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-Subunit Gene to the Follicle-Stimulating Hormone and Chorionic Gonadotropin-ß Subunit Genes: Production of a Bifunctional Protein
Departments of Molecular Biology and Pharmacology and Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri 63110
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit assembled to a
hormone-specific ß-subunit. The subunits combine noncovalently early
in the secretory pathway and exist as heterodimers but not as
multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and
several of the -subunit sequences required for association with
either the LHß or FSHß subunits are different. Thus, it is
intriguing that no ternary complexes are observed for LH and FSH
in vivo (e.g. two different ß-assembled to a
single -subunit). To examine whether the -subunit can interact
with more than one ß-subunit, and to study the conformational
relationships between the ligand and the receptor, we constructed a
vector encoding two tandemly arranged ß-subunits fused to a single
-subunit gene (FSHß-CGß-). This approach permitted
structure-function analyses of /ß domain complexes without the
possibility of subunit dissociation. We reported previously that the
CGß or FSHß subunit gene can be genetically fused to the -gene
and the resulting single chains (CGß and FSHß, respectively)
were biologically active. Here we demonstrate that a triple-domain
single chain bearing the configuration FSHß-CGß- is efficiently
secreted from transfected Chinese hamster ovary (CHO) cells and
exhibits high-affinity receptor binding to both FSH and LH/hCG
receptors, comparable to the native heterodimers. These results
indicate that the -subunit can interact with each ß-subunit in the
same complex and that an -domain fused to a ß- domain can still
interact with an additional ß-subunit. The data also demonstrate the
remarkable flexibility of the receptor to accommodate the increased
bulkiness of the triple-domain ligand. In addition, the formation of
intrachain FSH- and CG-like complexes observed in a triple-domain
single chain suggests that the -subunit can resonate,
i.e. shuttle between -ß heterodimeric intermediates
during the early stages of synthesis and accumulation in the
endoplasmic reticulum. Such model compounds could be useful as
substrates to generate a new class of analogs in which the ratio of the
LH/FSH activity is varied. This could aid in the design of analogs that
could be used to mimic the in vivo hormonal profiles. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit and a hormone-specific ß-subunit (1). The assembly of the
glycoprotein hormone subunits, which occurs in the endoplasmic
reticulum (ER) (2, 3, 4), is vital to the function of these hormones: The
conformation of the heterodimer is essential for controlling secretion,
hormone-specific posttranslational modifications, and signal
transduction. Although the -subunit is common to the four hormones,
it appears that the contact sites in the subunit for the gonadotropin
ß-subunits are not the same (5, 6). This raises the possibility that
an -subunit can interact intracellularly with more than one
ß-subunit at steady state. For example, both LH and FSH are
synthesized in the same cell and, presumably, the -subunit comingles
with a mixed population of ß- subunits. It is unclear then what
determines the fate of a newly synthesized -subunit that establishes
the physiological ratio of LH and FSH in the reproductive cycle; there
could be a compartmentalization of the nascent ER pools, or
alternatively, the -subunit can coinsert with a specific ß subunit
in the ER.
Because ternary complexes comprised of two ß-subunits and one
-subunit would likely be unstable–such molecules have not been
detected in vivo–we and others devised a strategy to
examine this problem using a single-chain gonadotropin model in which
the ß- and -subunits are genetically linked (7, 8, 9, 10). We showed
that these variants were secreted and displayed biological activity
comparable to the corresponding heterodimers (7, 9). A distinct
advantage for using this single-chain model is that it permits
structure-function analyses of ß/ domain complexes without the
possibility of subunit dissociation.
To assess the ability of the -subunit to interact with more than one
ß- subunit, we constructed a gene encoding two tandemly arranged
ß-subunits fused to a single -subunit gene. We report here that a
triple-domain single chain of the structure FSHß-CGß- generates
FSH and hCG heterodimeric-like epitopes. Moreover, this protein
exhibits high-affinity receptor binding to both hCG/LH and FSH
receptors. These results show that the -subunit can interact with
more than one ß-subunit within the same complex.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(7) and FSHß (9), were used as substrates.
In constructing the CGß single chain (double domain), the carboxy
terminus of CGß was fused directly to the N terminus of the
-subunit without an added spacer sequence (Fig. 1A
; Ref. 7). The CGß subunit (length
145 amino acids) is distinguished among the other ß-subunits by the
presence of a C-terminal extension with four serine-linked
oligosaccharides (CTP). This sequence is a suitable linker, since it is
serine/proline rich and thus lacks significant secondary structure, and
would provide sufficient distance between Cys-110 and the first
disulfide bond in the linked -subunit. The FSHß subunit (length
111 amino acids) contains a shorter C-terminal region that could result
in more interference between the cysteine 20–104 loop and the adjacent
-subunit sequences in the single chain. Thus, the CTP sequence was
used as a linker in constructing the FSHß single chain (double
domain) (Fig. 1B; Ref. 9). In the case of the triple-domain single
chains, the CTP sequence is inserted between the FSHß and the
adjacent subunit (Fig. 1, C and D).
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and
FSHß) or three (FSHß-CGß- and CGß-FSHß-) subunit
domains from stably transfected Chinese hamster ovary (CHO) cells was
examined by metabolic labeling with [35S] cysteine, and
the proteins were immunoprecipitated from cell lysate and medium with
polyclonal antiserum (Fig. 2). The
apparent molecular masses of the FSHß and CGß single
chains are 53 ± 0.4 kDa and 51 ± 0.1 kDa, respectively. It
is clear that both triple-domain variants, FSHß-CGß- and
CGß-FSHß-, are secreted (Fig. 2; lanes 5–8), and their apparent
molecular masses are 88 ± 0.6 kDa and 88 ± 0.9 kDa,
respectively, which correspond to the presence of an additional subunit
domain and CTP cassette. The shift in mobility observed between the
lysate/medium forms is due to the processing of Asn-linked
oligosaccharides and the addition of carbohydrates to serine acceptor
sites in the CTP (11). It is noteworthy that no smaller species
corresponding to a heterodimeric-like protein is detected.
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). As previously observed, the CGß
(7) and FSHß (9) single chains were secreted efficiently
(t1/2 = 90 min and t1/2 = 100 min,
respectively). The secretion of the mutants was considerable slower;
the t1/2 of CGß-FSHß- and FSHß-CGß- are 205
min and 325 min, respectively. While CGß and CGß-FSHß- were
quantitatively secreted (>80% recovery), only 30–50% of the
FSHß-CGß- chimera is recovered. These data show that the
orientation of the two ß-subunits affects secretion and recovery, and
for the FSHß-CGß- chain, a significant fraction was
degraded.
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). In addition to the triple-domain
single chains, we probed the double-domain single chains, CGß
and FSHß, as controls. As shown previously (7, 9), the ß- and
-domains in CGß (molecular mass = 51 ± 0.5 kDa) and
FSHß (molecular mass = 53 ± 0.5 kDa) retained the
majority of the heterodimeric interactions which was reflected by the
signals detected by dimer-specific mAbs B109 (CG) (panel A, lane 1) and
117 (FSH) (panel B, lane 2). As expected, no signals were detected for
FSHß and CGß in the CG and FSH immunoblots, respectively.
FSHß-CGß- was immunoreactive with both B109 (panel A, lane 3;
molecular mass = 83 ± 1.9 kDa) and 117 (panel B, lane 3;
molecular mass = 83 ± 1.3 kDa) antibodies. However, while no
detectable signal was seen when CGß-FSHß- was probed with mAb
B109 (panel A, lane 4), this mutant was reactive to mAb 117 (panel B,
lane 4; Mr = 83 ± 1.8 kDa). Together, these data
show that both the CGß and FSHß subunits form heterodimeric-like
interactions with the -subunit in the FSHß-CGß- mutant but
only the FSHß component is dimer configured in the CGß-FSHß-
variant. Similar to the results obtained with the reduced SDS-PAGE
(Fig. 2
), no significant accumulation of bands corresponding to a
heterodimer-related protein was seen. Slower migrating species were
detected on the blots (Fig. 4; Mr = 134 ± 6
kDa), and although the nature of these proteins is not clear, such
aggregates are observed during the purification of native hCG (12, 13).
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mutant was 25.3 ± 1.8 µg/ml and
19.4 ± 0.7 µg/ml, respectively. For the CGß-FSHß-
chimera, the FSH and hCG content was 22.5 ± 1.9 µg/ml and
15.1 ± 1.2 µg/ml, respectively. The ELISA contains
subunit-specific monoclonal antibodies; the FSH and hCG centers in the
FSHß-CGß- calculated by this assay were 31 µg/ml and 55
µg/ml, respectively. Although there is some difference in the
immunoreactivity of the FSH/hCG components as determined by these
methods (likely due to the use of different antibodies and standards),
the results obtained by two independent immunological assays are within
reasonable agreement. Moreover, the extent of binding of an equivalent
amount of the triple-domain chimera and the heterodimer, as determined
by ELISA, was measured by the BIACORE (BIACORE Products, Life Sciences,
Uppsala, Sweden). No significant difference in the extent or kinetics
of binding was observed when the reactivity of the triple domain was
compared with that of the corresponding heterodimers (data not shown).
This corroborates the reliability of the immunoassay determinations and
confirms the expected 1:1 molar ratio of the ß-subunits arranged in
the chimera. Taken together, these data verify that the
immunoreactivity of the chimera is similar to the corresponding
heterodimers.
Each chimera was tested independently for binding to the FSH and LH/hCG
receptors using transfected CHO cells expressing either the human
LH/hCG or human FSH receptors (Fig. 5 and
Table 1). The FSHß-CGß- mutant
displayed high-affinity binding to the LH/hCG receptors, although the
binding affinity was reduced 4- and 8-fold relative to the hCG
heterodimer and CGß, respectively. In the case of the FSH receptor
binding, there was a 4- to 5-fold decrease in binding by this triple
domain chimera compared with the FSH heterodimer and FSHß (Fig. 5, A and B, and Table 1). In contrast to these results, CGß-FSHß-
exhibited little binding to either the hLH/hCG or hFSH receptor (Fig. 5
and Table 1).
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, A and B, and Table 1) paralleled the
receptor binding affinity, there was some apparent uncoupling in
binding and cAMP activation.
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DISCUSSION |
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had a greater binding affinity than CGß-FSHß- to both the FSH
and hCG receptors, its secretion rate and recovery was less. These data
further support the hypothesis that for the glycoprotein hormone
family, the subunit-specific epitopes responsible for intracellular
behavior are uncoupled from those required for bioactivity. Regarding
the intracellular differences, since the -subunit is at the carboxyl
end in both chimeras, this apparently precludes a direct affect of this
domain in the secretory pathway. Previously we showed that the
uncombined FSHß subunit was secreted poorly whereas the free CGß
subunit was secreted quantitatively (14). Because the FSHß subunit
occupies the amino-terminal end of the FSHß-CGß- chimera, it may
behave similarly to the free FSHß subunit where exposure of epitopes
to one or more chaperones impedes secretion. By contrast, the
noncombined CGß subunit was secreted efficiently consistent with the
secretion of the mutant bearing CG at the amino terminal. These data
imply that the amino-terminal region of the ß-subunits influences the
intracellular behavior of the chimeras.
It is curious that in CGß-FSHß- chimera, only the FSHß domain
formed heterodimeric-like epitopes; no signals characteristic of a CG
dimer were detected with mAb B109. These data suggest that the FSHß
subunit is less flexible than the CGß subunit since the latter
permitted the amino-terminal FSHß domain to interact with the
-subunit in the FSHß-CGß- chimera. In the case of
CGß-FSHß-, only the adjacent FSHß subunit domain, but not the
CGß domain, interacted with the -subunit to establish a
dimeric-like association.
Given the increased size of the triple-domain structure, it was
surprising that the complexes were biologically active. Although
determinants from both - and ß-subunits are critical for
bioactivity, our previous studies suggested that ligands with different
conformations can also bind to the receptor (15, 16). In addition,
others have reported that ligand conformation can be altered during
binding (17, 18). The high-affinity binding of the triple-domain
complex supports this hypothesis. That the receptor productively
interacts with three subunit domain gonadotropins suggests that the
receptor has sufficient flexibility to accommodate a much larger
ligand.
Although the trimeric entity exhibits both hCG and FSH dimeric-like epitopes, we cannot exclude the possibility that the higher molecular mass forms also contributed to the observed bioactivity. However, we consider this unlikely since in the case of the double-domain single chain, there was no correlation with biological activity and the extent of aggregation in a series of tethered gonadotropin mutants (15, 16).
A critical question arising from these observations is whether
the FSHß-CGß- chimera possesses dual activity, i.e.
does a single form of this protein exhibit both FSH and CG activity? If
such a molecule is dually active, it implies that an -subunit can
serve two ß-subunits simultaneously and achieve a heterodimeric-like
configuration for each hormonal unit, as exemplified by
FSHß-CGß-. Consistent with this suggestion is the evidence for
different contact sites in the -subunit for the FSHß and CGß
subunits (5, 6). It is likely that such dual interactions would not be
stable but rather transitory heterodimeric complexes pro
tem. This would be analogous to a resonance interaction in which
the -subunit phases between both ß-subunits. However, we cannot
exclude the existence of at least two distinct stable species with
heterodimeric-like features in the chimeric population, CGß/ or
FSHß/, i.e. no dually active species. That both the hCG
and FSH bio- and immunoreactivities in the FSHß-CGß- chimera are
comparable implies that a stochastic generation of equal distribution
of the two stable, dimer-like conformers would have to occur. On the
other hand, it is unnecessary to assume a priori that the
native heterodimeric configuration is formed in this chimera. As
discussed above, single-chain mutants were biologically active despite
changes in the quaternary structure (15, 16, 19). Thus, if the subunits
were properly folded but not configured strictly as native
heterodimers, the protein was biologically active. If the -subunit
interacts simultaneously with two ß-subunits, this would imply that a
multimeric gonadotropin could be generated in the secretory pathway.
This point is especially relevant to FSH/LH because both are
synthesized in the pituitary gonadotropes and presumably the two
ß-subunits and -subunit comingle in the same ER pools. Thus,
complexes of , FSHß, and LHß could exist during the early stages
of synthesis and accumulation in the ER before the segregation of the
subunits to form native hormone.
The ability to design dually active single-chain analogs (17) may be useful as therapeutics in addition to structure-function issues. For example, there could be more effective control of the half-life and duration of activity of gonadotropins. If LH and FSH were given as single entities, there is the potential for unexpected difference in the in vivo response. This point is important given the variations in metabolic clearance by the patient population.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-COOH. We wanted the C-terminal end of
the -subunit to be free since this region is critical for the
receptor binding (20, 21, 22, 23). To generate mutants, the previously reported
single chains, CGß (7) and FSHß (9), were used as substrates
(Fig. 1, A and B).
Two triple-domain tethers were constructed: 1) CGß-FSHß- (Fig. 1C) and 2) FSHß-CGß- (Fig. 1D). CGß-FSHß- was engineered
in a series of cut-and-paste experiments. To construct
FSHß-CGß-, PCR was used with the following primers (5' 
3'):
1. CAA GCA GTA TTC AAT TTC TGT CTC; contains a newly created EcoRI site in the intron of FSHß gene.
2. CGG CTC CTT GGA TTG TGG GAG GAT CGG; this sequence encodes amino acids 141–145 and 1–4 of the CGß subunit.
3. CCG ATC CTC CCA CAA TCC AAG GAG CCG; corresponds to amino acids 141–145 and 1–4 of the CGß subunit.
4. CTG ACC AGA GAG GTC GAC CAC CCT TCC; contains a SalI site in the CGß intron.
Primers 1 and 2 generated a fragment containing the EcoRI
site of the FSHß gene, exon 3 of the FSHß subunit, amino acids
118–145 of the CGß subunit (CTP), and amino acids 1–4 of the CGß
subunit. A fragment corresponding to amino acids 141–145, 1–4 of the
CGß subunit, and the SalI site in CGß intron 2 was
synthesized using primers 3 and 4. Primers 1 and 4 were used to ligate
the above fragments by overlapping PCR, and this product contained the
EcoRI/SalI site in the FSHß intron, CTP, CGß
exon 2, and the SalI site in the intron between exons 2 and
3 of the CGß subunit. After EcoRI/SalI
digestion, this product was ligated into the
EcoRI/SalI site of FSHß [BlueScript KS(+)]
and after subcloning, the vector was digested with
BamHI/SalI, and the resulting fragment was
subcloned into pM2HA (7). This construct was then digested
with SalI. In a separate reaction pM2HA bearing
the CGß single chain (7) was digested with SalI. The
fragment containing CGß exon 3 and the entire coding sequence of the
-subunit was inserted into the SalI site containing the
FSHß-CTP-CGß-exon 2 sequence. The final product,
FSHß-CTP-CGß-, was sequenced to verify that no errors were
introduced during the construction.
Transfection and Cell Culture
The tethered variants were transfected into CHO cells using the
calcium phosphate method as previously described (7). Stable clones
were selected approximately 11 days later using the neomycin analog
G418 (250 µg/ml). The clones were maintained in Ham’s F-12 medium
[supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml),
and 2 mM glutamine] containing 5% FBS and G418 (125
µg/ml) at 37 C in a humidified atmosphere of 5% CO2/95%
air.
Metabolic Labeling
To examine the synthesis of single-chain chimeras, CHO cells
expressing mutants were labeled for 6 h in Ham’s F-12 medium
containing dialyzed BSA, [35S]cysteine (Amersham Pharmacia Biotech, Arlington Heights, IL), or a mixture of
[35S]cysteine and methionine (Promix; Amersham Pharmacia Biotech) (15, 16). Aliquots of cell lysate and medium
were immunoprecipitated with polyclonal antiserum directed against the
common -subunit, which was raised in the laboratory. The reduced
proteins were resolved on 10% or 12.5% SDS-PAGE. To determine the
secretion kinetics of chimeras, cells were pulse labeled and chased up
to 24 h. Lysates and media were immunoprecipitated with
-antiserum, followed by SDS-PAGE. The secretion t1/2 of
chimeras was calculated as the time (min) when half of the maximal
secreted hormone was detected in the media.
Western Blot
Media samples were resolved on 12.5% SDS-PAGE under nonreduced
conditions without heating. Blotting was performed on a nitrocellulose
membrane and visualized with the chemiluminescence detection system
(Tropix Inc., Bedford, MA) according to the manufacturer’s protocol.
The blots were probed with the hCG conformational sensitive mAb B109
(provided by Dr. Steven Birken, Columbia University Medical School, New
York) that recognizes primarily the heterodimer but not the monomeric
ß-subunit (24) and an hFSH-specific monoclonal antibody (mAb 117),
which has a 100-fold greater affinity for the FSH heterodimer than to
the noncombined FSHß subunit (25).
Receptor Binding and Signal Transduction
Collection media were concentrated using either a Centric Prep
concentrator (Amicon Inc., Beverly, MA) or an Ultra-free centrifuge
filter device (Millipore Corp., Bedford, MA). The
triple-domain single chains were quantitated using two different
methods. In the first method the amount of FSH and hCG was determined
with a RIA kit (Diagnostic Products, Los Angeles, CA),
which uses either hFSHß or hCGß polyclonal antisera. These assays
score the ß-subunit whether or not it is configured in a
heterodimeric-like association with the -subunit. The second
approach employs a sandwich-type ELISA method and includes two sets of
monoclonal antibodies. The FSH assay contained an FSHß mAb as the
capture reagent and an mAb for detection. The hCG assay included a
capture mAb and an hCGß-specific mAb for detection. All mAbs
reacted with both intact heterodimer and noncombined subunits in
solution. Thus, the assay minimizes the conformational influences on
detection. To further verify the concentrations of the FSH and hCG
centers in the chimera and heterodimers determined by the ELISA, we
characterized the immunoassays with a BIACORE system (26, 27). We used
the same amount of the triple-domain single chain and wild-type
heterodimers with BIACORE chips each containing an FSHß and CGß
subunit-specific mAbs. These mAbs recognize different epitopes from
those used in the ELISA.
Receptor binding was determined using stably transfected CHO cell lines
expressing either the hLH/hCG or FSH receptors. The cells were
incubated (4 x 105 cells per tube) with ligands and
[125I]hCG or [125I]FSH (100,000 cpm/tube),
respectively, for 16–18 h at room temperature. The binding reaction
was terminated by washing the cells twice with PBS containing 0.1%
BSA, and the radioactivity was quantified in a 
-counter. Each
experiment was performed three times with duplicate tubes.
The total (extra-and intracellular) amount of cAMP was determined using
the Adenyl Cyclase Activation Flash Plate kit (NEN Life Science Products, Boston, MA) as per the manufacturer’s instructions.
CHO cells (5 x 104 cells per well) expressing either
the LH/hCG or FSH receptors were incubated with ligand for 2 h at
room temperature, [125I]cAMP was added, and the cells
were incubated for an additional 16–18 h at room temperature. The
Flash Plates were read in a Packard Top -Counter (Packard
Instruments, Downers Grove, IL). Each experiment was performed
three to four times with duplicate wells. The cAMP content was
expressed in picomoles/ml.
Purified hCG (CR127) and recombinant FSH were obtained from the NIH and N.V. Organon (Oss, The Netherlands), respectively. The binding affinity (IC50) and the half-maximal adenylate cyclase stimulation (EC50) were calculated for each concentration-response curve (hCG and FSH) for each ligand.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Albina Jablonka-Shariff was supported by National Research Service Award Fellowship HD-08301. This work was supported by N.V. Organon.
1 Current Address: Department of Obstetrics and Gynecology, Rokko
Island Hospital, 658-0032 Kobe, Japan. 
2 Current address: Nishi Kobe Medical Center, 650-0017 Kobe,
Japan.
3 N.V. Organon, 5340 BH Oss, The Netherlands.
Received for publication March 22, 1999. Revision received June 21, 1999. Accepted for publication July 20, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and signaling in human chorionic gonadotropin. Mol Endocrinol 6:914–919This article has been cited by other articles:
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T. Hiro'oka, D. Maassen, P. Berger, and I. Boime Disulfide Bond Mutations in Follicle-Stimulating Hormone Result in Uncoupling of Biological Activity from Intracellular Behavior Endocrinology, December 1, 2000; 141(12): 4751 - 4756. [Abstract] [Full Text] [PDF]
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D. Ben-Menahem, A. Jablonka-Shariff, R. K. Hyde, M. R. Pixley, S. Srivastava, P. Berger, and I. Boime The Position of the alpha and beta Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes J. Biol. Chem., August 3, 2001; 276(32): 29871 - 29879. [Abstract] [Full Text] [PDF]
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88 kDa (n = 2 experiments). The
extended band width of the FSHß
