Heparin and Activin-Binding Determinants in Follistatin and FSTL3
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内分泌学杂志 2005年第1期
Reproductive Endocrine Unit (Y.S., A.L.S.) and Endocrine Unit (H.T.K.), Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Henry T. Keutmann, Endocrine Unit, Wellman 501, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Keutmann@helix.mgh.harvard.edu.
Abstract
Local regulation of pituitary FSH secretion and many other cellular processes by follistatin (FS) can be ascribed to its potent ability to bind and bioneutralize activin, in conjunction with binding to cell surface heparan-sulfate proteoglycans through a basic heparin-binding sequence (HBS; residues 75–86) in the first of the three FS domains. The FS homolog, FSTL3, also binds activin, but lacks any HBS and cannot associate with cell surfaces. We have used mutational analyses to define the determinants for heparin binding and activin interaction in FS and to determine the effects of conferring heparin binding to FSTL3. Mutants expressed from 283F cells were tested for cell surface and heparin affinity binding, for competititive activin binding and for bioactivity by suppression of pituitary cell FSH secretion. Replacement of the HBS or the full-length FS-domain 1 abolished cell surface binding but enhanced activin binding 4- to 8-fold. Surface binding was partially reduced after mutation of either lysine pair 75/76 or 81/82 and eliminated after mutation of both pairs. The 75/76 mutation reduced activin binding and, therefore, pituitary cell bioactivity by 5-fold. However, insertion of the HBS into FSTL3 did not restore heparin binding or pituitary-cell bioactivity. These results show that 1) the residues within the HBS are necessary but not sufficient for heparin binding, and 2) the HBS also harbors determinants for activin binding. Introduction of the full domain from FS conferred heparin binding to FSTL3, but activin binding was abolished. This implies an evolutionary safeguard against surface binding by FSTL3, supporting other evidence for physiological differences between FS and FSTL3.
Introduction
THE ABILITY OF FOLLISTATIN (FS) to regulate pituitary FSH secretion can be ascribed to its potent ability to bind and bioneutralize activin (1). Extensive evidence has affirmed that, rather than presenting activin to its receptor, FS sequesters activin nearly irreversibly to prevent stimulation of FSH secretion (2, 3, 4, 5). Although present in the circulation, colocalization of FS and activin within the pituitary, ovary, and many other tissues have drawn attention to its role as a local regulator of activin action in a wide range of processes influencing cellular secretion, development and differentiation (see Ref. 6 for recent review).
FS’s local actions have been widely attributed to its interaction with cell surface proteoglycans, in common with many extracellular proteins and growth factors (7). FS binding to heparin and heparin-like homologs on either synthetic solid supports or natural cell surfaces was recognized at an early stage (8, 9, 10). This capability has been postulated to enable FS to modulate activin of autocrine origin and also may provide a barrier to access by exogenous activin from the circulation (11).
Architecturally, FS is a typical mosaic protein derived from exon shuffling. After a 63-residue N-terminal segment, the majority of the molecule consists of three successive 10-cysteine, 73- to 77-residue FS domains (FSD 1, 2, and 3) (12). At the C terminus, alternative splicing and/or posttranslational processing generate FS isoforms of 288, 300–303, and 315 residues in length.
The binding determinants for heparin have been localized to a highly basic 12-residue segment (residues 75–86) in the first FS domain (Fig. 1); natural and synthetic peptides including this sequence bind to both sulfate-cellulose and heparin-Sepharose affinity columns (10). The sequence includes two sets of repeating lysine or arginine residues in a pattern (BBXB) proposed as a consensus sequence for heparin binding in a number of proteins (7, 13, 14). Recently, the crystal structure of FS domain I in association with small heparin analogs has been completed (15), showing in more detail the contacts involved in this interaction.
FIG. 1. Primary structure of FS showing the three FSDs aligned at their cysteine residues, preceded by 63-residue N-terminal domain (FSND) and followed by the C-terminal domain (FSCD) present in the extended FS-315 isoform. The heparin-binding sequence (residues 75–86) in FSD-1 is underlined. The FS domain sequences are divided into their N-terminal (EGF-like) and C-terminal (Kazal-like) subdomains. The two FS domains and C-terminal extension of FSTL3 are also shown.
FS-like 3 (FSTL3), a closely related homolog of FS also known as FS-related gene product or FS-related protein, has been also localized to a variety of tissues and cell types (16, 17, 18, 19, 20). FSTL3 resembles FS in structure, but lacks a third FS domain and, importantly, is devoid of an obvious heparin-binding sequence or other means for binding to cell surfaces. As a consequence, although it binds exogenously supplied activin with an affinity approaching that of FS, its ability to suppress FSH secretion in response to endogenously produced activin in cultured pituitary cells is markedly diminished compared with FS (19).
Structure-activity studies to date have identified the importance in FS-activin interaction of the N-terminal domain (21, 22) as well as of C-terminal structural elements in the first two FS domains (23). Several earlier reports have suggested that the heparin-binding sequence itself may also play a role in activin binding. However, these findings have depended extensively upon the FS isoform and the assay system employed (2, 21, 24, 25, 26). In the only study directly implicating the heparin-binding sequence in activin interaction (25), binding by FS-288 to an activin-coupled affinity column was diminished after mutation of single lysines at position 75 or 82.
The mutational analyses of FS-288 described here use domain and sequence exchanges and point mutations of key components of the heparin-binding region to define in greater detail the determinants for heparin binding in FS, as well as factors accounting for its absence in FSTL3. The results also provide additional new evidence for the importance of this region in activin binding and, in conjunction with cell surface association, in determining the downstream biological effects of FS.
Materials and Methods
Reagents
Pure recombinant human FS-288 was obtained courtesy of the National Hormone and Pituitary Project, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Health (Bethesda, MD). Partially purified FS for coating of plates in the binding assays was prepared by affinity chromatography of expressed FS-288 on a solid support containing polyclonal anti-FS antibody 7FS30 (27). Recombinant human activin A for iodination was purchased from R&D Systems (Minneapolis, MN).
Preparation of FS/FSTL3 mutants
Coding sequences of FS-288 (a gift of Dr. S. Shimasaki, University of California at San Diego, La Jolla, CA) and FSTL3 (Millennium Pharmaceuticals, Cambridge, MA) were subcloned in frame into the mammalian expression vector pcDNA3.1/myc-His (Invitrogen, Carlsbad, CA). The resulting construct (pFS288mycH and pFSTL3mycH) was then used as a template for mutagenesis (22, 23). Chimeras of FS and FSTL3 with exchanges of domain I or substituted heparin-binding sequences were generated by PCR amplification of the appropriate sequence using extended primers containing sequence homologues to the desired neighbor region at their 5' end. These overlapping sequences were then fused by a second PCR and cloned back into the expression vector. Site-directed mutagenesis of individual residues within FS domain 1 was carried out as described (22) using the QuikChange kit (Stratagene, La Jolla, CA). Mutant sequences were verified by bidirectional sequencing at the DNA sequencing core facility of Massachusetts General Hospital.
Expression of recombinant proteins
The pFS288 or FSTL3mycHis vectors were transfected into HEK-293-F cell suspension cultures in Freestyle serum-free medium (Invitrogen Life Technologies) as described (23). Secretion, typically at levels of 10–20 μg/ml, was monitored by solid-phase immunochemiluminescent assay (SPICA) for FS and/or immunoassay for C-terminal myc tag for FS or FSTL3 as noted below. The recombinant FS was isolated from medium by nickel-Sepharose affinity chromatography (QIAGEN, Valencia CA) via the C-terminal poly-His tag. After stepwise elution with imidazole, products appearing between 50 and 300 mM were concentrated and exchanged into Dulbecco’s PBS by filter centrifugation. Conditioned medium from nontransfected cells was processed similarly for use as a control preparation in all assays.
Quantitation of secreted proteins
FS concentrations in media and concentrated affinity eluates were established by two independent immunological assays: 1) a two-site SPICA (27, 28) and 2) a solution-phase assay directed toward the C-terminal myc tag as detailed in ref (23). A synthetic peptide incorporating the myc epitope linked by a poly-Gly spacer to an N-terminal tyrosine for (125I) labeling, was used as radioligand and reference standard. The concentrations obtained by the two methods were in agreement for all FS mutants studied, assuring that the mutations did not disrupt quantitation. FSTL3 preparations were quantified by the myc RIA as described above, and concentrations were verified by reduced Western blotting against myc-tagged wild-type FS, using the anti-myc antibody (clone 4A6, Upstate Biologicals, Lake Placid, NY) for detection.
Binding to cell surface heparan-sulfate proteoglycans
COS cells were plated in DMEM supplemented with 10% fetal bovine serum (Invitrogen) into 24-well trays and cultured to confluence. FS or FSTL3 preps were added at 50 ng/ml for 2 h at 25C in fresh medium containing 0.1% BSA, washed, and replaced with medium containing 75,000 cpm 125I-activin A alone, or together with 10 μg/ml heparan sulfate (Sigma, St. Louis, MO). After 1 h incubation at 25 C, cells were rinsed, harvested, and counted to detect FS-bound activin as previously described (19).
Heparin affinity chromatography
Partially purified FS preparations (1 ml, 500-1000 ng) were applied to columns of heparin-agarose gel (Affi-Gel, Bio-Rad, Hercules, CA; 0.3-ml bed volume) in 0.01 M phosphate/0.15 M NaCl (pH 7.2) in presence of 0.1% BSA. Columns were washed (0.5 ml) then eluted with 1.5 ml of the same phosphate/BSA buffer. Aliquots of applied and eluted FS were measured by SPICA and/or myc-tag assay to determine the proportion retained on the column.
Activin binding assay
Binding of expressed FS to labeled activin was determined by competition assay as described (2). Mutant or wild-type FS were incubated with (125I)-labeled activin in binding buffer (10 mM PBS, 0.1% gelatin, 0.05% Tween; 200 μl) for 2 h at 20 C, then added to 96-well plates (Immulon-2; Dynatech Laboratories, Chantilly, VA) coated with 25 ng of affinity-purified FS288. After incubation at 20 C for 90 min, wells were washed and counted in a -counter. Each mutant preparation was assayed in at least three independent experiments. Relative potencies were calculated by comparison of half-maximal inhibition of labeled activin binding by mutant FS or FSTL3 with that of wild-type FS.
Bioassay for pituitary FSH secretion
Assay for suppression of basal FSH secretion in cultured rat anterior pituitary cells was based on the method of Scott et al. (29) and described in detail in Ref. 22 . Dispersed cells were incubated at 37 C in 95% air/5% CO2 for 72 h and reincubated in 0.5 ml fresh media containing the various FS and control preparations at the specified concentrations. After 72 h, the conditioned media were assayed for rat FSH using reagents and protocols provided by Dr. A. F. Parlow through the National Hormone and Pituitary Program, NIDDK. Potencies were determined using four-parameter logistic fit (NIHRIA) (2).
Results
Replacement of heparin-binding sequence
Because each of the FS domains is an autonomous folding unit with exclusively internal disulfides, exchanges of whole domains or domain segments can be carried out with normal expression and secretion (23). Two mutants of full-length FS-288 were used to study the effects of outright replacement of the FSD-1 (75–86) heparin-binding sequence (HBS): 1) substitution by the corresponding segment from FS-domain 2 (residues 148–159) that lacks any heparin-binding capability (designated FS/HBS) and 2) replacement of the entire FS domain 1 by domain 1 from FSTL3 (designated FS/FSTL-D1).
Neither mutant bound to the surface of a plated COS cell monolayer, as found with FSTL3 itself and in contrast to the strong binding observed for full-length, wild-type FS-288 (Fig. 2). This would be expected from loss of the heparin-binding region. The effects on activin binding were unanticipated, however; both mutants bound activin with 4- to 8-fold greater affinity than wild-type FS-288 (Fig. 3A; Table 1), with qualitatively different inhibition curves, suggesting distortion of a multicomponent contact surface. Suppression of pituitary cell FSH secretion was also observed (Fig. 3B), consistent with impaired ability to bind pituitary cell surfaces and inhibit endogenous activin secretion. The mutant responses in this assay are still considerably greater than that of FSTL3 (19), in keeping with their inherently higher activin binding.
FIG. 2. Cell surface binding by wild-type FS and FSTL3 and their heparin-binding sequence chimeras and mutants, determined by incubation of FS preparations with cultured HEK-283 cells followed by binding with (125I)-labeled activin-A. Heparan-sulfate-dependent surface binding of each FS preparation (n = 3–7 determinations) is expressed as the ratio of total activin bound to activin bound in presence of heparin (nonspecific binding). Starred preparations bound significantly higher than basal (no added FS) (P < 0.01 by standard t test).
FIG. 3. A, Inhibition of labeled activin binding by chimera replacing 75–86 heparin-binding sequence with corresponding segment from FSD-2. Binding potency is 4- to 8-fold greater than unmodified wild-type FS-288 as estimated from a qualitatively different dose-dilution curve. B, Pituitary cell FSH suppression by the 75–86 chimera. Compared with its binding activity, the bioactivity of the mutant is diminished due to loss of ability to bind the cell surface and suppress endogenous activin. Similar curves were obtained using chimera replacing the full FSD-1 with domain 1 from FSTL3.
TABLE 1. Activin-binding activity of follistatin/FSTL3 domain 1 mutants
Mutations of lysine motifs within the heparin-binding sequence
The Lys/Arg-rich (75–86) sequence (KKCRMNKKNKPR; Fig. 1) contains important determinants for heparin binding (10, 15) and, potentially, activin-binding as well. Point mutations were targeted to the two basic motifs (BBXB) known to be associated with heparin-binding (7, 14, 30) to determine their relative importance to heparin interaction by FS. Because in each motif at least one of the first two basic residues are absent from FSTL3 (Fig. 1), whereas the third is conserved, we mutated the respective paired lysines 75/76 and 81/82 to alanine. As shown in Fig. 2, partial cell surface binding was retained by either mutant. This suggests that one or the other of these consensus clusters is sufficient for surface binding, although with reduced heparin-binding efficacy. After modification of both motifs, cell surface binding was eliminated (Lys-76,81,82-Ala; Fig. 2).
The paired-basic residue mutations differed in their effects on activin binding, as shown in Fig. 4A and Table 1. Alanine substitution for the Lys-75,76 doublet reduced activin binding by 5-fold, whereas the Lys-81,82 substitution resulted in a 2-fold enhancement. The respective mutants suppressed pituitary FSH secretion (Fig. 4B). The potency of the Lys-81,82 mutant relative to activin binding was somewhat reduced compared with its activin binding, attributable to its diminished cell surface binding, but was not significantly lower than wild-type FS-288. This represents additional evidence that each mutant retained sufficient surface-binding capability for suppression of endogenous activin.
FIG. 4. A, Activin binding inhibition by FS-288 preparations bearing alanine mutations at paired basic residues within the 75–86 heparin-binding sequence. B, Suppression of pituitary cell FSH secretion by the same mutants. The potency of the K-75,76A mutant was significantly lower (ID50 = 3.48 ± 0.86 nM; p < 0.05) than wild-type FS-288 (1.13 ± 0.20 nM), whereas the K-81,82A was not significantly different (2.11 ± 0.81 nM).
Mutation of Arg-86
Basic sequences including a cationic residue corresponding to Arg-86 in FS have also been implicated in heparin binding (7, 31), and this residue appears to be a contact site with small heparin analogs in the crystal structure of FSD-1 (15). After mutation of Arg-86 to Ala, heparin-binding activity was retained as measured by both cell surface association (Fig. 2) and by binding to a heparin affinity matrix (Fig. 5) as used in earlier studies of the heparin-binding region (10). Therefore, this position alone is not required for heparin binding.
FIG. 5. Heparin affinity chromatography of chimeras introducing FS heparin-binding sequence into FSTL3. Provision of full-length FSD-1 confers full binding (90–95% retention on resin) to FSTL3, similar to wild-type FS288 and its R86A mutant.
Introduction of heparin-binding regions into FSTL3
The apparent localization of heparin-binding determinants to a single region within FS-domain 1 raised the possibility that capability for heparin interaction might be readily conferred upon FSTL3. Hence, the (75–86) heparin-binding sequence from FS was substituted into the corresponding site in the 218-residue FSTL3 molecule analogous to FS-288. The expressed protein (FSTL3/HBS) did not bind to cell surfaces, and binding to heparin affinity matrix was also low (Fig. 5). It bound activin with an affinity 50% that of wild-type FS-288 (Table 1) but was inactive in the pituitary cell bioassay (Fig. 6), consistent with its lack of cell surface binding. Similar results were obtained after substitution into the C-terminal-extended 256-residue FSTL3 molecule comparable with FS-315 (data not shown).
FIG. 6. Pituitary cell FSH suppression by FSTL3 with substituted heparin-binding sequence (75–86) from FS (FSTL3/HBS). The mutant potency was less than 1% that of wild-type FS; the somewhat higher potency of FSTL3 itself may reflect the inherently lower binding activity of the mutant preparation.
We therefore replaced the full domain 1 sequence in FSTL3 with FSD-1 from FS (designated FSTL3/FSD1). The protein was expressed normally but did not bind activin (Table 1). Although this precluded direct measurement of cell surface association using labeled activin, binding to heparin-Sepharose affinity matrix was comparable to that observed for wild-type FS-288 (Fig. 5). This implies the involvement in heparin binding of residues in FSD-1 outside the (75–86) sequence, available in FS but absent from FSTL3 unless provided by substitution of the full-length domain from FS itself.
Discussion
The biological activity of FS is ultimately dependent upon two binding processes: interaction with activin ligand and association with cell surface heparan-sulfate proteoglycans. Our results emphasize how both are influenced strongly by amino acids in and around the classical heparin-binding sequence, residues 75–86 in the N-terminal (EGF-like) subdomain of FSD-1, originally identified by Inouye et al. (10). The importance of this sequence is reiterated at the outset of these studies by the loss of cell surface binding by FS-288 after replacement by the corresponding 12-residue segment from FS-domain 2 or by the full-length domain 1 from the FS homolog, FSTL3. Despite this, our inability to confer cell surface binding by introduction of the (75–86) sequence into FSTL3 indicates that the (75–86) region is necessary but not sufficient for heparin interaction; other sites in the FS sequence must also be required.
Heparin-binding determinants have been studied in a variety of proteins functionally reliant upon binding to cell surfaces or extracellular matrix. A number of these sites involve conformational determinants, in which individual lysines distant in the linear sequence are oriented appropriately by folding (13, 32, 33). Nonetheless, in FS the linear, contiguous nature of the polybasic heparin-binding motifs is apparent from the previously reported ability of reduced, carboxymethylated FS to bind heparin (10). This would be consistent also with the highly flexible, surface-exposed nature of the N-terminal subdomain, as revealed by the recently published crystal structure of FSD-1 (15).
Within the 75–86 heparin-binding sequence itself, the clusters of lysine/arginine residues are characteristic of heparin-binding sequences in many proteins (7). These include the BBXB motif (B = Lys or Arg) represented twice in FS by residues 75–78 and 81–84. The mutation of either Lys-75,76 or Lys-81,82 each reduced, but did not abolish, cell surface binding, which was eliminated only when both motifs were modified, as in the Lys-75,81,82-Ala mutant. This is consistent with an additive role for each motif, but also—because either of the paired-lysine mutants could still suppress pituitary FSH secretion—may imply a redundancy in heparin-binding determinants that underscores the importance of surface binding to the physiological actions of FS.
Another basic residue, Arg-86, was found to be among the prominent contact points for the small heparan-sulfate analogs, sucrose octasulfate, or D-myo-inositol hexasulfate, used in the crystal structure of ligand-bound FSD-1 (15). However, the R86A mutant bound to both cell surfaces and to a heparin-agarose affinity column, and Arg-86 is also conserved in FSTL3, which does not bind heparin. Therefore, if this position participates in heparin binding, it must do so in conjunction with other residues in the sequence.
A useful comparison of heparan-sulfate and ligand/ receptor binding sites is found among the chemokines, many of which contain BBXB or similar polybasic clusters. RANTES (regulated on activation normal T cell expressed and secreted), for example, contains two motifs, closely spaced as in FS (14). In contrast to FS, mutation of the basic residues in the first sequence (RKNR; residues 44–47) alone is sufficient to eliminate heparin binding, whereas mutation of the second (KKWVR; residues 55–59) has no effect (14). The 44–47 sequence also overlaps the binding site for the chemokine receptor CCR1, although in this case, the presence of heparin suppresses receptor interaction.
Because insertion of the 75–86 sequence into FSTL3 failed to restore cell surface or heparin affinity binding, other sites, available only in the context of the FS sequence, must also contribute to heparin interaction. The crystal structure of FSD-1 in presence of the small heparan sulfate analogs noted above (15) affirms the primary importance of the sequence, but several additional contacts were also observed within the C-terminal Kazal-like subdomain, beyond the 75–86 sequence. The influence of the C-terminal subdomain on heparin binding is likewise shown by our own findings in which mutations affecting hydrophobic residues within this region impair heparin binding, most likely through disruptive effects on overall domain conformation (23).
The role of the heparin binding sequence itself in activin binding and bioactivity has been a matter of some debate, depending in part upon the FS isoform and assay system employed. Assays of activin binding by the different isoforms (FS-288, FS-303, and FS-315) for the most part show similar affinities (2, 24), although one report shows FS-288 to inhibit activin binding and transcriptional activity more potently than FS-315 (34). Assays dependent upon binding to pituitary cell surfaces to suppress endogenous FSH consistently show the C-terminal extended forms to be less active (21, 24, 25, 26). This has been attributed to interference with heparin-binding determinants by the acidic C-terminal extension in FS-303 and FS-315 (10, 25, 26).
A direct role for components of the heparin-binding region in activin binding was first reported by Sumitomo et al. (25). FS-288 could be eluted from an activin-coupled affinity column under milder conditions (4 M urea instead of 2 M guanidine-HCl) after mutation of either Lys-75 or Lys-82 to alanine. Pituitary cell FSH suppression was reduced 2-fold, although—as with our own Lys-75,76 and Lys-81,82 mutants—this could have been a result of impaired heparin binding rather than a direct effect on activin binding. However, little information has been added subsequently until the current studies, which have benefited also from structure-function comparisons with FSTL3 (17, 18, 19, 35) and the availability of the FSD-1 crystal structure (15).
Using the more quantitative measurement of competitive activin binding, the present results showing significantly diminished binding activity of the Lys-75,76 mutant, together with the affinity-enhancing effects of substitution for the full (75–86) or FSD-1 sequences, clearly point toward involvement of this region in FS-activin interaction. Our earlier results (22) provide evidence for the role of hydrophobic interactions in activin binding, identifying two essential tryptophan residues, at positions 4 and 36 within the N-terminal domain of FS. Because lysine can act as a hydrophobic residue through its methylene side-chain, one or more of these may contribute additional hydrophobic contacts. Alternatively, the terminal amino groups could add a charged component to the binding process as surmised previously (25).
The ability of the same region to bind separate ligands, as suggested by these data, may seem contradictory. There is ample evidence, however, that FS can bind activin and heparin or cell surface heparan sulfate simultaneously (25, 26, 36, 37). In one report (37), activin actually enhanced binding of heparin to both FS-288 and FS-315. It is possible that different portions of a given amino acid can be contacted by the respective ligands: activin to a hydrophobic side-chain, for example, and heparan sulfate to a charged group, or to a backbone amino group as found for least one contact in the crystal structure (15). This configuration may be facilitated by dimerization of FS if predictions of a 2:1 molar complex with activin prove accurate (38). We also cannot rule out the possibility that one or more of these residues interact with other regions in FS to modify the shape or conformation of activin-binding components elsewhere in the molecule. These issues will ultimately need to be resolved by a crystal structure of the FS-activin complex.
The biological importance of heparin binding in distinguishing FS from FSTL3 was affirmed by the effects of inserting the 75–86 sequence into FSTL3. Despite retaining its ability to bind exogenous activin, the FSTL3/HBS mutant was unable to suppress FSH secretion in response to endogenous activin in cultured pituitary cells, consistent with its lack of cell surface binding. We were able to confer heparin binding by provision of the full FS-domain 1 from FS, but this introduced other changes that eliminated the ability of the FSTL3 protein to bind activin. Taken together, these observations may exemplify an evolutionary safeguard to prevent acquisition of surface binding by FSTL3. Finally, FS itself appears to have gained cell surface binding at some cost in activin binding affinity, based on the enhanced activin binding observed after replacement of the heparin-binding region or substitution of domain 1 from FSTL3.
In summary, the local action of FS in regulating pituitary FSH secretion and many other endocrine and developmental functions is a vital consequence of the heparan-sulfate and activin-binding capability conferred by sequences in FS-domain 1. The physiological actions of FSTL3, on the other hand, are far less defined, but its inability to bind cell surfaces (even when provided with the 75–86 sequence from FS) undoubtedly confer biological properties differing significantly from those of FS. FSTL3’s distinctive nuclear localization within cells (39), for example, is not likely to involve heparin binding. The involvement of FSTL3 in suppressing the activin homolog myostatin has been described (20); that may call for a broader distribution best served by a protein free in solution. The continued parallel investigation of structure, cell biology, and ligand interactions will be essential to efforts to distinguish the functional role of these two interesting regulatory proteins.
Acknowledgments
We acknowledge with gratitude Leslie Johnson, Amy Schoen, and Alicia Zaske for their expert technical assistance in the cell cultures and assays used in these studies, Ashok Khatri of the Massachusetts General Hospital Endocrine Unit/AIDS Center Biopolymer Core facility for preparation of synthetic peptide standards and ligands for our RIAs, and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, directed by Dr. A. F. Parlow, for preparing and providing the reagents for the rat FSH RIA.
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Address all correspondence and requests for reprints to: Henry T. Keutmann, Endocrine Unit, Wellman 501, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Keutmann@helix.mgh.harvard.edu.
Abstract
Local regulation of pituitary FSH secretion and many other cellular processes by follistatin (FS) can be ascribed to its potent ability to bind and bioneutralize activin, in conjunction with binding to cell surface heparan-sulfate proteoglycans through a basic heparin-binding sequence (HBS; residues 75–86) in the first of the three FS domains. The FS homolog, FSTL3, also binds activin, but lacks any HBS and cannot associate with cell surfaces. We have used mutational analyses to define the determinants for heparin binding and activin interaction in FS and to determine the effects of conferring heparin binding to FSTL3. Mutants expressed from 283F cells were tested for cell surface and heparin affinity binding, for competititive activin binding and for bioactivity by suppression of pituitary cell FSH secretion. Replacement of the HBS or the full-length FS-domain 1 abolished cell surface binding but enhanced activin binding 4- to 8-fold. Surface binding was partially reduced after mutation of either lysine pair 75/76 or 81/82 and eliminated after mutation of both pairs. The 75/76 mutation reduced activin binding and, therefore, pituitary cell bioactivity by 5-fold. However, insertion of the HBS into FSTL3 did not restore heparin binding or pituitary-cell bioactivity. These results show that 1) the residues within the HBS are necessary but not sufficient for heparin binding, and 2) the HBS also harbors determinants for activin binding. Introduction of the full domain from FS conferred heparin binding to FSTL3, but activin binding was abolished. This implies an evolutionary safeguard against surface binding by FSTL3, supporting other evidence for physiological differences between FS and FSTL3.
Introduction
THE ABILITY OF FOLLISTATIN (FS) to regulate pituitary FSH secretion can be ascribed to its potent ability to bind and bioneutralize activin (1). Extensive evidence has affirmed that, rather than presenting activin to its receptor, FS sequesters activin nearly irreversibly to prevent stimulation of FSH secretion (2, 3, 4, 5). Although present in the circulation, colocalization of FS and activin within the pituitary, ovary, and many other tissues have drawn attention to its role as a local regulator of activin action in a wide range of processes influencing cellular secretion, development and differentiation (see Ref. 6 for recent review).
FS’s local actions have been widely attributed to its interaction with cell surface proteoglycans, in common with many extracellular proteins and growth factors (7). FS binding to heparin and heparin-like homologs on either synthetic solid supports or natural cell surfaces was recognized at an early stage (8, 9, 10). This capability has been postulated to enable FS to modulate activin of autocrine origin and also may provide a barrier to access by exogenous activin from the circulation (11).
Architecturally, FS is a typical mosaic protein derived from exon shuffling. After a 63-residue N-terminal segment, the majority of the molecule consists of three successive 10-cysteine, 73- to 77-residue FS domains (FSD 1, 2, and 3) (12). At the C terminus, alternative splicing and/or posttranslational processing generate FS isoforms of 288, 300–303, and 315 residues in length.
The binding determinants for heparin have been localized to a highly basic 12-residue segment (residues 75–86) in the first FS domain (Fig. 1); natural and synthetic peptides including this sequence bind to both sulfate-cellulose and heparin-Sepharose affinity columns (10). The sequence includes two sets of repeating lysine or arginine residues in a pattern (BBXB) proposed as a consensus sequence for heparin binding in a number of proteins (7, 13, 14). Recently, the crystal structure of FS domain I in association with small heparin analogs has been completed (15), showing in more detail the contacts involved in this interaction.
FIG. 1. Primary structure of FS showing the three FSDs aligned at their cysteine residues, preceded by 63-residue N-terminal domain (FSND) and followed by the C-terminal domain (FSCD) present in the extended FS-315 isoform. The heparin-binding sequence (residues 75–86) in FSD-1 is underlined. The FS domain sequences are divided into their N-terminal (EGF-like) and C-terminal (Kazal-like) subdomains. The two FS domains and C-terminal extension of FSTL3 are also shown.
FS-like 3 (FSTL3), a closely related homolog of FS also known as FS-related gene product or FS-related protein, has been also localized to a variety of tissues and cell types (16, 17, 18, 19, 20). FSTL3 resembles FS in structure, but lacks a third FS domain and, importantly, is devoid of an obvious heparin-binding sequence or other means for binding to cell surfaces. As a consequence, although it binds exogenously supplied activin with an affinity approaching that of FS, its ability to suppress FSH secretion in response to endogenously produced activin in cultured pituitary cells is markedly diminished compared with FS (19).
Structure-activity studies to date have identified the importance in FS-activin interaction of the N-terminal domain (21, 22) as well as of C-terminal structural elements in the first two FS domains (23). Several earlier reports have suggested that the heparin-binding sequence itself may also play a role in activin binding. However, these findings have depended extensively upon the FS isoform and the assay system employed (2, 21, 24, 25, 26). In the only study directly implicating the heparin-binding sequence in activin interaction (25), binding by FS-288 to an activin-coupled affinity column was diminished after mutation of single lysines at position 75 or 82.
The mutational analyses of FS-288 described here use domain and sequence exchanges and point mutations of key components of the heparin-binding region to define in greater detail the determinants for heparin binding in FS, as well as factors accounting for its absence in FSTL3. The results also provide additional new evidence for the importance of this region in activin binding and, in conjunction with cell surface association, in determining the downstream biological effects of FS.
Materials and Methods
Reagents
Pure recombinant human FS-288 was obtained courtesy of the National Hormone and Pituitary Project, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Health (Bethesda, MD). Partially purified FS for coating of plates in the binding assays was prepared by affinity chromatography of expressed FS-288 on a solid support containing polyclonal anti-FS antibody 7FS30 (27). Recombinant human activin A for iodination was purchased from R&D Systems (Minneapolis, MN).
Preparation of FS/FSTL3 mutants
Coding sequences of FS-288 (a gift of Dr. S. Shimasaki, University of California at San Diego, La Jolla, CA) and FSTL3 (Millennium Pharmaceuticals, Cambridge, MA) were subcloned in frame into the mammalian expression vector pcDNA3.1/myc-His (Invitrogen, Carlsbad, CA). The resulting construct (pFS288mycH and pFSTL3mycH) was then used as a template for mutagenesis (22, 23). Chimeras of FS and FSTL3 with exchanges of domain I or substituted heparin-binding sequences were generated by PCR amplification of the appropriate sequence using extended primers containing sequence homologues to the desired neighbor region at their 5' end. These overlapping sequences were then fused by a second PCR and cloned back into the expression vector. Site-directed mutagenesis of individual residues within FS domain 1 was carried out as described (22) using the QuikChange kit (Stratagene, La Jolla, CA). Mutant sequences were verified by bidirectional sequencing at the DNA sequencing core facility of Massachusetts General Hospital.
Expression of recombinant proteins
The pFS288 or FSTL3mycHis vectors were transfected into HEK-293-F cell suspension cultures in Freestyle serum-free medium (Invitrogen Life Technologies) as described (23). Secretion, typically at levels of 10–20 μg/ml, was monitored by solid-phase immunochemiluminescent assay (SPICA) for FS and/or immunoassay for C-terminal myc tag for FS or FSTL3 as noted below. The recombinant FS was isolated from medium by nickel-Sepharose affinity chromatography (QIAGEN, Valencia CA) via the C-terminal poly-His tag. After stepwise elution with imidazole, products appearing between 50 and 300 mM were concentrated and exchanged into Dulbecco’s PBS by filter centrifugation. Conditioned medium from nontransfected cells was processed similarly for use as a control preparation in all assays.
Quantitation of secreted proteins
FS concentrations in media and concentrated affinity eluates were established by two independent immunological assays: 1) a two-site SPICA (27, 28) and 2) a solution-phase assay directed toward the C-terminal myc tag as detailed in ref (23). A synthetic peptide incorporating the myc epitope linked by a poly-Gly spacer to an N-terminal tyrosine for (125I) labeling, was used as radioligand and reference standard. The concentrations obtained by the two methods were in agreement for all FS mutants studied, assuring that the mutations did not disrupt quantitation. FSTL3 preparations were quantified by the myc RIA as described above, and concentrations were verified by reduced Western blotting against myc-tagged wild-type FS, using the anti-myc antibody (clone 4A6, Upstate Biologicals, Lake Placid, NY) for detection.
Binding to cell surface heparan-sulfate proteoglycans
COS cells were plated in DMEM supplemented with 10% fetal bovine serum (Invitrogen) into 24-well trays and cultured to confluence. FS or FSTL3 preps were added at 50 ng/ml for 2 h at 25C in fresh medium containing 0.1% BSA, washed, and replaced with medium containing 75,000 cpm 125I-activin A alone, or together with 10 μg/ml heparan sulfate (Sigma, St. Louis, MO). After 1 h incubation at 25 C, cells were rinsed, harvested, and counted to detect FS-bound activin as previously described (19).
Heparin affinity chromatography
Partially purified FS preparations (1 ml, 500-1000 ng) were applied to columns of heparin-agarose gel (Affi-Gel, Bio-Rad, Hercules, CA; 0.3-ml bed volume) in 0.01 M phosphate/0.15 M NaCl (pH 7.2) in presence of 0.1% BSA. Columns were washed (0.5 ml) then eluted with 1.5 ml of the same phosphate/BSA buffer. Aliquots of applied and eluted FS were measured by SPICA and/or myc-tag assay to determine the proportion retained on the column.
Activin binding assay
Binding of expressed FS to labeled activin was determined by competition assay as described (2). Mutant or wild-type FS were incubated with (125I)-labeled activin in binding buffer (10 mM PBS, 0.1% gelatin, 0.05% Tween; 200 μl) for 2 h at 20 C, then added to 96-well plates (Immulon-2; Dynatech Laboratories, Chantilly, VA) coated with 25 ng of affinity-purified FS288. After incubation at 20 C for 90 min, wells were washed and counted in a -counter. Each mutant preparation was assayed in at least three independent experiments. Relative potencies were calculated by comparison of half-maximal inhibition of labeled activin binding by mutant FS or FSTL3 with that of wild-type FS.
Bioassay for pituitary FSH secretion
Assay for suppression of basal FSH secretion in cultured rat anterior pituitary cells was based on the method of Scott et al. (29) and described in detail in Ref. 22 . Dispersed cells were incubated at 37 C in 95% air/5% CO2 for 72 h and reincubated in 0.5 ml fresh media containing the various FS and control preparations at the specified concentrations. After 72 h, the conditioned media were assayed for rat FSH using reagents and protocols provided by Dr. A. F. Parlow through the National Hormone and Pituitary Program, NIDDK. Potencies were determined using four-parameter logistic fit (NIHRIA) (2).
Results
Replacement of heparin-binding sequence
Because each of the FS domains is an autonomous folding unit with exclusively internal disulfides, exchanges of whole domains or domain segments can be carried out with normal expression and secretion (23). Two mutants of full-length FS-288 were used to study the effects of outright replacement of the FSD-1 (75–86) heparin-binding sequence (HBS): 1) substitution by the corresponding segment from FS-domain 2 (residues 148–159) that lacks any heparin-binding capability (designated FS/HBS) and 2) replacement of the entire FS domain 1 by domain 1 from FSTL3 (designated FS/FSTL-D1).
Neither mutant bound to the surface of a plated COS cell monolayer, as found with FSTL3 itself and in contrast to the strong binding observed for full-length, wild-type FS-288 (Fig. 2). This would be expected from loss of the heparin-binding region. The effects on activin binding were unanticipated, however; both mutants bound activin with 4- to 8-fold greater affinity than wild-type FS-288 (Fig. 3A; Table 1), with qualitatively different inhibition curves, suggesting distortion of a multicomponent contact surface. Suppression of pituitary cell FSH secretion was also observed (Fig. 3B), consistent with impaired ability to bind pituitary cell surfaces and inhibit endogenous activin secretion. The mutant responses in this assay are still considerably greater than that of FSTL3 (19), in keeping with their inherently higher activin binding.
FIG. 2. Cell surface binding by wild-type FS and FSTL3 and their heparin-binding sequence chimeras and mutants, determined by incubation of FS preparations with cultured HEK-283 cells followed by binding with (125I)-labeled activin-A. Heparan-sulfate-dependent surface binding of each FS preparation (n = 3–7 determinations) is expressed as the ratio of total activin bound to activin bound in presence of heparin (nonspecific binding). Starred preparations bound significantly higher than basal (no added FS) (P < 0.01 by standard t test).
FIG. 3. A, Inhibition of labeled activin binding by chimera replacing 75–86 heparin-binding sequence with corresponding segment from FSD-2. Binding potency is 4- to 8-fold greater than unmodified wild-type FS-288 as estimated from a qualitatively different dose-dilution curve. B, Pituitary cell FSH suppression by the 75–86 chimera. Compared with its binding activity, the bioactivity of the mutant is diminished due to loss of ability to bind the cell surface and suppress endogenous activin. Similar curves were obtained using chimera replacing the full FSD-1 with domain 1 from FSTL3.
TABLE 1. Activin-binding activity of follistatin/FSTL3 domain 1 mutants
Mutations of lysine motifs within the heparin-binding sequence
The Lys/Arg-rich (75–86) sequence (KKCRMNKKNKPR; Fig. 1) contains important determinants for heparin binding (10, 15) and, potentially, activin-binding as well. Point mutations were targeted to the two basic motifs (BBXB) known to be associated with heparin-binding (7, 14, 30) to determine their relative importance to heparin interaction by FS. Because in each motif at least one of the first two basic residues are absent from FSTL3 (Fig. 1), whereas the third is conserved, we mutated the respective paired lysines 75/76 and 81/82 to alanine. As shown in Fig. 2, partial cell surface binding was retained by either mutant. This suggests that one or the other of these consensus clusters is sufficient for surface binding, although with reduced heparin-binding efficacy. After modification of both motifs, cell surface binding was eliminated (Lys-76,81,82-Ala; Fig. 2).
The paired-basic residue mutations differed in their effects on activin binding, as shown in Fig. 4A and Table 1. Alanine substitution for the Lys-75,76 doublet reduced activin binding by 5-fold, whereas the Lys-81,82 substitution resulted in a 2-fold enhancement. The respective mutants suppressed pituitary FSH secretion (Fig. 4B). The potency of the Lys-81,82 mutant relative to activin binding was somewhat reduced compared with its activin binding, attributable to its diminished cell surface binding, but was not significantly lower than wild-type FS-288. This represents additional evidence that each mutant retained sufficient surface-binding capability for suppression of endogenous activin.
FIG. 4. A, Activin binding inhibition by FS-288 preparations bearing alanine mutations at paired basic residues within the 75–86 heparin-binding sequence. B, Suppression of pituitary cell FSH secretion by the same mutants. The potency of the K-75,76A mutant was significantly lower (ID50 = 3.48 ± 0.86 nM; p < 0.05) than wild-type FS-288 (1.13 ± 0.20 nM), whereas the K-81,82A was not significantly different (2.11 ± 0.81 nM).
Mutation of Arg-86
Basic sequences including a cationic residue corresponding to Arg-86 in FS have also been implicated in heparin binding (7, 31), and this residue appears to be a contact site with small heparin analogs in the crystal structure of FSD-1 (15). After mutation of Arg-86 to Ala, heparin-binding activity was retained as measured by both cell surface association (Fig. 2) and by binding to a heparin affinity matrix (Fig. 5) as used in earlier studies of the heparin-binding region (10). Therefore, this position alone is not required for heparin binding.
FIG. 5. Heparin affinity chromatography of chimeras introducing FS heparin-binding sequence into FSTL3. Provision of full-length FSD-1 confers full binding (90–95% retention on resin) to FSTL3, similar to wild-type FS288 and its R86A mutant.
Introduction of heparin-binding regions into FSTL3
The apparent localization of heparin-binding determinants to a single region within FS-domain 1 raised the possibility that capability for heparin interaction might be readily conferred upon FSTL3. Hence, the (75–86) heparin-binding sequence from FS was substituted into the corresponding site in the 218-residue FSTL3 molecule analogous to FS-288. The expressed protein (FSTL3/HBS) did not bind to cell surfaces, and binding to heparin affinity matrix was also low (Fig. 5). It bound activin with an affinity 50% that of wild-type FS-288 (Table 1) but was inactive in the pituitary cell bioassay (Fig. 6), consistent with its lack of cell surface binding. Similar results were obtained after substitution into the C-terminal-extended 256-residue FSTL3 molecule comparable with FS-315 (data not shown).
FIG. 6. Pituitary cell FSH suppression by FSTL3 with substituted heparin-binding sequence (75–86) from FS (FSTL3/HBS). The mutant potency was less than 1% that of wild-type FS; the somewhat higher potency of FSTL3 itself may reflect the inherently lower binding activity of the mutant preparation.
We therefore replaced the full domain 1 sequence in FSTL3 with FSD-1 from FS (designated FSTL3/FSD1). The protein was expressed normally but did not bind activin (Table 1). Although this precluded direct measurement of cell surface association using labeled activin, binding to heparin-Sepharose affinity matrix was comparable to that observed for wild-type FS-288 (Fig. 5). This implies the involvement in heparin binding of residues in FSD-1 outside the (75–86) sequence, available in FS but absent from FSTL3 unless provided by substitution of the full-length domain from FS itself.
Discussion
The biological activity of FS is ultimately dependent upon two binding processes: interaction with activin ligand and association with cell surface heparan-sulfate proteoglycans. Our results emphasize how both are influenced strongly by amino acids in and around the classical heparin-binding sequence, residues 75–86 in the N-terminal (EGF-like) subdomain of FSD-1, originally identified by Inouye et al. (10). The importance of this sequence is reiterated at the outset of these studies by the loss of cell surface binding by FS-288 after replacement by the corresponding 12-residue segment from FS-domain 2 or by the full-length domain 1 from the FS homolog, FSTL3. Despite this, our inability to confer cell surface binding by introduction of the (75–86) sequence into FSTL3 indicates that the (75–86) region is necessary but not sufficient for heparin interaction; other sites in the FS sequence must also be required.
Heparin-binding determinants have been studied in a variety of proteins functionally reliant upon binding to cell surfaces or extracellular matrix. A number of these sites involve conformational determinants, in which individual lysines distant in the linear sequence are oriented appropriately by folding (13, 32, 33). Nonetheless, in FS the linear, contiguous nature of the polybasic heparin-binding motifs is apparent from the previously reported ability of reduced, carboxymethylated FS to bind heparin (10). This would be consistent also with the highly flexible, surface-exposed nature of the N-terminal subdomain, as revealed by the recently published crystal structure of FSD-1 (15).
Within the 75–86 heparin-binding sequence itself, the clusters of lysine/arginine residues are characteristic of heparin-binding sequences in many proteins (7). These include the BBXB motif (B = Lys or Arg) represented twice in FS by residues 75–78 and 81–84. The mutation of either Lys-75,76 or Lys-81,82 each reduced, but did not abolish, cell surface binding, which was eliminated only when both motifs were modified, as in the Lys-75,81,82-Ala mutant. This is consistent with an additive role for each motif, but also—because either of the paired-lysine mutants could still suppress pituitary FSH secretion—may imply a redundancy in heparin-binding determinants that underscores the importance of surface binding to the physiological actions of FS.
Another basic residue, Arg-86, was found to be among the prominent contact points for the small heparan-sulfate analogs, sucrose octasulfate, or D-myo-inositol hexasulfate, used in the crystal structure of ligand-bound FSD-1 (15). However, the R86A mutant bound to both cell surfaces and to a heparin-agarose affinity column, and Arg-86 is also conserved in FSTL3, which does not bind heparin. Therefore, if this position participates in heparin binding, it must do so in conjunction with other residues in the sequence.
A useful comparison of heparan-sulfate and ligand/ receptor binding sites is found among the chemokines, many of which contain BBXB or similar polybasic clusters. RANTES (regulated on activation normal T cell expressed and secreted), for example, contains two motifs, closely spaced as in FS (14). In contrast to FS, mutation of the basic residues in the first sequence (RKNR; residues 44–47) alone is sufficient to eliminate heparin binding, whereas mutation of the second (KKWVR; residues 55–59) has no effect (14). The 44–47 sequence also overlaps the binding site for the chemokine receptor CCR1, although in this case, the presence of heparin suppresses receptor interaction.
Because insertion of the 75–86 sequence into FSTL3 failed to restore cell surface or heparin affinity binding, other sites, available only in the context of the FS sequence, must also contribute to heparin interaction. The crystal structure of FSD-1 in presence of the small heparan sulfate analogs noted above (15) affirms the primary importance of the sequence, but several additional contacts were also observed within the C-terminal Kazal-like subdomain, beyond the 75–86 sequence. The influence of the C-terminal subdomain on heparin binding is likewise shown by our own findings in which mutations affecting hydrophobic residues within this region impair heparin binding, most likely through disruptive effects on overall domain conformation (23).
The role of the heparin binding sequence itself in activin binding and bioactivity has been a matter of some debate, depending in part upon the FS isoform and assay system employed. Assays of activin binding by the different isoforms (FS-288, FS-303, and FS-315) for the most part show similar affinities (2, 24), although one report shows FS-288 to inhibit activin binding and transcriptional activity more potently than FS-315 (34). Assays dependent upon binding to pituitary cell surfaces to suppress endogenous FSH consistently show the C-terminal extended forms to be less active (21, 24, 25, 26). This has been attributed to interference with heparin-binding determinants by the acidic C-terminal extension in FS-303 and FS-315 (10, 25, 26).
A direct role for components of the heparin-binding region in activin binding was first reported by Sumitomo et al. (25). FS-288 could be eluted from an activin-coupled affinity column under milder conditions (4 M urea instead of 2 M guanidine-HCl) after mutation of either Lys-75 or Lys-82 to alanine. Pituitary cell FSH suppression was reduced 2-fold, although—as with our own Lys-75,76 and Lys-81,82 mutants—this could have been a result of impaired heparin binding rather than a direct effect on activin binding. However, little information has been added subsequently until the current studies, which have benefited also from structure-function comparisons with FSTL3 (17, 18, 19, 35) and the availability of the FSD-1 crystal structure (15).
Using the more quantitative measurement of competitive activin binding, the present results showing significantly diminished binding activity of the Lys-75,76 mutant, together with the affinity-enhancing effects of substitution for the full (75–86) or FSD-1 sequences, clearly point toward involvement of this region in FS-activin interaction. Our earlier results (22) provide evidence for the role of hydrophobic interactions in activin binding, identifying two essential tryptophan residues, at positions 4 and 36 within the N-terminal domain of FS. Because lysine can act as a hydrophobic residue through its methylene side-chain, one or more of these may contribute additional hydrophobic contacts. Alternatively, the terminal amino groups could add a charged component to the binding process as surmised previously (25).
The ability of the same region to bind separate ligands, as suggested by these data, may seem contradictory. There is ample evidence, however, that FS can bind activin and heparin or cell surface heparan sulfate simultaneously (25, 26, 36, 37). In one report (37), activin actually enhanced binding of heparin to both FS-288 and FS-315. It is possible that different portions of a given amino acid can be contacted by the respective ligands: activin to a hydrophobic side-chain, for example, and heparan sulfate to a charged group, or to a backbone amino group as found for least one contact in the crystal structure (15). This configuration may be facilitated by dimerization of FS if predictions of a 2:1 molar complex with activin prove accurate (38). We also cannot rule out the possibility that one or more of these residues interact with other regions in FS to modify the shape or conformation of activin-binding components elsewhere in the molecule. These issues will ultimately need to be resolved by a crystal structure of the FS-activin complex.
The biological importance of heparin binding in distinguishing FS from FSTL3 was affirmed by the effects of inserting the 75–86 sequence into FSTL3. Despite retaining its ability to bind exogenous activin, the FSTL3/HBS mutant was unable to suppress FSH secretion in response to endogenous activin in cultured pituitary cells, consistent with its lack of cell surface binding. We were able to confer heparin binding by provision of the full FS-domain 1 from FS, but this introduced other changes that eliminated the ability of the FSTL3 protein to bind activin. Taken together, these observations may exemplify an evolutionary safeguard to prevent acquisition of surface binding by FSTL3. Finally, FS itself appears to have gained cell surface binding at some cost in activin binding affinity, based on the enhanced activin binding observed after replacement of the heparin-binding region or substitution of domain 1 from FSTL3.
In summary, the local action of FS in regulating pituitary FSH secretion and many other endocrine and developmental functions is a vital consequence of the heparan-sulfate and activin-binding capability conferred by sequences in FS-domain 1. The physiological actions of FSTL3, on the other hand, are far less defined, but its inability to bind cell surfaces (even when provided with the 75–86 sequence from FS) undoubtedly confer biological properties differing significantly from those of FS. FSTL3’s distinctive nuclear localization within cells (39), for example, is not likely to involve heparin binding. The involvement of FSTL3 in suppressing the activin homolog myostatin has been described (20); that may call for a broader distribution best served by a protein free in solution. The continued parallel investigation of structure, cell biology, and ligand interactions will be essential to efforts to distinguish the functional role of these two interesting regulatory proteins.
Acknowledgments
We acknowledge with gratitude Leslie Johnson, Amy Schoen, and Alicia Zaske for their expert technical assistance in the cell cultures and assays used in these studies, Ashok Khatri of the Massachusetts General Hospital Endocrine Unit/AIDS Center Biopolymer Core facility for preparation of synthetic peptide standards and ligands for our RIAs, and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, directed by Dr. A. F. Parlow, for preparing and providing the reagents for the rat FSH RIA.
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