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• W2103798941 abstract "Activins are members of the transforming growth factor-β family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-β2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins. Activins are members of the transforming growth factor-β family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-β2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins. transforming growth factor-β activin receptor activin receptor-like kinase bone morphogenetic protein follistatin follicle-stimulating hormone growth and differentiation factor human cartilage-derived morphogenetic protein osteogenic protein radioimmunoassay TGF-β receptor vaccinia virus polyacrylamide gel electrophoresis bis-sulfosuccinimidyl suberate The TGF-β1 family consists of a large group of structurally related, but functionally diverse polypeptides that control the growth and differentiation of many cell types in vitro and in vivo (1Massagué J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 2Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. I. Springer-Verlag, Heidelberg1990: 419-472Google Scholar, 3Matzuk M.M. Kumar T.R. Vassalli A. Bickenbach J.R. Roop D.R. Jaenisch R. Bradley A. Nature. 1995; 374: 354-356Crossref PubMed Scopus (513) Google Scholar, 4Hogan B.L.M. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar). TGF-βs, activins, and bone morphogenetic proteins (BMPs) exert their biological effects through binding to two types of serine/threonine kinase receptors, termed type I (± 53 kDa) and type II (± 70 kDa) receptors (1Massagué J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 5Mathews L.S. Endocr. Rev. 1994; 15: 310-325Crossref PubMed Scopus (273) Google Scholar, 6Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1730) Google Scholar). Type I and II receptors can form high affinity receptor complexes at the cell surface and this is necessary for signal transduction (7Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.-F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1369) Google Scholar, 8Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2112) Google Scholar, 9Inagaki M. Moustakas A. Lin H.Y. Lodish H.F. Carr B.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5359-5363Crossref PubMed Scopus (184) Google Scholar, 10Attisano L. Carcamo J. Ventura F. Weis F.M.B. Massagué J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (602) Google Scholar, 11Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar). Overexpressed type II receptors can bind ligand in the absence of type I receptor with moderate affinity, while it is generally accepted that type I receptors require type II receptors to bind ligand in the high affinity receptor complex. The type II receptor phosphorylates the type I receptor after ligand binding, and the latter propagates the signal to downstream effectors, the Smad proteins (see Ref. 12Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3343) Google Scholar). TGF-β members are biologically active as dimers. Like other members of the TGF-β family, the activins are synthesized as large precursor proteins consisting of a signal peptide, a glycosylated prodomain and a mature domain. The maturation of activin requires intracellular cleavage by protein convertases, such as furin, at the basic cleavage site which separates the mature chain from the prodomain (13Huylebroeck D. Van Nimmen K. Waheed A. von Figura K. Marmenout A. Fransen L. De Waele P. Jaspar J.-M. Franchimont P. Stunnenberg H. Van Heuverswijn H. Mol. Endocrinol. 1990; 4: 1153-1165Crossref PubMed Scopus (28) Google Scholar, 14Roebroek A. Creemers J. Pauli I. Bogaert T. Van de Ven W. EMBO J. 1993; 12: 1853-1870Crossref PubMed Scopus (53) Google Scholar). Removal of the prodomains from the precursor dimer is necessary for biological activity of the mature 25-kDa dimer, since unprocessed high molecular weight forms of activin A display no biological activity (13Huylebroeck D. Van Nimmen K. Waheed A. von Figura K. Marmenout A. Fransen L. De Waele P. Jaspar J.-M. Franchimont P. Stunnenberg H. Van Heuverswijn H. Mol. Endocrinol. 1990; 4: 1153-1165Crossref PubMed Scopus (28) Google Scholar,15Mason A.J. Farnworth P.G. Sullivan J. Mol. Endocrinol. 1996; 10: 1055-1065Crossref PubMed Scopus (71) Google Scholar, 16Wittbrodt J. Rosa F.M. Genes Dev. 1994; 8: 1448-1462Crossref PubMed Scopus (104) Google Scholar). Thus far, TGF-β2, TGF-β3, and BMP-7 (also called osteogenic protein-1 (OP-1)) have been crystallized, and the three-dimensional structures of the mature, dimeric molecules have been elucidated (17Daopin S. Piez K. Ogawa Y. Davies D. Science. 1992; 257: 369-373Crossref PubMed Scopus (375) Google Scholar, 18Schlunegger M. Grütter M. Nature. 1992; 358: 430-434Crossref PubMed Scopus (285) Google Scholar, 19Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (248) Google Scholar). These proteins share a common three-dimensional polypeptide folding pattern, although their amino acid sequence identity is limited to 36% (BMP-7 compared with TGF-β2). Hence, it is likely that this structure is the prototype for the whole family and might be extrapolated to activins as well. The common fold of the monomer is defined by seven cysteines that are conserved throughout the family. Six of these form intrachain disulfide bonds and make up the cystine knot, while the seventh cysteine forms an interchain disulfide bond that stabilizes the dimer (17Daopin S. Piez K. Ogawa Y. Davies D. Science. 1992; 257: 369-373Crossref PubMed Scopus (375) Google Scholar, 18Schlunegger M. Grütter M. Nature. 1992; 358: 430-434Crossref PubMed Scopus (285) Google Scholar, 19Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (248) Google Scholar). By analogy with a left hand, the monomeric structure consists of the N-terminal thumb region, two antiparallel pairs of β-strands that build up four fingers, two loops that connect the fingers (loop 1 connects finger 1 and 2; loop 2 connects finger 3 and 4), and a long α-helix at the heel of the hand (see Fig. 1). This prototype structure defines four solvent-accessible, flexible and divergent regions, which may contain putative receptor-binding sites, i.e. the N terminus, loop 1, loop 2, and the C-terminal end of the long α-helix (17Daopin S. Piez K. Ogawa Y. Davies D. Science. 1992; 257: 369-373Crossref PubMed Scopus (375) Google Scholar, 18Schlunegger M. Grütter M. Nature. 1992; 358: 430-434Crossref PubMed Scopus (285) Google Scholar, 19Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (248) Google Scholar). Amino acids important for biological activity have been defined by limited structure-function analysis of TGF-β members and by molecular characterization of naturally occurring mutations that cause drastic phenotypes in different organisms. Mutation analysis revealed that the nine cysteines, including the seven conserved cysteines in the family, of mature activin A are essential for either the biosynthesis or the (full) biological activity of activin A (20Mason A.J. Mol. Endocrinol. 1994; 8: 325-332PubMed Google Scholar), and that a phenylalanine to isoleucine substitution at position 21 of activin B creates a dominant-interfering protein (16Wittbrodt J. Rosa F.M. Genes Dev. 1994; 8: 1448-1462Crossref PubMed Scopus (104) Google Scholar). In mature TGF-β1, the C-terminal portion (amino acids 83–112) has recently been defined as necessary for high affinity binding to the TGF-β receptor type II (TβRII) (21Qian S.W. Burmester J.K. Tsang M.L.-S. Weatherbee J.A. Hinck A.P. Ohlsen D.J. Sporn M.B. Roberts A.B. J. Biol. Chem. 1996; 271: 30656-30662Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition, frameshift and/or point mutations (i.e.replacement of the first conserved cysteine by tyrosine) in hCDMP-1 (human cartilage-derived morphogenetic protein), and growth and differentiation factor (GDF)-8, have been identified in human chondrodysplasia and double muscling in cattle, respectively (22Thomas J.T. Lin K. Nandedkar M. Camargo M. Cervenka J. Luyten F.P. Nat. Genet. 1996; 12: 315-317Crossref PubMed Scopus (348) Google Scholar, 23Thomas J.T. Kilpatrick M.W. Lin K. Erlacher L. Lembessis P. Costa T. Tsipouras P. Luyten F.P. Nat. Genet. 1997; 17: 58-64Crossref PubMed Scopus (286) Google Scholar, 24McPherron A.C. Lee S.-J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12457-12461Crossref PubMed Scopus (1592) Google Scholar). However, no specific receptor binding determinants are known for any TGF-β member. Detailed mutagenesis studies of TGF-β family members would provide insight into how such mutations affect their biological activities, and this may facilitate the development of therapeutic agents that can be used in TGF-β-related diseases. In order to identify amino acids important for receptor binding and biological activity, we started structure-function analysis of activin A by introducing single amino acid substitutions in the mature domain, in regions that are thought to be involved in receptor interaction (18Schlunegger M. Grütter M. Nature. 1992; 358: 430-434Crossref PubMed Scopus (285) Google Scholar). In this way, we identified two amino acids in activin A which are important for its biological activity and its interaction with the type II receptor: Asp-27 and Lys-102, located in loop 1 and 2, respectively. Oligonucleotide-directed mutagenesis was performed using plasmid PTZ18R, which contains a mouse activin A cDNA cloned in the sense orientation with respect to the T7 promoter, and the Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad). Mutations were introduced using the single-stranded sense oligonucleotides listed in TableI.Table IMutant activins and oligonucleotides used for mutagenesisE3A:5′-GTAGGCGGGGCTTGGAGTGCGACGGCAAGG-3′D5A:5′-GGCTTGGAGTGCGCCGGCAAGG-3′K7A:5′-GGAGTGCGACGGCGCCGTCAACATTTGCTG-3′K13A5′-CAACATTTGCTGTGCCAAACAGTTCTTTGTC-3′K13T:5′-CAACATTTGCTGTACCAAACAGTTCTTTGTC-3′K14A:5′-CAACATTTGCTGTAAGGCACAGTTCTTTGTC-3′K14E:5′-CAACATTTGCTGTAAGGAACAGTTCTTTGTC-3′K21A:5′-CTTTGTCAGCTTCGCGGACATTGGC-3′K21T:5′-CTTTGTCAGCTTCACGGACATTGGC-3′K21E:5′-CTTTGTCAGCTTCGAGGACATTGGC-3′D22A:5′-CAGTTCTTTGTCAGCTTTAAAGCCATTGGCTGG-3′D27A:5′-GGCTGGAATGCCTGGATCATTGCTCCC-3′D27K:5′-GCTTCAAGGATATCGGCTGGAATAAATGGATCATTGC-3′V18I/S19D:5′-GAAACAGTTCTTTATCGATTTCAAGGACATTGGCTGG-3′H47A:5′-GAGTGCCCAAGCGCCATAGCAGGTACCTCTGGGTC-3′H71A:5′-CACTACCGCATGCGGGGTGCCAGCCCCTTTGC-3′H71N:5′-CACTACCGCATGCGGGGTAACAGCCCCTTTGC-3′H71K:5′-CACTACCGCATGCGGGGTAAGAGCCCCTTTGC-3′H71-:5′-CACTACCGCATGCGGGGTAGCCCCTTTGCCAACCTT-3′H71-/S72-:5′-CACTACCGCATGCGGGGTCCCTTTGCCAACCTTAAG-3′K85A:5′-GCTGTGTGCCCACAGCGCTGAGACCCATG-3′R87A:5′-GTGCCCACCAAGCTTGCACCCATGTCCATG-3′D95A:5′-GCTGTATTACGCTGATGGCCAAAACATC-3′D96A:5′-GCTGTATTACGATGCCGGCCAAAACATC-3′K102A:5′-GTCAAAACATCATCGCAAAGGATATCCAAAACATG-3′K102E:5′-GTCAAAACATCATCGAAAAGGATATCCAAAACATG-3′K102R:5′-GTCAAAACATCATCAGAAAGGATATCCAAAACATG-3′K103A:5′-CATCATCAAAGCGGATATCCAAAACATGATTGTGG-3′Q106A:5′-CATCAAAAAGGATATCGCAAACATGATTGTG-3′E111A:5′-CATGATTGTGGCGGAGTGTGGCTGCAGCTAGAGTCGCC-3′E112A:5′-CATGATTGTGGAGGCATGCGGCTGCTCCTAG-3′G114K:5′-CATGATTGTGGAGGAGTGTAAGTGCTCCTGAATTCGCCAGGTCCC-3′ Open table in a new tab Mutant TGF-β2L1 was constructed using two complementary oligonucleotides representing the DNA sequence of loop 1 of TGF-β2: 5′-CGATTTCAAGAGAGATCTAGGGTGGAAATGGATACACGAACCCT-3′ and 5′-CCGGAGGGTTCGTGTATCCATTTCCACCCTAGATCTCTCTTGAAAT-3′ This sequence was cloned into an activin mutant construct in which aClaI site and a MroI site had been introduced upstream and downstream, respectively, of the loop 1 (Val-18 to Pro-32) sequence. Mutant activin V18I/S19D/N26I/D27G was generated by partial double annealing via the nine 3′-nucleotides of 5′-GAAACAGTTCTTTATCGATTTCAAGGACATTGGCTGGATTGGCTGG-3′. This generated two extra mutations (N26I and D27G) in addition to those generated by oligonucleotide V18I/S19D. All mutations, listed in Fig. 1 B, were confirmed by DNA sequencing. This expression system has been described previously (13Huylebroeck D. Van Nimmen K. Waheed A. von Figura K. Marmenout A. Fransen L. De Waele P. Jaspar J.-M. Franchimont P. Stunnenberg H. Van Heuverswijn H. Mol. Endocrinol. 1990; 4: 1153-1165Crossref PubMed Scopus (28) Google Scholar), but it was applied in a slightly modified manner. Subconfluent HeLa or PK15 cells (10-cm2dishes) were infected with a recombinant vaccinia virus expressing phage T7 RNA polymerase (multiplicity of infection: 5) for 1 h at 24 °C. These cells were then transfected with T7 promoter-containing plasmids encoding the wild type and mutant activins A, using DOTAP (Boehringer Mannheim). For the heterodimerization assay, cells were cotransfected with T7 plasmids encoding zebrafish activin B (a gift from F. Rosa, U368 INSERM, Ecole Normale Supérieure, Paris, France). Cells were incubated with this DNA/DOTAP mix for 6 h at 37 °C in an atmosphere containing 5% CO2. Cells were then washed with methionine-free minimum essential medium before starvation in this medium for 1 h at 37 °C. The cells were next pulse-labeled for 1 h by addition of 1 ml of the same medium containing 50 μCi of [35S]methionine and [35S]cysteine (ICN). The cells were chased by addition of 1 ml of Dulbecco's modified Eagle's medium supplemented with 20 μg/ml bovine serum albumin and a 10-fold higher concentration of cold methionine than is normally present in this medium. After 14 h at 37 °C, the medium was collected and centrifuged, and the supernatant was frozen. Samples were prepared for electrophoresis after precipitation of the proteins with trichloroacetic acid, as described previously (13Huylebroeck D. Van Nimmen K. Waheed A. von Figura K. Marmenout A. Fransen L. De Waele P. Jaspar J.-M. Franchimont P. Stunnenberg H. Van Heuverswijn H. Mol. Endocrinol. 1990; 4: 1153-1165Crossref PubMed Scopus (28) Google Scholar). The follicle-stimulating hormone (FSH) assay was performed as described (25Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.-H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (461) Google Scholar). Briefly, primary rat pituitary cells were cultured for 2 days in serum-free medium (as specified in Ref. 25Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.-H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (461) Google Scholar) containing dilutions of the activin A mutant proteins. The medium was then collected, and the FSH concentration was determined by radioimmunoassay (RIA). Each mutant activin was added to three wells, and the RIA for FSH was performed in duplicate using the FSH-RIA kit (NIDDKD, National Institutes of Health, Rockville, MD) according to Denef et al. (26Denef C. Maertens P. Allaerts W. Mignon A. Robberecht W. Swennen L. Carmeliet P. Methods Enzymol. 1989; 168: 47-71Crossref PubMed Scopus (88) Google Scholar). Xenopusembryos were obtained by in vitro fertilization (27Smith J.C. Slack J.M.W. J. Embryol. Exp. Morphol. 1983; 78: 299-317PubMed Google Scholar). They were maintained in 10% Normal Amphibian Medium (28Slack J.M.W. J. Embryol. Exp. Morphol. 1984; 80: 289-319PubMed Google Scholar) and staged according to Nieuwkoop and Faber (29Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis. Daudlin, Amsterdam1967Google Scholar). Animal pole regions were dissected from mid-blastula (stage 8) embryos (30Smith J.C. Hartley D. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 181-204Google Scholar) and cultured in 75% Normal Amphibian Medium containing 0.1% (w/v) bovine serum albumin and wild type or mutant activin (2.5 ng/ml). A preliminary assessment of mesoderm induction was based on the elongation of the animal caps. Animal pole regions were then frozen on dry ice, and expression of the mesoderm-specific gene Brachyury (Xbra) (31Smith J.C. Price B.M.J. Green J.B.A. Weigel D. Herrmann B.G. Cell. 1991; 67: 79-87Abstract Full Text PDF PubMed Scopus (833) Google Scholar) was assayed by RNase protection analysis as described by Jones et al. (32Jones C.M. Kuehn M.R. Hogan B.L.M. Smith J.C. Wright C.V.E. Development. 1995; 121: 3651-3662Crossref PubMed Google Scholar). Wild type and mutant activins, and follistatin were iodinated using a modified chloramine-T method (33De Winter J.P. ten Dijke P. de Vries C.J.M. van Achterberg T.A.E. Sugino H. de Waele P. Huylebroeck D. Verschueren K. van den Eijnden-van Raaij A.J.M. Mol. Cell. Endocrinol. 1996; 116: 105-114Crossref PubMed Scopus (173) Google Scholar). Two μg of protein (in 10 μl of 30% acetonitrile, 0.1% trifluoroacetic acid) were diluted with 10 μl of 600 mm sodium phosphate (pH 7.5) and 5 μl of Na125I (0.25 mCi; Amersham Pharmacia Biotech) and 5 μl of phosphate-buffered saline (137 mm NaCl, 2.7 mmKCl, 6.5 mm Na2HPO4 and 1.5 mm KH2PO4). To initiate the radioiodination, 10 μl of chloramine-T (100 μg/ml in 50 mm sodium phosphate (pH 7.5); Sigma) was added. After 2 min, the iodination was stopped by addition of 20 μl of 50 mm N-acetyl-l-tyrosine (Sigma), 200 μl of 60 mm sodium iodide, and 200 μl of 10m ultrapure urea (Life Technologies). Subsequently, the reaction mixture was passed over a Sephadex G-25 column (Amersham Pharmacia Biotech), which was equilibrated and eluted with phosphate-buffered saline containing 0.1% (w/v) hemoglobin (Sigma). Peak fractions, with specific activities of 30–100 μCi/μg of protein, were routinely obtained, pooled, and stored at −80 °C. Radioiodinated follistatin288 (FS288) was cross-linked to cold wild type and mutant activins using bis-sulfo-succinimidyl suberate (BS3; Pierce) (33De Winter J.P. ten Dijke P. de Vries C.J.M. van Achterberg T.A.E. Sugino H. de Waele P. Huylebroeck D. Verschueren K. van den Eijnden-van Raaij A.J.M. Mol. Cell. Endocrinol. 1996; 116: 105-114Crossref PubMed Scopus (173) Google Scholar). Approximately 2 ng of iodinated FS288 (5 μl) was incubated with 500 μl of activin-containing conditioned medium prepared as described above. After 2 h of incubation at 4 °C on a rotary shaker, 125 μl of 5 mm BS3 in HEPES-buffered saline (150 mm NaCl and 20 mmHEPES; Life Technologies, Inc.) was added and the reaction was incubated for 1 h at 4 °C. Activin/follistatin complexes were purified using wheat germ agglutinin-agarose (Sigma) beads (33De Winter J.P. ten Dijke P. de Vries C.J.M. van Achterberg T.A.E. Sugino H. de Waele P. Huylebroeck D. Verschueren K. van den Eijnden-van Raaij A.J.M. Mol. Cell. Endocrinol. 1996; 116: 105-114Crossref PubMed Scopus (173) Google Scholar). They were separated by SDS-PAGE under reducing conditions and visualized by autoradiography. PK15 cells (28-cm2dishes) were transfected with different combinations of activin receptors using the vaccinia virus-T7 system as described above. On the second day, the cells were washed with ice-cold binding medium (HEPES-buffered Dulbecco's modified Eagle's medium (pH 7.5) containing 0.2% (w/v) bovine serum albumin) for 10 min. Cells were incubated with 150 pm labeled activin A in 1.5 ml of binding medium for 2 h at 4 °C. For competition studies, cells were incubated with a constant amount (150 pm) of125I-labeled wild type activin and (simultaneously added) different amounts of cold wild type or mutant activins. Then, iodinated activin was removed by gently and repeatedly washing the cells with ice-cold HEPES-buffered saline containing 0.9 mmCaCl2. Activin was cross-linked by incubation in 1.5 ml of HEPES-buffered saline containing 1 mm BS3 for 30 min at 4 °C. The reaction was then quenched for 5 min at 4 °C by addition of 150 μl of 10× quench solution (10×: 10 mm Tris (pH 7.5), 2 mm EDTA, and 200 mm glycine). The cells were scraped from the plates in 1 ml of detachment buffer (10 mm Tris (pH 7.4), 1 mmEDTA, 10% (v/v) glycerol, 0.5 μg of aprotinin/ml, 0.5 μg of leupeptin/ml, and 0.3 mm phenylmethylsulfonyl fluoride), and collected by centrifugation (5 min at 4 °C). The pellet was then dissolved in 50 μl of solubilization buffer (10 mm Tris (pH 7.4), 1 mm EDTA, 125 mm NaCl, 1% (v/v) Triton X-100, 0.5 μg of aprotinin/ml, 0.5 μg of leupeptin/ml, and 0.3 mm phenylmethylsulfonyl fluoride), followed by incubation for 40 min on ice. Proteins were separated by SDS-PAGE and visualized by autoradiography. The large scale production of wild type and mutant activins was performed using a baculovirus expression system. The mutant cDNA was inserted in the baculotransfer vector pVL1393 under transcriptional control of the baculoviral polyhedrin promotor. Recombinant baculovirus was generated by homologous recombination in Spodoptera frugiperda cells (Sf9) cotransfected with the recombinant transfer construct and BaculogoldTM virus AcNPV DNA (PharMingen). Recombinant virus was plaque-purified and amplified to high titer stock for production. Activin was purified from conditioned medium of recombinant baculovirus infected Sf9 cells, harvested 72 h after infection. Purification of wild type and mutant activins was performed by use of an optimized four-step purification protocol in which the conditioned medium is diafiltrated and concentrated in the presence of 6m urea and loaded onto an anion exchange column (Fractogel-(EMD)-TMAE, Merck). The flow-through is then loaded on a Protein Pack Sulfonyl (Millipore) cation exchange column. The 150 mm NaCl fraction is then adjusted to 10% acetonitrile, 0.1% trifluoroacetic acid (v/v) and separated by RPC-4 (Fractogel-butyl) reversed phase chromatography. Mutant activins are recovered in the 30–34% acetonitrile fraction (34Smith J.C. Price B.M.J. Van Nimmen K. Huylebroeck D. Nature. 1990; 345: 729-731Crossref PubMed Scopus (565) Google Scholar) and further purified on a RPC-8 (Brownlee octyl) column run as a polishing step. Quantification of these pure activins was obtained through amino acid composition analysis. Some mutations in structurally important regions of TGF-β family members have been reported to lead to improper biosynthesis of these ligands (15Mason A.J. Farnworth P.G. Sullivan J. Mol. Endocrinol. 1996; 10: 1055-1065Crossref PubMed Scopus (71) Google Scholar, 16Wittbrodt J. Rosa F.M. Genes Dev. 1994; 8: 1448-1462Crossref PubMed Scopus (104) Google Scholar, 20Mason A.J. Mol. Endocrinol. 1994; 8: 325-332PubMed Google Scholar, 35Hawley S.H.B. Wünnenberg-Stapleton K. Hashimoto C. Laurent N. Watabe T. Blumberg B.W. Cho K.W.Y. Genes Dev. 1995; 9: 2923-2935Crossref PubMed Scopus (303) Google Scholar, 36Brunner A.M. Lioubin M.N. Marquardt H. Malacko A.R. Wang W.-C. Shapiro R.A. Neubauer M. Cook J. Madisen Purchio A.F. Mol. Endocrinol. 1992; 6: 1691-1700Crossref PubMed Scopus (50) Google Scholar). In our study, this was also the case when severe changes in activin A were introduced. For example, the substitution of loop 1 of activin A by the equivalent region of TGF-β2 led to undetectable protein expression levels in the vaccinia virus-T7 system, both in the secreted and in the intracellular fraction (data not shown). It is likely that intracellular degradation occurred, as has been suggested for most cysteine mutants of activin A and TGF-β1 (20Mason A.J. Mol. Endocrinol. 1994; 8: 325-332PubMed Google Scholar, 36Brunner A.M. Lioubin M.N. Marquardt H. Malacko A.R. Wang W.-C. Shapiro R.A. Neubauer M. Cook J. Madisen Purchio A.F. Mol. Endocrinol. 1992; 6: 1691-1700Crossref PubMed Scopus (50) Google Scholar). To avoid synthesis and intracellular trafficking problems due to structural changes, we anticipated that the majority of mutant activins used in this study should be generated by single amino acid substitution. Four solvent-accessible regions can be deduced from the three-dimensional structure of TGF-β2 and BMP-7 (17Daopin S. Piez K. Ogawa Y. Davies D. Science. 1992; 257: 369-373Crossref PubMed Scopus (375) Google Scholar, 18Schlunegger M. Grütter M. Nature. 1992; 358: 430-434Crossref PubMed Scopus (285) Google Scholar, 19Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (248) Google Scholar): the N terminus, loop 1, loop 2, and the C-terminal segment of the long α-helix (18; see also Fig. 1). These regions are the most flexible structures in the dimer, and their sequences are divergent throughout the family, which marks them as good candidates for receptor interaction. Most of the mutations introduced are single alanine substitutions at charged residues in these domains (Fig. 1 A). A large panel of 39 activin A mutants was constructed by oligonucleotide-directed mutagenesis (Fig.1 B). Synthesis of this large panel of activin polypeptides was first analyzed in HeLa cells using the T7 vaccinia virus-based expression system. As suggested previously, these cells have sufficient levels of endogenous furin to support correct and efficient processing of the activin A precursor (13Huylebroeck D. Van Nimmen K. Waheed A. von Figura K. Marmenout A. Fransen L. De Waele P. Jaspar J.-M. Franchimont P. Stunnenberg H. Van Heuverswijn H. Mol. Endocrinol. 1990; 4: 1153-1165Crossref PubMed Scopus (28) Google Scholar). Synthesized proteins were visualized by metabolic labeling followed by SDS-PAGE. We assessed both the maturation of activin A mutants to a 25-kDa dimer as well as their capacity to heterodimerize with zebrafish activin B; a secreted activin AB heterodimer can be resolved in SDS-PAGE because activin A homodimers have a slower migration than activin B homodimers. Nearly all activin A mutant polypeptides were processed like the wild type precursor and they heterodimerized efficiently (and predominantly) with activin B, as observed previously (Ref. 37Ling N. Ying S.-Y. Ueno N. Shimasaki S. Esch F. Hotta M. Guillemin R. Nature. 1986; 321: 779-782Crossref PubMed Scopus (948) Google Scholar and data not shown; only activin dimers of D27K, K102A, K102E and K102R, respectively, are shown in Fig.2). This indicates that the overall structure of the precursor polypeptides and their intracellular folding and dimerization in the rough endoplasmic reticulum are not altered. As well as analyzing their ability to dimerize, the ability of the mutants to bind follistatin (FS), an antagonistic binding protein of activin, was tested by cross-linking. All mutant activins tested (including K102A and K102E), formed complexes with FS288 like wild type activin (shown for K102A and K102E in Fig. 3). This again suggests that their overall structure is not dramatically altered, if at all.Figure 3Binding of mutant activins to follistatin. Approximately 2 ng of iodinated FS288 (in 5 μl of phosphate-buffered saline) was incubated with 500 μl of vaccinia virus-T7 produced proteins. After cross-linking of the activin/follistatin complexes, they were purified using wheat germ agglutinin-Sepharose (33De Winter J.P. ten Dijke P. de Vries C.J.M. van Achterberg T.A.E. Sugino H. de Waele P. Hu" @default.
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• W2103798941 title "Identification of Two Amino Acids in Activin A That Are Important for Biological Activity and Binding to the Activin Type II Receptors" @default.
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