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Reproductive Endocrine Unit and National Center for Infertility Research, Massachusetts General Hospital (D.V.T., Y.S., A.S.), Boston, Massachusetts 02144; Microbia, Inc. (D.A.H.), Cambridge, Massachusetts 02139; and Millennium Pharmaceuticals (W.E.H.), Cambridge, Massachusetts 02144
Address all correspondence and requests for reprints to: Dr. Drew V. Tortoriello, Columbia University College of Physicians and Surgeons, Division of Molecular Genetics, Ross Berrie Medical Sciences Pavilion, 1150 St. Nicholas Avenue, New York, New York 10032. E-mail: dvtortoriello{at}pol.net
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A domain structure for FS has been proposed based upon the discovery that its exons encode distinct amino acid sequences that demonstrate a high degree of evolutionary conservation (2). The most salient feature of this structure is a series of 3 consecutive FS domains. Although encoded by separate exons, each FS domain is distinguished by an identical alignment of 10 cysteine residues. The 315-amino acid splice variant of FS contains an acidic C-terminal domain that may impede adherence to cell surfaces by neutralizing the basic heparin binding motif present in the first FS domain (10). FS also has a hydrophobic signal domain that facilitates entry of the nascent protein into the endoplasmic reticulum and is proteolytically cleaved before secretion of the mature peptide. The functional importance of FS, however, probably resides in its N-terminal domain, which has recently been implicated as the site responsible for the majority of FS’s ability to bind activin, with 2 tryptophan residues at positions 4 and 36 being especially important in this capacity (12, 13).
A novel gene was recently cloned from a B cell leukemia line and was
named FLRG (FS-related gene) based upon primary sequence homology to FS
(14). Its five exons encode a signal domain, an N-terminal
domain, two cysteine-rich FS domains, and a C-terminal domain, yielding
an overall modular architecture remarkably similar to that of FS (Fig. 1
).
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As several extracellular matrix proteins, such as agrin, SPARC (secreted protein acidic and rich in cysteines), and testican (20, 21, 22), that are not known to bind TGFß superfamily ligands, have previously been termed products of follistatin-related genes based solely upon their possession of one or more FS domains, we refer to FLRG as FS-related protein (FSRP), to emphasize its uniquely high level of homology with regard to sequence, structure, and perhaps function.
We explored the hypothesis that FSRP serves a functionally redundant role with FS by first directly comparing their affinities and binding kinetics with activin and the closely related TGFß superfamily members BMP-6 and BMP-7. As the extent and site of a protein’s production as well as its subcellular destination can also be predictive of function, we investigated FSRP mRNA and protein biosynthesis in human tissues and cell lines. Our results indicate that FSRP, although clearly an activin-binding structural homolog of FS, has maximal expression in different tissues from FS, is located in the nucleus of all cells examined, and is only infrequently secreted. These findings suggest that FSRP may serve biological roles distinct from those of FS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ligand binding studies
Binding studies were conducted in 96-well microtiter plates that
were coated with 25 ng FSRP-Fc or FS288 in 100 µl 0.1 M
carbonate buffer (pH 9.6) and incubated overnight at 25 C on an orbital
shaker as previously described (4). Unbound protein was
removed by washing three times for 5 min each time with 200 µl wash
buffer (0.01% Tween in 10 mM PBS solution). Blocking
solution (200 µl; 3% BSA and 0.01% Tween in 10 mM PBS)
was then added to each well, and the trays were incubated at 25 C for
2 h on an orbital shaker. The wells were rinsed three times with
wash buffer, and 150 µl assay buffer (0.01 M PBS, 0.1%
gelatin, and 0.05% Tween) containing approximately 100,000 cpm
radiolabeled ligand were added to each well. Ligands were iodinated to
a specific activity of approximately 35 µCi/µg using
lactoperoxidase as previously described (23). Nonspecific
binding was determined by the addition of 100- to 500-fold excess
unlabeled ligand to some wells. This radioactivity was subtracted from
the radioligand only wells to give specific ligand binding. To
determine the association rate, the binding reactions were removed at
the indicated times, the wells were washed, and bound radioligand was
determined in a 
-counter. As steady state was reached by 2 h,
the dissociation rate was determined by removing unbound radioligand at
3 h, washing the wells, and then adding a 300-fold excess of
unlabeled ligand in 150 µl assay buffer. At the indicated times, the
binding reaction was removed, and the wells were washed and then
counted for bound radioactivity. The equilibrium association rate
constant (Ka) was determined from these kinetic experiments using
linearized binding data and least squares regression to determine
association and dissociation rates as previously described
(23), using the formula
Ka =
k1/k-1, where
k1 is the association rate, and
k-1 is the dissociation rate.
To estimate their relative direct binding of FS and FSRP, equal amounts (counts per min) of radiolabeled activin, BMP-7, or BMP-6 were added to FS- or FSRP-coated wells. Nonspecific binding was determined in the presence of a 100- to 500-fold excess of unlabeled ligand and was subtracted from the total to give specific binding, expressed as a percentage of the total counts added.
Northern analysis
For Northern analysis of FSRP expression in cell lines,
approximately 20 µg total RNA/cell line were denatured at 70 C in a
glyoxal/dimethylsulfoxide buffer (NorthernMax-Gly kit, Ambion, Inc., Austin, TX) and electrophoretically separated in a 1%
agarose gel at a constant 120 V. RNA was transferred to a positively
charged nylon membrane (BrightStar-Plus, Ambion, Inc.,
Austin, TX) using downward transfer for 2 h and microwave
cross-linked. The membrane was hybridized overnight to a 472-bp
radiolabeled double-stranded DNA human FSRP probe at approximately
5 x 106 cpm/ml in a roller bottle at 42 C.
After washing twice at 42 C, the membrane was exposed to film for
12 h at -80 C, then stripped by boiling in
diethylpyrocarbonate-water containing 0.1% SDS for 5 min and
subsequently rehybridized to double stranded DNA probes specific for
human FS (512 bp) or human ß-actin (213 bp).
The Northern probes were generated as follows. DNA template (100 ng)
and 5 µg random hexamers were boiled for 3 min, iced, and then added
to a reaction mix containing 2.5 µl 0.5 mM deoxy (d)-NTPs
(without dCTP), 2.5 µl 10 x Klenow fragment buffer, 5
µl 3000 Ci/mmol [
-32P]dCTP, and 1 µl
Klenow fragment (5 U). After incubating for 3 h at 25 C, the
reaction was stopped by the addition of 1 µl 0.5 M EDTA,
3 µl 10 mg/ml tRNA, and 100 µl TRIS-EDTA buffer. The unbound
radioactivity was removed by centrifuging the reaction mix through a
Sephadex G-50 column. The DNA probes were then denatured by incubating
with 1.5 ml 10 mM EDTA at 90 C for 10 min and mixed with
500 µl hybridization solution before adding them to the membrane for
overnight hybridization.
The oligomer used as template for the FSRP probe was generated by PCR of the cloned human FSRP-coding sequence. The forward FSRP primer spanned the first intron, and its sequence was CGCCCGGTGGTGTTTG. The reverse primer was located within exon 4, and its sequence was GCGCTGCCCGTCTGGTCC. FSRP cDNA was then amplified with 35 cycles at 94, 58, and 72 C and was purified using a PCR purification kit (Promega Corp., Madison, WI). The oligomers used for the FS and ß-actin probes were generated by performing PCR on human granulosa cell cDNA using previously described primer sets (24).
To compare the expression of FSRP and FS in human tissues, the DNA FSRP and FS probes were hybridized sequentially to a Human Multiple Tissue Expression Array (CLONTECH Laboratories, Inc., Palo Alto, CA), which contains poly(A)+ RNA from various human tissues spotted at equal densities, as calibrated by five different housekeeping genes. Autoradiograms were scanned using Adobe PhotoShop software (Abacus Concepts, Inc., Berkeley, CA), and the band densities were quantified using NIH Image software.
FSRP RIA
Iodinated pure recombinant human FSRP-FLAG was used as a
radioligand with our polyclonal FSRP antibody. In 300 µl, 25 ng
antibody in 1:400 normal rabbit serum, 50,000 cpm radioligand, and
FSRP-FLAG standard or unknown sample were added and incubated overnight
at 22 C. Goat antirabbit antiserum (1:16 in PBS) and 2.5% (final
concentration) polyethylene glycol were added for 2 h, after which
the tubes were centrifuged, aspirated, and counted in a -counter.
The minimum detectable dose was approximately 5 ng/ml, and the
intraassay coefficient of variation was 6.5%. All samples from a
single experiment were analyzed in a single assay.
Immunocytochemistry
Cells grown on coverslips in six-well trays were treated
sequentially with 4% paraformaldehyde, 0.1% Triton-X, and 0.2%
gelatin solution, and then incubated in PBS with polyclonal anti-FSRP
antibody (2.5 µg/ml) for 1 h at 22 C. Additional coverslips
containing LGC were also incubated with a specific monoclonal anti-FS
antibody (7FS30, 3 µg/ml) to compare the intracellular distributions
of FS and FSRP in the same primary cell type. The coverslips were then
placed into PBS containing 1:5000 FITC-conjugated donkey antirabbit or
donkey antimouse IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 22 C.
In a separate experiment CHO cells permanently transfected with FSRP-FLAG cDNA using Effectene (QIAGEN, Valencia, CA) were analyzed by immunocytochemistry with the anti-FSRP antibody or 10 µg/ml monoclonal anti-FLAG M2 primary antibody (Sigma, St. Louis, MO) and 1:5000 rhodamine-conjugated donkey antimouse IgG (Santa Cruz Biotechnology, Inc.). Coverslips were mounted over a droplet of Vectashield (Vector Laboratories, Inc., Burlingame, CA), and inspected and photographed using a Nikon epifluorescent microscope (Melville, NY).
Western blot of fractionated cell extracts
Fractions selectively enriched for cytoplasmic proteins, soluble
nuclear proteins, or insoluble nuclear proteins were extracted from CHO
FSRP-FLAG cells according to previously published methods
(25). Five micrograms of each fraction from the CHO
FSRP-FLAG cells and 20 µg wild-type CHO whole cell extract underwent
reducing (lithium dodecyl sulfate) PAGE in a 12% bis-Tris gel (NuPAGE
System, Novex, San Diego, CA) and were then transferred to
a polyvinylidene difluoride membrane. The primary antibody was either
the polyclonal anti-FSRP antibody (2 µg/ml) or the monoclonal
anti-FLAG M2 antibody (7.5 µg/ml). The secondary antibody was either
donkey antimouse or donkey antirabbit conjugated to horseradish
peroxidase (each 1:20,000).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Alignment of the N-terminal domains of FSRP and FS reveals 32%
sequence identity between FS and FSRP, including the six conserved
cysteines. The tryptophans (W) at positions 4 and 36, which have been
shown to be responsible for 95–99% of the activin-binding activity of
FS (13), are also conserved in FSRP. Their high degree of
structural similarity coupled with their shared abilities to bind
activin suggest that FSRP and FS comprise their own separate
follistatin subfamily.
Activin binding specificity and kinetics
As shown in Fig. 2A, specific
[125I]activin binding to both FSRP and FS
reached 90% of the maximum after 20 min and achieved a steady state by
2 h. The addition of a 100-fold excess of unlabeled activin after
3 h revealed similarly slow dissociation rates of approximately
5% after 2 h and 10–20% by 3 h. As it is possible that
some FSRP-activin complexes lifted off the plate during the 3-h
incubation, the actual dissociation rate may be even less than
depicted. As Scatchard analysis is not applicable to incompletely
reversible binding reactions, equilibrium constants of dissociation
were estimated by dividing the on rate by the off rate using linearized
data from Fig. 2A. The Kd for FS was 4.63 x
10-10, whereas for FSRP, a
constant of 1.13 x
10-9 was observed. Thus,
the affinity of FS for activin under these assay conditions was
approximately 2.4-fold greater than that of FSRP.
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depicts the relative direct binding of equal amounts (counts
per min) of radiolabeled ligand to solid phase FS or FSRP. Activin
binding to both FS and FSRP was more than 20-fold greater than BMP-7
and 40-fold greater than BMP-6. This suggests that both FS and FSRP
specifically bind activin, as their relative affinities for even the
structurally similar BMPs were quite low.
mRNA expression
FSRP and FS mRNA expression was quantified in a wide variety of
human adult and fetal tissues using a commercial array containing
normalized amounts of poly(A)+ RNA. As shown in
Fig. 3, both genes were expressed in
nearly all tissues, but the sites of maximal expression were largely
distinct. Testicular expression of both genes was quite high, so
densitometric analyses were normalized to this tissue. Other than in
the testis, however, FSRP expression was maximal in placenta, heart,
and pancreas, whereas FS expression was maximal in ovary, pituitary,
kidney, and fetal heart and liver. This largely nonoverlapping pattern
of maximal expression suggests that in at least some tissues these two
proteins do not serve redundant functions.
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). Both
FSRP and FS mRNA were also detected in primary human LGCs.
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depicts
the RIA dilution curve for medium conditioned by HeLa cells, which is
parallel to the standard curve, suggesting antibody specificity.
Neither FS288 nor FS315 (100 ng) cross-reacted in this assay. In
addition, preincubation of FSRP with excess human activin did not
affect FSRP measurements, demonstrating that the RIA is capable of
detecting both free and bound FSRP (data not shown).
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Immunocytochemistry
As FSRP mRNA was detected in all cells examined to date, but
secreted protein was detected only in the minority, we next assessed
FSRP protein production using immunocytochemistry with the same
FSRP-specific antibody as that used in the RIA. FSRP
immunoreactivity was detected in all cells examined. However, rather
than showing a cytoplasmic or endoplasmic reticular/Golgi pattern
commonly observed for secreted proteins such as FS, in all cases FSRP
immunoreactivity was predominantly confined to the nucleus (Fig. 6, A–C). Preincubation of the anti-FSRP
antibody with a 10-fold excess of recombinant FSRP-FLAG completely
abrogated antibody staining (Fig. 6D), indicating that this antibody is
specific for an epitope on FSRP. In primary LGCs, which produce both FS
and FSRP mRNA, FS immunoreactivity was detected exclusively in the
cytoplasm (Fig. 7A). In contrast, FSRP
immunoreactivity was again localized to the nucleus (Fig. 7B).
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) detected FSRP-FLAG within the
nucleus, whereas in untransfected cells, no immunoreactivity was
detected with either antibody (Fig. 8B).
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, lane 2). Untagged FSRP has
previously been identified as a 32-kDa protein by Western analysis
(1), which shifts to its expected Mr
of 27.6 kDa after deglycosylation. Therefore, our 34-kDa immunoreactive
band is appropriate for glycosylated FSRP possessing a 10-amino acid
FLAG tag. Cytoplasmic and soluble nuclear protein from CHO FSRP-FLAG
cells (Fig. 8C, lanes 1 and 3) and a 4-fold excess of whole cell
protein extract from untransfected CHO cells (lane 4) showed little or
no such immunoreactivity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As the relative affinities of FS and FSRP for activin as well FSRP’s activin binding kinetics were unknown, we investigated these parameters in our evaluation of their potential for functional redundancy. We found that activin binding to both FS and FSRP was rapid and nearly irreversible, but the affinity for activin was approximately 2.4-fold greater for FS than for FSRP, suggesting that FSRP might be slightly less potent than FS in binding and neutralizing activin. In addition, the preferential ligand for both FS and FSRP appears to be activin, which, in a direct binding assay, was able to bind both FS and FSRP with at least 20-fold greater magnitude than its closely related TGFß family counterparts, BMP-6 and BMP-7 (26).
Although both FSRP and FS mRNA are located in a wide and largely overlapping range of human adult and fetal tissues, their sites of peak expression are different, with FSRP expression being exceptionally high in the placenta, testis, and cardiovascular tissue, whereas FS was highest in the ovary and pituitary. Their relative gene expression was also different among fetal tissues and several human cell lines. These observations suggest that FSRP and FS are differentially regulated spatially and temporally.
Based upon identification of an N-terminal signal sequence and no known nuclear localization signals, FSRP was predicted to be a secretory glycoprotein, analogous to FS, by both PSORT II (27) and TargetP V1.0 (28). Indeed, FSRP has been detected by Western blotting in the conditioned medium of transfected COS-7 cells as well as certain tumoral cell lines (14). However, when we scrutinized several other cell lines that produced FSRP mRNA by Northern analysis, secreted FSRP was only detectable from confluent HeLa, JEG, and CHO cells permanently transfected with human FSRP-FLAG cDNA. In addition, follicular fluid aspirated from in vitro fertilization patients was negative for FSRP, a finding in stark contrast to FS, which is present at concentrations greater than 200 ng/ml in this fluid (29).
Several possible explanations exist for our failure to detect secreted FSRP in cells known to produce its mRNA. It is possible that certain cell lines secrete very low amounts of FSRP that are below our RIA’s 5 ng/ml limit of detection. In addition, FSRP may be proteolytically processed after secretion and thereby escape detection by our polyclonal FSRP antibody. Alternatively, human FSRP may only be secreted under conditions of exceptionally high expression. This explanation seems plausible given that FSRP was detected in medium conditioned by JEG and HeLa cells, which we noted to produce among the highest levels of FSRP mRNA by Northern analysis.
To determine whether FSRP protein was being synthesized in cells that were negative for secretion by our RIA, we performed immunocytochemistry. Our analyses clearly indicate that at least a portion of the synthesized FSRP is located within the nucleus or associated with the nuclear membrane. This is supported by our finding that FSRP-FLAG, as detected with both an anti-FSRP and an anti-FLAG antibody, is also localized to the nucleus. Western analyses of protein extracts from CHO FSRP-FLAG cells corroborate the immunocytochemical studies and further demonstrate that FSRP-FLAG is concentrated in the insoluble nuclear protein fraction, a fraction enriched for nuclear membrane-associated proteins. Importantly, the nuclear localization of FSRP-FLAG in transfected CHO cells demonstrates that the nuclear transport of human FSRP is a characteristic intrinsic to its amino acid sequence, which although very similar to FS in terms of overall domain structure, shares only approximately 40% identity with FS.
Unfortunately, our anti-FSRP antibody was unable to detect FSRP, nuclear or otherwise, on slides of fixed human tissues despite its ability to specifically recognize FSRP in immunocytochemistry, RIA, and Western blotting. We believe this is due to antigen masking, a not uncommon consequence of the fixing and processing of tissue for immunohistochemistry. It is worth emphasizing, however, that the nuclear localization of FSRP was seen prominently in monolayers of human granulosa cells, which are primary human cells in culture and not an immortalized cell line. The nuclear pattern of FSRP was also demonstrable in cultured nonluteinized granulosa cells (data not shown). As these cells were aspirated from early follicular phase antral follicles, they were never exposed to supraphysiological concentrations of gonadotropins in vivo and are therefore even further representative of normal human tissue.
Although it is possible that the nuclear localization of FSRP in cell lines is secondarily induced by in vitro culture conditions, we believe this to be improbable, as this was a universal finding in the cell lines screened, including both secretors and nonsecretors of FSRP. Moreover, we were able to detect nuclear FSRP in human granulosa cells that were in culture for only 48 h. Nonetheless, the finding that a protein so structurally similar to the secretory glycoprotein FS is targeted to the nucleus under any conditions is an intriguing discovery, suggesting a novel intracellular function and transport mechanism.
At this point, the precise pattern and regulation of FSRP’s intracellular trafficking pattern are unknown. Although other proteins have been observed to translocate from the endoplasmic reticulum to the cytoplasm, this process usually involves removal of incorrectly folded proteins (30), or results from alternate splicing and processing of the signal peptide (31). However, one possible explanation for our observations is that transport of FSRP out of the endoplasmic reticulum and into the nucleus requires binding of a chaperone whose concentration is limited. Thus, when FSRP is overexpressed, the chaperone becomes saturated, and excess FSRP gets secreted. Alternatively, as a functional nuclear localization signal has recently been identified in the activin ßA-subunit precursor (32), it is possible that FSRP may bind activin A en route to the nucleus. In fact, both ßA-subunit and an immunoreactive follistatin-like protein have been separately observed in the nuclei of rat spermatogenic cells using immunocytochemical approaches (32, 33).
In conclusion, despite their high degree of structural homology and mutual affinity for activin, additional evidence suggests that FSRP may not be functionally redundant to FS. Despite significant overlap, their sites of maximal mRNA expression are distinct in human tissues, and although FSRP clearly has a signal sequence and the capacity to be secreted, the majority of cell lines we tested secreted no detectable FSRP. FSRP was universally detectable, however, as a nuclear protein by immunocytochemistry and Western blotting. These intriguing findings suggest that FSRP may have unique intracellular functions distinct from those of FS.
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Footnotes |
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Abbreviations: BMP, Bone morphogenic protein; FLRG, FS-related gene; FSRP, follistatin-related protein; FS, follistatin; LGC, luteinized granulosa cells; SPARC, secreted protein acidic and rich in cysteines.
Received February 9, 2001.
Accepted for publication April 16, 2001.
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