Reconstitution and Analysis of Soluble Inhibin and Activin Receptor Complexes in a Cell-free System
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m408090200
ISSN1083-351X
AutoresElisabetta C. del Re, Yisrael Sidis, David Fabrizio, Herbert Y. Lin, Alan L. Schneyer,
Tópico(s)Kruppel-like factors research
ResumoActivins and inhibins compose a heterogeneous subfamily within the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-β type III receptor (TβRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII·sTβRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TβRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses. Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-β type III receptor (TβRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII·sTβRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TβRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses. Activins A and B, members of the transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; ActR, activin receptor; TβR, TGF-β receptor; ECD, extracellular domain; s, soluble; PBS, phosphate-buffered saline; BMP, bone morphogenetic protein. superfamily of growth and differentiation factors, have a variety of actions in both embryonic and adult tissues, including tissue fate determination in vertebrate embryos, regulation of follicle-stimulating hormone biosynthesis in pituitary gonadotrophs, and regulation of gametogenesis in ovaries and testes (reviewed in Ref. 1Welt C. Sidis Y. Keutmann H. Schneyer A. Exp. Biol. Med. 2002; 227: 724-752Crossref PubMed Scopus (282) Google Scholar). Although five vertebrate activin genes have been identified (βA–βE), only activins A (βA subunit homodimer) and B (βB subunit homodimer) have experimental evidence supporting biological roles in vivo (2Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Crossref PubMed Scopus (663) Google Scholar), although a role for activin C was recently proposed in the liver (3Wada W. Maeshima A. Zhang Y.Q. Hasegawa Y. Kuwano H. Kojima I. Am. J. Physiol. 2004; 287: E247-E254Crossref PubMed Scopus (22) Google Scholar). When examined in vitro, recombinant activins A and B appear to have similar biological activities (4Mason A.J. Berkemeier L.M. Schmelzer C.H. Schwall R.H. Mol. Endocrinol. 1989; 3: 1352-1358Crossref PubMed Scopus (119) Google Scholar, 5Sidis Y. Tortoriello D.V. Holmes W.E. Pan Y. Keutmann H.T. Schneyer A.L. Endocrinology. 2002; 143: 1613-1624Crossref PubMed Scopus (51) Google Scholar). However, in vivo, disruption of the mouse βA gene results in early neonatal lethality with craniofacial defects and lack of whiskers, suggesting that activin A is critical for normal mammalian development (6Matzuk M.M. Kumar T.R. Vassilli A. Bickenbach R.R. Roop D.R. Jaenisch R. Bradley A. Nature. 1995; 374: 354-356Crossref PubMed Scopus (516) Google Scholar). In contrast, disruption of the βB gene is not lethal but results in fertility defects in homozygous knockout females related to inability to nurse newborn pups (7Vassalli A. Matzuk M.M. Gardner H.A. Lee K.F. Jaenisch R. Genes Dev. 1994; 8: 414-427Crossref PubMed Scopus (345) Google Scholar). Interestingly, replacement of the βA mature coding sequence with that from the βB gene, resulting in activin B production wherever activin A or B would be synthesized in wild-type animals, rescues many of the skeletal abnormalities of the activin A knockout but still results in fertility defects and growth retardation, indicating that activin B cannot completely compensate for activin A even when expressed in an identical fashion (8Brown C.W. Li L. Houston-Hawkins D.E. Matzuk M.M. Mol. Endocrinol. 2003; 17: 2404-2417Crossref PubMed Scopus (45) Google Scholar, 9Brown C.W. Houston-Hawkins D.E. Woodruff T.K. Matzuk M.M. Nat. Genet. 2000; 25: 453-457Crossref PubMed Scopus (177) Google Scholar). It therefore appears that the two activins subserve at least some non-overlapping functions in the adult, although the mechanism whereby this differential activity is manifested remains unclear. One possible explanation is that distinct subsets of activin receptors may differentially bind activins A and B, at least in some tissues. The wide distribution of activin A and B expression in the adult suggests that they primarily act as autocrine or paracrine signals. On the other hand, inhibin, a heterodimeric molecule composed of one of the activin βA or βB subunits linked to a distantly related α subunit to produce inhibin A or B, respectively, was originally discovered as an endocrine regulator of follicle-stimulating hormone biosynthesis, being synthesized primarily in gonadal tissues and acting on pituitary gonadotrophs (reviewed in Ref. 10Vale W. Rivier C. Hsueh A. Campen C. Meunier H. Bicsak T. Vaughan J. Corrigan A. Bardin W. Sawchenko P. Spiess J. Rivier J. Recent Prog. Horm. Res. 1988; 44: 1-34PubMed Google Scholar). Specific assays for human dimeric inhibins A and B indicate that they are differentially produced during the menstrual cycle and that alterations of inhibin concentrations are inversely associated with changes in follicle-stimulating hormone concentrations in a variety of fertility abnormalities in both men and women (reviewed in Ref. 1Welt C. Sidis Y. Keutmann H. Schneyer A. Exp. Biol. Med. 2002; 227: 724-752Crossref PubMed Scopus (282) Google Scholar). In addition, animal studies have indicated that inhibin may also have autocrine/paracrine activities within the gonads, including influencing steroidogenesis and gametogenesis (11Woodruff T.K. Lyon R.J. Hansen S.E. Rice G.C. Mather J.P. Endocrinology. 1990; 127: 3196-3205Crossref PubMed Scopus (251) Google Scholar). However, disruption of the inhibin α subunit in the mouse, which would be expected to delete both inhibins A and B, results in the appearance of aggressive gonadal tumors that kill most animals before reproductive defects can be fully assessed (12Matzuk M.M. Finegold M.J. Su J.J. Hsueh A.J.W. Bradley A. Nature. 1992; 360: 313-319Crossref PubMed Scopus (832) Google Scholar). Interestingly, activin A and B levels are severely exaggerated in these mice (13Matzuk M.M. Finegold M.J. Mather J.P. Krummen L. Lu L. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8817-8821Crossref PubMed Scopus (334) Google Scholar). These studies suggest that inhibin acts as a tumor suppressor and may also regulate, at least to some degree, activin biosynthesis and release, emphasizing the complex interrelationship between inhibin and activin biosynthesis and action in numerous tissues and physiological systems. Like other TGF-β superfamily members, activins signal via a heteromeric complex of type II and type I receptors. Activin binds directly to one of two identified type II receptors, ActRII or ActRIIB, which recruits one of two type I receptors, ActRIA or ActRIB (also known as Alk2 (activin-like kinase-2) and Alk4, respectively). Following ligand binding, the type I receptors are phosphorylated by the type II receptors to initiate the intracellular signaling cascade (reviewed in Refs. 14Mathews L.S. Endocr. Rev. 1994; 15: 310-341Crossref PubMed Scopus (274) Google Scholar and 15Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4867) Google Scholar). Although inhibin can bind to activin type II receptors, this binding is at least 10-fold lower compared with activin (16Mathews L.S. Vale W.W. Cell. 1991; 65: 973-982Abstract Full Text PDF PubMed Scopus (680) Google Scholar) and thus does not appear to be sufficient to account for the endocrine activity of inhibin. This situation was recently clarified when a high affinity inhibin receptor comprising ActRII and the TGF-β type III receptor (TβRIII; betaglycan) was proposed (17Lewis K.A. Gray P.C. Blount A.L. MacConell L.A. Wiater E. Bilezikjian L.M. Vale W. Nature. 2000; 404: 411-414Crossref PubMed Scopus (509) Google Scholar). Presumably, when ActRII is complexed with inhibin and TβRIII, ActRII is inhibited from associating with activin and ActRI, thereby disrupting activin signaling. However, because these studies were conducted in transfected cells, they could not exclude the participation of other as yet uncharacterized cell-surface proteins in this binding complex. Thus, determination of the affinity and specificity for each possible activin- or inhibin-binding complex would benefit from pure soluble receptors that could be analyzed individually or in complexes, thereby clarifying results from cell-based experiments. We have recently shown that a purified fusion protein containing the TβRII extracellular domain (ECD) linked to human Fc is a useful tool to explore receptor complex specificity and ligand binding affinity (18del Re E. Babitt J.L. Pirani A. Schneyer A.L. Lin H.Y. J. Biol. Chem. 2004; 279: 22765-22772Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We have now created soluble (s) ActRII and ActRIIB ECD-Fc fusion proteins (sActRII-Fc and sActRIIB-Fc, respectively) and explored their affinity and specificity for activin and inhibin. In addition, purified sTβRIII-Fc fusion protein in combination with sActRII-Fc or sActRIIB-Fc formed high affinity inhibin-binding complexes in solution, confirming earlier cell-based studies. Finally, in contrast to TGF-β receptors, we did not observe any potentiation of activin binding to sActRII-Fc or sActRIIB-Fc by soluble activin type I receptors, consistent with the non-cooperative model proposed from the crystal structure of ActRIIB (19Greenwald J. Groppe J. Gray P. Wiater E. Kwiatkowski W. Vale W. Choe S. Mol. Cell. 2003; 11: 605-617Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Thus, our studies using purified soluble receptors demonstrate the specific requirements for high affinity binding of activins and inhibins to different complexes of activin type II and type I and TGF-β type III receptors, without the confounding variables that whole cell systems necessarily confer. Production of Soluble Receptors—The ECDs of ActRII (residues 1–135) (20Donaldson C.J. Mathews L.M. Vale W.W. Biochem. Biophys. Res. Commun. 1995; 184: 310-316Crossref Scopus (79) Google Scholar) and ActRIIB (residues 1–134) (21Hilden K. Tuuri T. Eramaa M. Ritvos O. Blood. 1994; 83: 2163-2170Crossref PubMed Google Scholar) were amplified by PCR and cloned in-frame with the Fc tag of a modified Signal pIgplus vector (R&D Systems, Minneapolis, MN) as described previously for sTβRII-Fc (18del Re E. Babitt J.L. Pirani A. Schneyer A.L. Lin H.Y. J. Biol. Chem. 2004; 279: 22765-22772Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The ECD of TβRIII (22Wang X.F. Lin H.Y. Ng-Eaton E. Downward J. Lodish H.F. Weinberg R.A. Cell. 1991; 67: 797-805Abstract Full Text PDF PubMed Scopus (543) Google Scholar) was similarly cloned, and the glycosaminoglycans were eliminated by site-directed mutagenesis at S535A and S546A. The Fc tail of Signal pIgplus contains a free cysteine so that the resulting proteins are secreted as dimers (see “Results”). The receptor-Fc plasmids were transfected into 293 cells, which were cultured in Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (Invitrogen). All transfections were performed with Lipofectamine 2000 (Invitrogen). Stably transfected cells were selected with 1 mg/ml G418 and cultured in Dulbecco's modified Eagle's medium supplemented with 10% ultra-low IgG fetal bovine serum (Invitrogen) in 175-cm2 multifloor flasks (TPP, Transadingen). Recombinant receptor-Fc fusion proteins were purified by protein A affinity chromatography. Tissue culture medium (1 liter/batch) was adjusted to pH 8.2 and applied to a HiTrap rProtein A FF column (Amersham Biosciences AB, Uppsala, Sweden) previously equilibrated with 10 mm phosphate-buffered saline (PBS). After protein loading, the column was washed with PBS to remove nonspecifically bound proteins and eluted in 100 mm glycine buffer (pH 3.0). Eluted fractions were immediately neutralized by addition of 1 m Tris-HCl (pH 9.0). The concentration of eluted protein was determined by bovine serum albumin protein assay (Pierce) and confirmed by amino acid analysis. Protein purity was assessed by 4–12% SDS-PAGE using precast gels (Invitrogen) followed by silver staining (Bio-Rad) according to the manufacturers' protocol. Receptor-Fc proteins were also analyzed by Western blotting. Each preparation was separated on 4–10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad). After transfer, the membrane was washed with PBS supplemented with 0.1% Tween 20 (PBST) and blocked overnight in 8% dry milk in PBST. The membrane was incubated with goat anti-ActRII, anti-ActRIIB, or anti-TβRIII antibody (R&D Systems) or with anti-human Fc antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and then with horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Chemiluminescence detection was performed with Renaissance Western blot chemiluminescent reagent (PerkinElmer Life Sciences). Mouse ActRIA-Fc and ActRIB-Fc proteins were purchased from R&D Systems. Iodination—Human activins A and B (R&D Systems) were iodinated by the lactoperoxidase method and purified by electrophoresis as described previously (5Sidis Y. Tortoriello D.V. Holmes W.E. Pan Y. Keutmann H.T. Schneyer A.L. Endocrinology. 2002; 143: 1613-1624Crossref PubMed Scopus (51) Google Scholar). Inhibins A and B (R&D Systems) were iodinated using the reduced chloramine-T method described previously for TGF-β (23Ruff E. Rizzino A. Biochem. Biophys. Res. Commun. 1986; 138: 714-719Crossref PubMed Scopus (58) Google Scholar) as adapted for inhibin (24Vaughan J.M. Vale W.W. Endocrinology. 1993; 132: 2038-2050Crossref PubMed Google Scholar) and purified by gel filtration chromatography. Ligand Binding Assays—Soluble recombinant human receptors were diluted in Tris-buffered saline/casein blocking buffer (BioFX, Owings Mills, MD) and incubated overnight on protein A-coated 96-well plates (Pierce) at 4 °C. Plates were then washed with wash buffer (BioFX) and blocked for 2 h at room temperature with Tris-buffered saline/casein blocking buffer. For competition binding assays, fixed amounts of radioligand (50,000–10,0000 cpm) was added to the receptor-coated plates together with increasing amounts (2 pm to 500 nm) of homologous or heterologous nonradioactive ligands as described under “Results” and incubated overnight at 4 °C. After washing three times with wash buffer, wells were separated and counted in a γ-counter. To allow inhibin receptor proteins to form complexes prior to binding assays, these experiments were performed with soluble binding assays. sActRII-Fc or sActRIIB-Fc with or without (control) sTβRIII-Fc proteins (as indicated in the figures) was diluted in Tris-buffered saline/casein blocking buffer and incubated overnight with a fixed amount of 125I-inhibin A or B (∼ 100,000 cpm) and increasing amounts (2 pm to 500 nm) of unlabeled inhibin at 4 °C. The samples were then plated on protein A-coated plates, incubated for 1 h at room temperature, washed, and counted as described above. The same protocol was employed to perform ActRII·ActRI complex binding experiments, except that iodinated activin A or B was used as radioligand. Assessment of Biological Activity—Human embryonic kidney 293 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum. Transient transfections were performed in 24-well trays using Effectene (QIAGEN Inc., Valencia, CA) and a total of 200 ng of DNA, including 80 ng of the Smad-responsive reporter CAGA-Luc (a gift from Dr. S. Dennler) (25Dennler S. Itoh S. Vivien D. Ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar), 20 ng of pRL-TK (Promega Corp., Madison, WI), and 100 ng of nonspecific plasmid DNA. Following 16 h of post-transfection incubation, cells were treated with fresh medium containing 0.1% bovine serum albumin and 0.18 nm activin alone or preincubated (60 min) with ActRIIA-Fc, ActRIIB-Fc, or FST288 (follistatin 288; obtained from the National Hormone and Peptide Program, National Institutes of Health) as indicated for an additional 16 h in duplicates. Cell extracts were assayed for luciferase activity and normalized to Renilla luciferase activity using the Dual-Luciferase reporter assay system (Promega Corp.). Each experiment was repeated three times. To evaluate the effect of the activin type I receptors on the activity of the activin type II receptors, 0.2 μg of commercially purchased ActRIA-Fc or ActRIB-Fc was preincubated with or without equal amounts of each of the type II receptors before addition to the cells as described above. Removal of the Fc Tag—To determine whether the presence of the Fc tag altered biological activity, sActRII-Fc or sActRIIB-Fc (0.5 μg) was incubated with or without 0.1 μg of Factor Xa (New England Biolabs Inc., Beverly MA) in 20 μl of reaction buffer containing 20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 2 mm CaCl2, and 0.1% bovine serum albumin for 6 h at 23 °C. Cleavage was verified by resolving 10 ng of the Factor Xa- or mock-treated receptors on a 12% Ready-Gel (Bio-Rad) under reducing conditions, followed by Western blotting as described above. Receptor Deglycosylation—To determine whether the reduced activity of sActRIIB-Fc relative to sActRII-Fc was due to the presence of carbohydrates, sActRIIA-Fc or sActRIIB-Fc (5 μg) was treated with or without 500 units of peptide N-glycosidase F (New England Biolabs Inc.) at 37 °C for 1 h. The completion of the deglycosylation reaction was confirmed by resolving 0.1 μg of the treated material on a Ready-Gel as described above and staining with Silver Stain Plus (Bio-Rad) following the manufacturer's protocol. Data Analysis—Binding data were analyzed by the NIHRIA program (26Rodbard D. Munson P.J. Delean A. Radioimmunoassay and Related Procedures in Medicine. 1. International Atomic Energy Agency, Vienna1978: 469-504Google Scholar) for parallelism and relative potency (ED50). For homologous assays, dissociation constants were determined by Scatchard analysis using the LIGAND program (27Munson P.J. Rodbard D. Anal. Biochem. 1979; 107: 220-228Crossref Scopus (7772) Google Scholar). Each curve was also analyzed by Prism (GraphPad Software, San Diego, CA), which gave nearly identical results. Data shown in tables and all dissociation constant estimates were derived from the LIGAND analysis, whereas Figs. 2 and 7 were produced by Prism. Results shown in figures are representative experiments, and data in tables represent the means ± S.E. of at least three replicates. Significance of differences in radiolabeled activin binding to mixtures of activin type II and type I receptors was assessed by Student's t test with p < 0.05 used to indicate significance. Results from bioassay experiments are expressed as the means ± S.E. of at least three replicates.Fig. 7Reconstitution of soluble high affinity inhibin receptors. A, investigation of radiolabeled inhibin A binding to sActRII-Fc, sTβRII (TBR II)-Fc, sTβRIII-Fc, and combinations of these receptors at the doses shown. Whereas 2000 cpm bound to sActRII-Fc alone, only 1000 cpm bound to sTβRIII-Fc alone or to TβRII used as a control (i.e. indicates nonspecific binding). When sActRII-Fc was mixed with increasing amounts of sTβRIII-Fc, substantial increases in inhibin binding were observed at doses above 13 nm. When sTβRII-Fc was substituted for sActRII-Fc, only nonspecific binding was observed, indicating that inhibin binding is specific for activin type II receptors complexed with TβRIII. B, binding competition curves and Scatchard analysis for sActRII-Fc·sTβRIII-Fc complexes showing high affinity binding for this soluble complex similar to affinities reported for the membrane-bound complex (17Lewis K.A. Gray P.C. Blount A.L. MacConell L.A. Wiater E. Bilezikjian L.M. Vale W. Nature. 2000; 404: 411-414Crossref PubMed Scopus (509) Google Scholar). C, competitive binding curves and Scatchard analysis for sActRIIB-Fc·TβRIII-Fc complexes showing 2-fold higher affinity for this combination than observed for sActRII-Fc·TβRIII-Fc complexes in B. The data shown are representative of three experiments, the means of which are shown in Table III. Inh A, inhibin A; Inh B, inhibin B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characterization of Soluble Activin Receptors—Recombinant sActRII-Fc and sActRIIB-Fc fusion proteins purified from conditioned medium were analyzed by silver staining of reduced SDS-polyacrylamide gels and found to be >95% pure. sActRII-Fc ran as a single band of ∼55,000 Da, whereas sActRIIB-Fc appeared as a doublet of ∼53,000 and 57,000 Da (Fig. 1A). sTβRIII-Fc was also a single band under reducing conditions with an apparent molecular mass of ∼120,000 Da (Fig. 1B); and consistent with deletion of glycosaminoglycans, none of the typical heterogeneity of molecular mass was observed. To characterize the multimeric nature of these recombinant proteins, they were analyzed by Western blotting with anti-human Fc antibody under reducing (Fig. 1C) and nonreducing (Fig. 1D) conditions. Although the relative molecular mass of each receptor-Fc fusion protein agrees with that obtained from the silver-stained gel when reduced (Fig. 1C), the fusion proteins are clearly dimeric under nonreducing conditions, most likely via disulfide bonding between the Fc chains. Affinity and Specificity of the Soluble Activin Receptor Fusion Proteins—The affinities of sActRII-Fc and sActRIIB-Fc for 125I-activin A or B were determined by Scatchard analysis, with representative experiments shown in Fig. 2 and means of at least three replicates shown in Table I. The affinity of sActRII-Fc for activin A was surprisingly high, averaging 49 pm (Table I) compared with 150–400 pm for the natural membrane-anchored receptor (28Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and 2–7 nm for the soluble receptor ECD with no tag (29Donaldson C.J. Vaughan J.M. Corrigan A.Z. Fischer W.H. Vale W.W. Endocrinology. 1999; 140: 1760-1766Crossref PubMed Google Scholar). In addition, this affinity was >60-fold greater than for activin B (3.2 nm) (Fig. 2 and Table I). In contrast, the affinity of sActRIIB-Fc for activin A (326 pm) was ∼7-fold lower than that of sActRII-Fc (Table I), but closer to that observed for the membrane-bound form of 100–380 pm (30Attisano L. Wrana J.L. Cheifetz S. Massague J. Cell. 1992; 68: 97-108Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 31Mathews L.S. Vale W.W. Receptor. 1993; 3: 173-181PubMed Google Scholar). Like sActRII-Fc, the affinity of sActRIIB-Fc for activin B was lower than for activin A (1.9 nm) but higher than the affinity of sActRII-Fc for activin B (Table I). These results demonstrate that the activin type II receptor-Fc fusion proteins are capable of high affinity binding to activin and are selective for activin A over activin B. Moreover, sActRII-Fc binds activin A at an affinity surpassing that of the intact membrane-anchored form of ActRII.Table IAffinity of sActRII-Fc and sActRIIB-Fc for activin A or BReceptorRadioligandCompetitorKd ± S.E.pmsActRII-FcActivin AActivin A49.3 ± 25sActRII-FcActivin BActivin B3240 ± 224sActRIIB-FcActivin AActivin A326 ± 134sActRIIB-FcActivin BActivin B1900 ± 370 Open table in a new tab The relative potencies of activins A and B compared with inhibins A and B were investigated for sActRII-Fc. When activin A was the radioligand (Fig. 3A and Table II), unlabeled activin A was the most potent competitor (ED50 = 0.73 ± 0.09 ng), whereas the potency of activin B was ∼4-fold lower (ED50 = 3.26 ± 1.29 ng). Inhibins A and B were 20-fold (ED50 = 15.8 ± 0.39 ng) and >30-fold (ED50 = 23.7 ± 2.59 ng) less potent, respectively, than activin A in competing with radiolabeled activin A. When radiolabeled activin B was examined in this system (Fig. 3B and Table II), unlabeled activin A was 8-fold more potent compared with unlabeled activin B (ED50 = 3.75 ± 1.02 ng), indicating that the difference in binding activity between activins A and B for each radioligand was not due to alterations from the iodination process, but was more likely related to aspects of ligand structure that are differentially recognized by sActRII-Fc and sActRIIB-Fc. Consistent with our observations using radiolabeled activin A, inhibins A and B were >20-fold less potent than unlabeled activin A in competing with radiolabeled activin B. These results indicate that sActRII-Fc is selective for activin A, followed by activin B (3–10-fold less) and inhibins A and B (20–30-fold less), similar to what was reported for the membrane-bound form of the receptor (28Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Table IIRelative potency of sActRII-Fc for activins A and B and inhibins A and BRadioligand/competitorED50Relative potencyngActivin AActivin A0.73 ± 0.091Activin B3.26 ± 1.290.23Inhibin A15.8 ± 0.390.05Inhibin B23.7 ± 2.590.03Activin BActivin A0.50 ± 0.251.46Activin B3.75 ± 1.020.19Inhibin A19.3 ± 6.020.04Inhibin B12.9 ± 2.620.06 Open table in a new tab Biological Activity of Soluble Activin Receptors—To investigate the ability of the soluble activin receptor-Fc proteins to modulate activin biological activity, we employed a previously described activin reporter assay consisting of the CAGA activin-TGF-β luciferase reporter transfected into 293 cells. As shown in Fig. 4A, treatment with 0.18 nm activin A resulted in >30-fold stimulation of reporter activity. This stimulation was inhibited dose-dependently by sActRII-Fc, which was 2-fold more active than the high affinity activin-neutralizing protein FST288. Interestingly, sActRIIB-Fc was ∼5-fold less effective in inhibiting activin A-stimulated reporter activity compared with sActRII-Fc. When the cells were treated with 0.12 nm activin B, the neutralization activity of the soluble receptors was similar to that observed for activin A (Fig. 4B), except that FST288 was less effective in inhibiting activin B compared with activin A as observed previously (32Schneyer A. Schoen A. Quigg A. Sidis Y. Endocrinology. 2003; 144: 1671-1674Crossref PubMed Scopus (62) Google Scholar). These results indicate that the soluble receptor-Fc fusion proteins are effective inhibitors of activin action in vitro. Moreover, given that both soluble activin receptors were purified to similar degrees (Fig. 1A), the difference in inhibitory potency of sActRII-Fc versus sActRIIB-Fc for activins A and B in this biological assay likely results from the difference in activin binding affinity (Table I). Effect of the Fc Tag and Glycosylation on ActRIIA/B Biological Activity—To determine whether the differences in the affinity and biological potencies of sActRII-Fc and sActRIIB-Fc reflect actual structural differences in the binding domains of the fusion proteins or differential effects of the Fc tag, we examined the effect of removing the tag on biological a
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