decreased basal FSH secretion, presumably by affecting the action of locally secreted activin B (42), and attenuated the stimulation of FSH secretion by exogenous activin A (Fig. 6Go). This effect of the soluble ActRII ECD was concentration dependent (data not shown).



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  		Figure 6. Stimulation of FSH secretion from rat anterior pituitary cells by activin A in the presence or absence of soluble ActRII ECD. Primary cultures of rat anterior pituitary cells were incubated in triplicate with increasing concentrations of activin A alone (•) or in the presence of ActRII ECD (0.5 µg; {circ}). Medium was collected after 72 h, and FSH secretion was measured by RIA. The values are the mean ± SEM from a representative experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
The binding properties and the biological activity of the type II activin receptor were evaluated by expressing a soluble form of the extracellular domain (ActRII ECD) of this protein in the baculovirus system. The recombinant ActRII ECD was secreted into the medium of High Five cells and was purified to homogeneity in three chromatographic steps. Our results indicate that the soluble ActRII ECD is a functional protein that retains the ability to bind both activin A and inhibin A with high affinity.

The recombinant ActRII ECD produced in the baculovirus system was a protein with an apparent mol wt of about 25,000, as visualized by Western blot analysis with an antibody directed against the N-terminus of the protein or by silver staining (Fig. 1Go, A and B, lane 4). While the predicted size of the protein is 13,450, the mol wt observed by mass spectrometry was 17,230. This difference was probably due to posttranslational modification of ActRII ECD, such as glycosylation.

The type II activin receptor has two potential N-glycosylation sites in its extracellular domain. The observed shift in the mobility of the protein on SDS-PAGE after treatment with N- and O-glycosidases confirmed the existence of posttranslational glycosylation of the secreted ActRII ECD. Based on the apparent mol wt of the smallest immunoreactive band after enzymatic treatment, it appears that N-glycosylation contributes about 4000 and O-glycosylation contributes about 1000 to the mass of baculovirus-generated ActRII ECD. Treatment with glycosidases removes carbohydrates to varying degrees, resulting in a mixture of deglycosylated products. Indeed, the doublet of bands after treatment with N- and O-glycosidases suggests that either the sugars have not been fully removed from the recombinant product or, alternatively and/or additionally, the expression product is composed of ActRII ECD proteins commencing and terminating at differing amino acids. N-Terminal sequencing of the purified ActRII ECD revealed that the majority of the proteins were processed to produce the N-terminal sequence Ala1-Ile-Leu-Gly-Arg5-Ser-Glu-Thr-, confirming cleavage after the signal peptide as we originally predicted (10). Ten to 20% of the ActRII ECD proteins were cleaved after Gly4. The difference between the mol wt of these two proteins was not observed on SDS-PAGE when the proteins were glycosylated, but may contribute to the variety of bands seen upon deglycosylation of the protein. Alternatively, as SDS-PAGE is not an accurate method for the estimation of the size of glycoproteins, it is possible that the discordance between the apparent and true mol wt of the expressed ActRII ECD, as determined by mass spectrometry, reflects the spurious behavior of many glycoproteins in SDS-PAGE.

Binding studies with purified ActRII ECD indicated that the soluble protein interacts with either activin A or inhibin A via the inhibin/activin ßA subunit. Affinity cross-linking experiments with either activin A or inhibin A provided evidence for the existence of ActRII ECD/ßA, ActRII ECD/ßAßA, and ActRII ECD/ßA{alpha} complexes. The ability of the soluble ActRII ECD to bind either activin A or inhibin A on its own also indicates that neither the transmembrane nor the intracellular domain of the full-length activin type II receptor is necessary for ligand binding. The soluble ActRII ECD binds activin A (or the ßA monomer) with an estimated Kd of 2–7 nM. This value is slightly greater than the Kd of 1.1 nM that has been reported for a membrane-bound kinase-deficient form of ActRII, lacking the serine/threonine kinase domain but retaining the transmembrane domain, transiently expressed in COS cells (29). The affinity of the full-length type II activin receptor for activin A has been reported to be 0.2–0.8 nM (8, 10, 11, 12, 43). Therefore, there is an order of magnitude difference in binding affinity between the soluble ActRII ECD and the full-length type II activin receptor. By contrast to the full-length type II activin receptor, for which activin A exhibits 10-fold greater affinity than does inhibin A (10), the EC50 values of the extracellular domain of the type II activin receptor for the two ligands are in the same range. The Pichia pasotris expressed ActRII ECD (48) binds activin A in the range reported here for the baculovirus expressed ActRII ECD and binds inhibin A with a Kd of 4–8 nM (our unpublished data). Recent results suggest that activin forms a complex with two type II receptors that may have higher binding energies than do the soluble complexes that we are studying here. The latter may explain the difference in the relative affinities of the two ligands to the soluble extracellular domain of the type II receptor compared with binding sites in the membrane environment.

The extracellular domain of the type II TGFß receptor showed an approximately 10-fold lower dissociation constant than the intact type II TGFß receptor; the soluble type II TGFß receptor was reported to have a Kd of 100 nM for TGFß1, as calculated using a BIAcore (33), or of approximately 0.2 nM for TGFß1 and approximately 0.5 nM for TGFß2, as measured by cross-linking experiments using radiolabeled TGFß1 (32). The published Kd values for the full-length membrane-bound type II TGFß receptors for the various TGFß ligands range from 0.01–0.05 nM (44, 45). Based on these observations, it was suggested that the differences in the affinities of the soluble TGFß extracellular domain and the full-length receptors may be due to the possibility that the type I receptor may cooperate with the type II receptor in vivo to affect the overall affinity of the receptor complex for TGFß (33). This explanation may also be pertinent to activin receptors. Future studies with the soluble extracellular domain of type I activin receptor will address the latter possibility.

The soluble ActRII ECD was biologically active in cultures of anterior pituitary cells. It is well established that activin A or activin B stimulates the secretion of FSH from the anterior pituitary (4, 46). Inhibin/activin ßB-subunit is present in gonadotropes (47), and activin B is a local product of pituitary cells, functioning as a local autocrine factor to regulate FSH secretion (42). Recombinant ActRII ECD decreased basal FSH secretion from cultured pituitary cells, probably by interfering with the actions of locally secreted activin B. Moreover, ActRII ECD interfered with the ability of exogenous activin A to stimulate FSH secretion.

The results of this study demonstrate that a soluble form of the extracellular domain of the type II activin receptor is sufficient for high affinity ligand binding. Moreover, this ActRII ECD is functionally active, suggesting that it would be a useful tool for further studies with activins. The protein could be used as a tool to modulate the effects of activin in experimental animals and ultimately in human beings. ActRII ECD could be used for structural studies to evaluate ligand-receptor interactions. This soluble protein could also be useful in binding studies to explore ligand binding requirements of the type II and type I receptor complexes, enabling detailed molecular analyses of ligand-receptor and receptor-receptor interactions in a cell-free system.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
Since submission of this manuscript, the ActRII ECD has been expressed in Pichia pastoris (48) and the x-ray crystallographic structure of the protein has been determined to be a three-finger toxin fold (49).


   Acknowledgments
 
We thank L. Bilezikjian for helpful comments on the manuscript, A. Craig for mass spectrometry, S. Guerra for manuscript preparation, Genentech, Inc. for providing recombinant activin A, and the National Hormone and Pituitary Program of the NIDDK and A. F. Parlow for providing FSH RIA kits.


   Footnotes
 
1 This work was supported in part by NIH Program Project Grant HD-13527, the Foundation for Research, and the Foundation for Medical Research, Inc. Back

2 Senior Investigator with the Foundation for Research, and the Foundation for Medical Research, Inc. Back

Received July 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

  1. Kingsley DM 1994 The TGF-ß superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133–146[Free Full Text]
  2. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321:776–779[CrossRef][Medline]
  3. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the ß-subunits from the two forms of inhibin. Nature 321:779–782[CrossRef][Medline]
  4. Vale W, Hsueh A, Rivier C, Yu J 1990 The inhibin/activin family of hormones and growth factors. In: Sporn MB, Roberts AB (eds) Handbook of Experimental Pharmacology. Springer-Verlag, New York, vol 95:211–248
  5. Vale W, Bilezikjian LM, Rivier C 1994 Reproductive and other roles of inhibins and activins. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1861–1878
  6. Hotten G, Neidhart H, Schneider C, Pohl J 1995 Cloning of a new member of the TGF-ß family: a putative new activin ß-C chain. Biochem Biophys Res Commun 206:608–613[CrossRef][Medline]
  7. Oda S, Nishimatsu S, Murakami K, Ueno N 1995 Molecular cloning and functional analysis of a new activin beta subunit: a dorsal mesoderm-inducing activity in Xenopus. Biochem Biophys Res Commun 210:581–588[CrossRef][Medline]
  8. Tsuchida K, Mathews LS, Vale WW 1993 Cloning and characterization of a transmembrane serine kinase that acts as an activin type-I receptor. Proc Natl Acad Sci USA 90:11242–11246[Abstract/Free Full Text]
  9. Lebrun J-J, Chen Y, Vale W 1997 Receptor serine kinases and signaling by activins and inhibins. In: Aono T, Sugino H, Vale WW (eds) Inhibin, Activin, and Follistatin: Regulatory Functions in System and Cell Biology. Springer-Verlag, New York, pp 1–20
  10. Mathews LS, Vale WW 1991 Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 65:973–982[CrossRef][Medline]
  11. Mathews LS, Vale WW, Kintner CR 1992 Cloning of a second type of activin receptor and functional characterization in Xenopus embryos. Science 255:1702–1705[Abstract/Free Full Text]
  12. Attisano L, Wrana J, Cheifetz S, Massague J 1992 Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors. Cell 68:97–108[CrossRef][Medline]
  13. ten Dijke P, Ichijo H, Franzen P, Schulz P, Saras J, Toyoshima H, Heldin CH, Miyazono K 1993 Activin receptor-like kinases: a novel subclass of cell-surface receptors with predicted serine/threonine kinase activity. Oncogene 8:2879–2887[Medline]
  14. He WW, Gustafson ML, Hirobe S, Donahoe PK 1993 Developmental expression of 4 novel serine threonine kinase receptors homologous to the activin transforming growth factor-beta type-II receptor family. Dev Dynam 196:133–142[Medline]
  15. Ebner R, Chen RH, Lawler S, Zioncheck T, Derynck R 1993 Determination of type I receptor specificity by the type II receptors for TGF-ß or activin. Science 262:900–902[Abstract/Free Full Text]
  16. Attisano L, Carcamo J, Ventura F, Weis FMB, Massague J, Wrana JL 1993 Identification of human activin and TGFß type-I receptors that form heteromeric kinase complexes with type-II receptors. Cell 75:671–680[CrossRef][Medline]
  17. Lin H, Wang X-F, Ng-Eaton E, Weinberg R, Lodish H 1992 Expression cloning of the TGF-ß type II receptor, a functional trans-membrane serine/threonine kinase. Cell 68:775–785[CrossRef][Medline]
  18. Hino M, Tojo A, Miyazono K, Miura Y, Chiba S, Eto Y, Shibai H, Takaku F 1989 Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells. J Biol Chem 264:10309–10314[Abstract/Free Full Text]
  19. Centrella M, McCarthy TL, Canalis E 1991 Activin-A binding and biochemical effects in osteoblast-enriched cultures from fetal-rat parietal bone. Mol Cell Biol 11:250–258[Abstract/Free Full Text]
  20. Mathews LS, Vale WW 1993 Characterization of type II activin receptors: binding, processing and phosphorylation. J Biol Chem 268:19013–19018[Abstract/Free Full Text]
  21. Wieser R, Wrana J, Massague J 1995 GS domain mutations that constitutively activate TßR-1, the downstream signaling component in the TGF-ß receptor complex. EMBO 14:2199–2008[Medline]
  22. Attisano L, Wrana JL 1996 Signal transduction by members of the transforming growth factor-ß superfamily. Cytokine Growth Factor Rev 7:327–339[CrossRef][Medline]
  23. Massague J, Hata A, Liu F 1997 TGF-ß signalling through the Smad pathway. Trends Cell Biol 7:187–192
  24. Chen Y, Bhushan A, Vale W 1997 Smad8 mediates the signaling of the receptor serine kinase. Proc Natl Acad Sci USA 94:12938–12943[Abstract/Free Full Text]
  25. Chen Y, Lebrun JJ, Vale W 1996 Regulation of transforming growth factor ß- and activin-induced transcription by mammalian Mad proteins. Proc Natl Acad Sci USA 93:12992–12997[Abstract/Free Full Text]
  26. Zhang Y, Musci T, Derynck R 1997 The tumor suppressor Smad4/DPC4 as a central mediator of Smad function. Curr Biol 7:270–276[CrossRef][Medline]
  27. Hemmati-Brivanlou A, Melton DA 1992 A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature 359:609–614[CrossRef][Medline]
  28. Dewinter JP, Devries CJM, Vanachterberg TAE, Ameerun RF, Feijen A, Sugino H, Dewaele P, Huylebroeck D, Verschueren K, Vandeneijndenvanraaij AJM 1996 Truncated activin type II receptors inhibit activin bioactivity by the formation of heteromeric complexes with activin type i receptors. Exp Cell Res 224:323–334[CrossRef][Medline]
  29. Tsuchida K, Vaughan JM, Wiater E, Gaddy-Kurten D, Vale WW 1995 Inactivation of activin-dependent transcription by kinase-deficient activin receptors. Endocrinology 136:5493–5503[Abstract]
  30. Wieser R, Attisano L, Wrana JL, Massagué J 1993 Signaling activity of transforming growth factor ß type II receptors lacking specific domains in the cytoplasmic region. Mol Cell Biol 13:7239–7247[Abstract/Free Full Text]
  31. Brand T, MacLellan WR, Schneider MD 1993 A dominant-negative receptor for transforming growth factors created by deletion of the kinase domain. J Biol Chem 268:11500–11503[Abstract/Free Full Text]
  32. Lin HY, Moustakas A, Knaus P, Wells RG, Henis YI, Lodish HF 1995 The soluble exoplasmic domain of the type II transforming growth factor (TGF)-ß receptor. J Biol Chem 270:2747–2754[Abstract/Free Full Text]
  33. Tsang ML, Zhou L, Zheng BL, Wenker J, Fransen G, Humphrey J, Smith JM, O’Connor-McCourt M, Lucas R, Weatherbee JA 1995 Characterization of recombinant soluble human transforming growth factor-ß receptor type II (rhTGF-ß sRII). Cytokine 7:389–397[CrossRef][Medline]
  34. Letourneur O, Goetschy JF, Horisberger M, Grutter MG 1996 Ligand-induced dimerization of the extracellular domain of the TGF-ß receptor type II. Biochem Biophys Res Commun 224:709–716[CrossRef][Medline]
  35. O’Reilly DR, Miller LK, Luckow VA 1992 Baculovirus Expression Vectors: A Laboratory Manual. Freeman, New York, pp 128–129
  36. Donaldson CJ, Vaughan JM, Corrigan AZ, Vale WW Expression, purification, and characterization of the ligand binding domain of the activin type II receptor. 76th Annual Meeting of The Endocrine Society, Anaheim CA, 1994, p 610 (Abstract)
  37. Hillenkamp F, Karas M, Beavis RC, Chait BT 1993 Matrix associated laser desorption ionization mass spectrometry of biopolymers. Anal Chem 63:1193A–1203A[CrossRef]
  38. Vaughan JM, Vale WV 1993 {alpha}2-Macroglobulin is a binding protein of inhibin and activin. Endocrinology 132:2038–2050[Abstract]
  39. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  40. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antisera generated against synthetic peptide fragments. In: Conn PM (ed) Methods in Enzymology. Academic Press, Orlando, vol 168:588–617
  41. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J 1983 Assay of coricotropin releasing factor. In: Conn PM (ed) Methods in Enzymology. Academic Press, New York, vol 103:565–577
  42. Corrigan AZ, Bilezikjian LM, Carroll RS, Bald LN, Schmelzer CH, Fendly BM, Mason AJ, Chin WW, Schwall RH, Vale W 1991 Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 128:1682–1684[Abstract]
  43. Donaldson CJ, Mathews LS, Vale WW 1992 Molecular cloning and binding properties of the human type II activin receptor. Biochem Biophys Res Commun 184:310–316[CrossRef][Medline]
  44. Massague J 1992 Receptors for the TGF-ß family. Cell 69:1067–1070[CrossRef][Medline]
  45. Massague J 1990 The transforming growth factor-ß family. Annu Rev Cell Biol 6:597–641[CrossRef]
  46. Mason AJ, Berkemeier LM, Schmelzer CH, Schwall RH 1989 Activin B: precursor sequences, genomic structure and in vitro activities. Mol Endocrinol 3:1352–1358[Abstract]
  47. Roberts V, Meunier H, Vaughan J, Rivier J, Rivier C, Vale W, Sawchenko P 1989 Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 124:552–554[Abstract]
  48. Greenwald J, Le V, Corrigan A, Fischer W, Komives E, Vale W, Choe S 1998 Characterization of the extracellular ligand-binding domain of the type II activin receptor. Biochem 37:16711–16718
  49. Greenwald J, Fischer WH, Vale WW, Choe S 1999 Three-finger toxin fold for the extracellular domain of the type II activin receptor serine kinase. Nat Struct Biol 6:18–22



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