Endocrinology, Vol 135, 667-674, Copyright © 1994 by Endocrine Society

ARTICLES

Characterization of unique binding kinetics of follistatin and activin or inhibin in serum

AL Schneyer, DA Rzucidlo, PM Sluss and WF Crowley Jr
National Center for Infertility Research, Massachusetts General Hospital, Boston 02114.

Serum binding proteins (BPs) have been identified for several peptide and protein hormones, and their presence has significant implications for the biological action of the hormone. Follistatin (FS) has been identified as an activin- and inhibin-BP in tissues, serum, and follicular fluid of several species, including humans. In this study, the binding kinetics of FS for activin and inhibin were characterized in human serum using gel filtration chromatography and compared to those of pure recombinant hormones using chromatography and a new solid phase assay. When complexed with radiolabeled activin or inhibin, FS eluted at a volume corresponding to a mol wt range of 67,000-150,000, an elution volume identical to the lower mol wt BP peak observed in serum. Furthermore, kinetic analyses of recombinant FS binding to activin using a solid phase assay revealed that 1) the FS-activin interaction is of high affinity, similar to or exceeding that estimated for activin binding to its receptor; 2) binding to activin is essentially irreversible at physiological pH; and 3) the potency of inhibin is approximately 500- to 1000-fold lower than that of activin in the FS binding assay. The lack of FS-[125I]activin complex reversibility observed in the solid phase assay was confirmed using a modified gel filtration chromatography protocol. Thus, preincubation of pure FS or serum with unlabeled activin for 2 h eliminated all binding of subsequently added labeled activin despite a much longer incubation period. However, when labeled activin was incubated with FS for 2 h, subsequent addition of unlabeled activin or inhibin was unable to displace labeled activin from FS, again demonstrating a lack of reversibility. Finally, to map this high affinity interaction, overlapping synthetic peptides were used to compete with labeled activin for FS binding. Two potential contact sites between FS and activin were identified, one near the N-terminus (amino acids 15-29) and the other near the C-terminus (amino acids 99-116). Given its apparently irreversible nature, high affinity, and ability to neutralize activin's biological activity, FS is quite different from the typical hormone-BP. These unique properties of FS undoubtedly attest to the potency of activin in many physiological and developmental settings and, therefore, to the importance of BPs, such as FS for regulating activin's bioactivity, distribution, and/or clearance.

This article has been cited by other articles:

				A. L. Schneyer, Y. Sidis, A. Gulati, J. L. Sun, H. Keutmann, and P. A. Krasney Differential Antagonism of Activin, Myostatin and Growth and Differentiation Factor 11 by Wild-Type and Mutant Follistatin Endocrinology, September 1, 2008; 149(9): 4589 - 4595. [Abstract] [Full Text] [PDF]

				C. J. Stoikos, C. A. Harrison, L. A. Salamonsen, and E. Dimitriadis A distinct cohort of the TGF{beta} superfamily members expressed in human endometrium regulate decidualization Hum. Reprod., June 1, 2008; 23(6): 1447 - 1456. [Abstract] [Full Text] [PDF]

				F. M. Reis, L. L. Nascimento, A. Tsigkou, M. C. Ferreira, S. Luisi, and F. Petraglia Activin A and Follistatin in Menstrual Blood: Low Concentrations in Women With Dysfunctional Uterine Bleeding Reproductive Sciences, May 1, 2007; 14(4): 383 - 389. [Abstract] [PDF]

				K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]

				Y. Sidis, A. Mukherjee, H. Keutmann, A. Delbaere, M. Sadatsuki, and A. Schneyer Biological Activity of Follistatin Isoforms and Follistatin-Like-3 Is Dependent on Differential Cell Surface Binding and Specificity for Activin, Myostatin, and Bone Morphogenetic Proteins Endocrinology, July 1, 2006; 147(7): 3586 - 3597. [Abstract] [Full Text] [PDF]

				P. G Farnworth, Y. Wang, P. Leembruggen, G. T Ooi, C. Harrison, D. M Robertson, and J. K Findlay Rodent adrenocortical cells display high affinity binding sites and proteins for inhibin A, and express components required for autocrine signalling by activins and bone morphogenetic proteins. J. Endocrinol., March 1, 2006; 188(3): 451 - 465. [Abstract] [Full Text] [PDF]

				S. Patella, D. J. Phillips, J. Tchongue, D. M. de Kretser, and W. Sievert Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G137 - G144. [Abstract] [Full Text] [PDF]

				F. Aoki, M. Kurabayashi, Y. Hasegawa, and I. Kojima Attenuation of Bleomycin-induced Pulmonary Fibrosis by Follistatin Am. J. Respir. Crit. Care Med., September 15, 2005; 172(6): 713 - 720. [Abstract] [Full Text] [PDF]

				Y. Sidis, A. L. Schneyer, and H. T. Keutmann Heparin and Activin-Binding Determinants in Follistatin and FSTL3 Endocrinology, January 1, 2005; 146(1): 130 - 136. [Abstract] [Full Text] [PDF]

				E. del Re, Y. Sidis, D. A. Fabrizio, H. Y. Lin, and A. Schneyer Reconstitution and Analysis of Soluble Inhibin and Activin Receptor Complexes in a Cell-free System J. Biol. Chem., December 17, 2004; 279(51): 53126 - 53135. [Abstract] [Full Text] [PDF]

				A. L. Schneyer, Q. Wang, Y. Sidis, and P. M. Sluss Differential Distribution of Follistatin Isoforms: Application of a New FS315-Specific Immunoassay J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5067 - 5075. [Abstract] [Full Text] [PDF]

				C. J. Jorgez, M. Klysik, S. P. Jamin, R. R. Behringer, and M. M. Matzuk Granulosa Cell-Specific Inactivation of Follistatin Causes Female Fertility Defects Mol. Endocrinol., April 1, 2004; 18(4): 953 - 967. [Abstract] [Full Text] [PDF]

				Y. Xia, Y. Sidis, and A. Schneyer Overexpression of Follistatin-Like 3 in Gonads Causes Defects in Gonadal Development and Function in Transgenic Mice Mol. Endocrinol., April 1, 2004; 18(4): 979 - 994. [Abstract] [Full Text] [PDF]

				H. T. Keutmann, A. L. Schneyer, and Y. Sidis The Role of Follistatin Domains in Follistatin Biological Action Mol. Endocrinol., January 1, 2004; 18(1): 228 - 240. [Abstract] [Full Text] [PDF]

				A. O. Bahathiq, R. L. Stewart, M. Wells, H. D. Moore, A. A. Pacey, and W. L. Ledger Production of Activins by the Human Endosalpinx J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5283 - 5289. [Abstract] [Full Text] [PDF]

				J. J. Hill, M. V. Davies, A. A. Pearson, J. H. Wang, R. M. Hewick, N. M. Wolfman, and Y. Qiu The Myostatin Propeptide and the Follistatin-related Gene Are Inhibitory Binding Proteins of Myostatin in Normal Serum J. Biol. Chem., October 18, 2002; 277(43): 40735 - 40741. [Abstract] [Full Text] [PDF]

				C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]

				M. Szweras, D. Liu, E. A. Partridge, J. Pawling, B. Sukhu, C. Clokie, W. Jahnen-Dechent, H. C. Tenenbaum, C. J. Swallow, M. D. Grynpas, et al. alpha 2-HS Glycoprotein/Fetuin, a Transforming Growth Factor-beta /Bone Morphogenetic Protein Antagonist, Regulates Postnatal Bone Growth and Remodeling J. Biol. Chem., May 24, 2002; 277(22): 19991 - 19997. [Abstract] [Full Text] [PDF]

				Y. Sidis, D. V. Tortoriello, W. E. Holmes, Y. Pan, H. T. Keutmann, and A. L. Schneyer Follistatin-Related Protein and Follistatin Differentially Neutralize Endogenous vs. Exogenous Activin Endocrinology, May 1, 2002; 143(5): 1613 - 1624. [Abstract] [Full Text] [PDF]

				V. Padmanabhan, D. Battaglia, M. B. Brown, F. J. Karsch, J. S. Lee, W. Pan, D. J. Phillips, and J. Van Cleeff Neuroendocrine Control of Follicle-Stimulating Hormone (FSH) Secretion: II. Is Follistatin-Induced Suppression of FSH Secretion Mediated via Changes in Activin Availability and Does It Involve Changes in Gonadotropin-Releasing Hormone Secretion? Biol Reprod, May 1, 2002; 66(5): 1395 - 1402. [Abstract] [Full Text]

				R. L. Jones, L. A. Salamonsen, Y. C. Zhao, J.-F. Ethier, A. E. Drummond, and J. K. Findlay Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization Mol. Hum. Reprod., April 1, 2002; 8(4): 363 - 374. [Abstract] [Full Text] [PDF]

				T. Fujiwara, Y. Sidis, C. Welt, G. Lambert-Messerlian, J. Fox, A. Taylor, and A. Schneyer Dynamics of Inhibin Subunit and Follistatin mRNA during Development of Normal and Polycystic Ovary Syndrome Follicles J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4206 - 4215. [Abstract] [Full Text] [PDF]

				D. V. Tortoriello, Y. Sidis, D. A. Holtzman, W. E. Holmes, and A. L. Schneyer Human Follistatin-Related Protein: A Structural Homologue of Follistatin with Nuclear Localization Endocrinology, August 1, 2001; 142(8): 3426 - 3434. [Abstract] [Full Text] [PDF]

				H.-J. Huang, J. C. Wu, P. Su, O. Zhirnov, and W. L. Miller A Novel Role for Bone Morphogenetic Proteins in the Synthesis of Follicle-Stimulating Hormone Endocrinology, June 1, 2001; 142(6): 2275 - 2283. [Abstract] [Full Text] [PDF]

				K.-C. Choi, S. K. Kang, C.-J. Tai, N. Auersperg, and P. C. K. Leung The Regulation of Apoptosis by Activin and Transforming Growth Factor-{beta} in Early Neoplastic and Tumorigenic Ovarian Surface Epithelium J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2125 - 2135. [Abstract] [Full Text]

				N. A. Klein, D. E. Battaglia, T. K. Woodruff, V. Padmanabhan, L. C. Giudice, W. J. Bremner, and M. R. Soules Ovarian Follicular Concentrations of Activin, Follistatin, Inhibin, Insulin-Like Growth Factor I (IGF-I), IGF-II, IGF-Binding Protein-2 (IGFBP-2), IGFBP-3, and Vascular Endothelial Growth Factor in Spontaneous Menstrual Cycles of Normal Women of Advanced Reproductive Age J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4520 - 4525. [Abstract] [Full Text]

				A. L. Schneyer, T. Fujiwara, J. Fox, C. K. Welt, J. Adams, G. M. Messerlian, and A. E. Taylor Dynamic Changes in the Intrafollicular Inhibin/Activin/Follistatin Axis during Human Follicular Development: Relationship to Circulating Hormone Concentrations J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3319 - 3330. [Abstract] [Full Text]

				D. J. Phillips, K. L. Jones, D. J. McGaw, N. P. Groome, J. J. Smolich, H. Pärsson, and D. M. de Kretser Release of Activin and Follistatin during Cardiovascular Procedures Is Largely due to Heparin Administration J. Clin. Endocrinol. Metab., July 1, 2000; 85(7): 2411 - 2415. [Abstract] [Full Text]

				H. Chong, S. A. Pangas, D. J. Bernard, E. Wang, J. Gitch, W. Chen, L. B. Draper, E. T. Cox, and T. K. Woodruff Structure and Expression of a Membrane Component of the Inhibin Receptor System Endocrinology, July 1, 2000; 141(7): 2600 - 2607. [Abstract] [Full Text] [PDF]

				M. A. Engelse, J. M. Neele, T. A. E. van Achterberg, B. E. van Aken, R. H. N. van Schaik, H. Pannekoek, and C. J. M. de Vries Human Activin-A Is Expressed in the Atherosclerotic Lesion and Promotes the Contractile Phenotype of Smooth Muscle Cells Circ. Res., November 12, 1999; 85(10): 931 - 939. [Abstract] [Full Text] [PDF]

				S. J. McPherson, S. L. Mellor, H. Wang, L. W. Evans, N. P. Groome, and G. P. Risbridger Expression of Activin A and Follistatin Core Proteins by Human Prostate Tumor Cell Lines Endocrinology, November 1, 1999; 140(11): 5303 - 5309. [Abstract] [Full Text]

				J. R. McFarlane, L. M. Foulds, A. E. O’Connor, D. J. Phillips, G. Jenkin, M. T. W. Hearn, and D. M. de Kretser Uterine Milk Protein, a Novel Activin-Binding Protein, Is Present in Ovine Allantoic Fluid Endocrinology, October 1, 1999; 140(10): 4745 - 4752. [Abstract] [Full Text]

				A. Delbaere, Y. Sidis, and A. L. Schneyer Differential Response to Exogenous and Endogenous Activin in a Human Ovarian Teratocarcinoma-Derived Cell Line (PA-1): Regulation by Cell Surface Follistatin Endocrinology, June 1, 1999; 140(6): 2463 - 2470. [Abstract] [Full Text]

				G. Wuytens, K. Verschueren, J. P. de Winter, N. Gajendran, L. Beek, K. Devos, F. Bosman, P. de Waele, M. Andries, A. J.M. v. d. E.-v. Raaij, et al. Identification of Two Amino Acids in Activin A That Are Important for Biological Activity and Binding to the Activin Type II Receptors J. Biol. Chem., April 2, 1999; 274(14): 9821 - 9827. [Abstract] [Full Text] [PDF]

				Y. Sidis,, T. Fujiwara,, L. Leykin,, K. Isaacson,, T. Toth,, and A. L. Schneyer Characterization of Inhibin/Activin Subunit, Activin Receptor, and Follistatin Messenger Ribonucleic Acid in Human and Mouse Oocytes: Evidence for Activin's Paracrine Signaling from Granulosa Cells to Oocytes Biol Reprod, October 1, 1998; 59(4): 807 - 812. [Abstract] [Full Text]

				D. S. McConnell, Q. Wang, P. M. Sluss, N. Bolf, R. H. Khoury, A. L. Schneyer, A. R. Midgley Jr., N. E. Reame, W. F. Crowley Jr., and V. Padmanabhan A Two-Site Chemiluminescent Assay for Activin-Free Follistatin Reveals That Most Follistatin Circulating in Men and Normal Cycling Women Is in an Activin-Bound State J. Clin. Endocrinol. Metab., March 1, 1998; 83(3): 851 - 858. [Abstract] [Full Text]

				N. Di Simone, H. A. Hall, C. Welt, and A. L. Schneyer Activin Regulates {beta}A-Subunit and Activin Receptor Messenger Ribonucleic Acid and Cellular Proliferation in Activin-Responsive Testicular Tumor Cells Endocrinology, March 1, 1998; 139(3): 1147 - 1155. [Abstract] [Full Text] [PDF]

				C. K. Welt, G. Lambert-Messerlian, W. Zheng, W. F. Crowley Jr., and A. L. Schneyer Presence of Activin, Inhibin, and Follistatin in Epithelial Ovarian Carcinoma J. Clin. Endocrinol. Metab., November 1, 1997; 82(11): 3720 - 3727. [Abstract] [Full Text] [PDF]

				Y. Shintani, M. Dyson, A. E. Drummond, and J. K. Findlay Regulation of Follistatin Production by Rat Granulosa Cells in Vitro Endocrinology, June 1, 1997; 138(6): 2544 - 2551. [Abstract] [Full Text] [PDF]

				M. Demetriou, C. Binkert, B. Sukhu, H. C. Tenenbaum, and J. W. Dennis Fetuin/alpha 2-HS Glycoprotein Is a Transforming Growth Factor-beta Type II Receptor Mimic and Cytokine Antagonist J. Biol. Chem., May 31, 1996; 271(22): 12755 - 12761. [Abstract] [Full Text] [PDF]

				K. Tsuchida, K. Y. Arai, Y. Kuramoto, N. Yamakawa, Y. Hasegawa, and H. Sugino Identification and Characterization of a Novel Follistatin-like Protein as a Binding Protein for the TGF-beta Family J. Biol. Chem., December 22, 2000; 275(52): 40788 - 40796. [Abstract] [Full Text] [PDF]

				Y. Sidis, A. L. Schneyer, P. M. Sluss, L. N. Johnson, and H. T. Keutmann Follistatin: Essential Role for the N-terminal Domain in Activin Binding and Neutralization J. Biol. Chem., May 18, 2001; 276(21): 17718 - 17726. [Abstract] [Full Text] [PDF]