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Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229
Address all correspondence and requests for reprints to: Dr. Prema Narayan, Department of Biochemistry and Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229. E-mail: narayan{at}bchiris.bmb.uga.edu
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Abstract |
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-subunit common among all
members of the glycoprotein hormone family, LH, FSH, and TSH, and a
unique ß-subunit responsible for receptor specificity. Biologically
active single chain analogs of these hormones have been engineered in
which the C-terminus of the ß-subunit was fused to the N-terminus of
the -subunit (N-ß--C) either with or without a linker such as
the hCGß C-terminal peptide (CTP). This tandem order of subunits was
chosen based on studies suggesting that the N-terminal region of hCGß
and particularly the C-terminal region of the -subunit are important
in receptor binding and activation. Single chain hCG (YhCG1) can, in
turn, be fused to the LH receptor to yield a hormone-receptor complex
that is biologically active in transfected cells. Herein, we report the
construction of a new single chain hCG analog (YhCG3) in which the
C-terminus of the -subunit is fused to the N-terminus of hCGß via
a CTP (N--CTP-ß-C). Compared with YhCG1, this analog binds
receptor with a 25- to 30-fold lower affinity, but, surprisingly, is
capable of stimulating intracellular cAMP levels to the same extent.
Furthermore, YhCG3 can be covalently linked to its receptor to produce
a biologically active complex that results in elevated levels of basal
cAMP in transfected cells. These results suggest that free N- and
C-termini of hCGß and the -subunit, respectively, are not
essential for receptor binding and activation and that YhCG3 is in a
more efficacious conformation for receptor activation than YhCG1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and a
hormone-specific ß-subunit. The functional activity of these hormones
is dependent on the efficient and correct assembly of the subunits into
the heterodimer. Recently, single chain or yoked analogs of these
hormones, in which the C-terminus of the ß-subunit was fused to the
N-terminus of the -subunit, have been engineered and proven to be
useful in structure-function studies and in the generation of hormones
with increased stability and activity (1, 2, 3, 4, 5, 6). In most cases, the
ß-subunit of hCG, FSH, LH, and TSH has been linked to the -subunit
via the C-terminal extension of 30 amino acids (amino acid residues
116–145), termed the C-terminal peptide (CTP), present only in hCG
(1, 2, 3, 4, 5). Functional hormone can also be obtained when the subunits are
directly linked to each other (3, 4) or by using 4, 5, or 6 repeating
units of serine-glycine as a linker (6). Moreover, it has been shown
that neither full-length hCGß nor is required to produce
biologically active single chain forms of hCG (1, 3, 6). Furthermore,
the single chain (yoked) hCG (YhCG1) can be fused to its receptor, the
LH receptor (LHR) to generate a complex, YHR, which in transfected
cells produces a constitutively active receptor leading to high
intracellular levels of cAMP (7).
hCG was the first single chain hormone to be prepared and characterized
(1, 2), and the arrangement of N-ß--C was chosen because of
reports in the literature suggesting that the N-terminal region of
hCGß and the C-terminal region of human participate in receptor
binding and activation. For example, studies involving synthetic
peptides (8, 9), site-directed mutagenesis (10), and site-specific
chemical modification (11) have implicated the N-terminal portion of
hCGß in LHR binding. The first seven amino acid residues of hCGß
can, however, be deleted without a significant loss of binding affinity
(12). A more critical concern in the design of the original single
chain gonadotropins was the purported role of the -subunit. Early
studies on LH and hCG showed that removal of amino acid residues from
by carboxypeptidase treatment, while not affecting recombination
with the ß subunit, diminished or essentially eliminated receptor
binding (13, 14, 15). More recently, this work was extended using
site-directed mutagenesis to prepare specific C-terminal deletion
mutants of and to replace selected amino acid residues with others
(16, 17, 18, 19). Although there are subtle differences in the reported
bioactivity of des-(89–92)-hCGß (17, 18), overall the results
confirm the importance of the C-terminal region of the -subunit in
LHR binding. Because these studies did not directly address the
requirement for a free -subunit C-terminus, these results on the N-
and C-termini of hCGß and , respectively, do not rule out the
possibility of a biologically active single chain gonadotropin of the
form N--ß-C. However, they strongly suggest that such a fusion of
the two subunits may result in diminished activity.
To test this hypothesis, we have constructed and characterized a new
single chain hormone analog, YhCG3, in which the -subunit is linked
to hCGß via the CTP (N--CTP-ß-C). In addition, we have
covalently linked YhCG3 to LHR to produce the hormone-receptor complex,
YHR3, and characterized its signaling properties. As might be expected,
YhCG3 binds receptor with a lower affinity than YhCG1, but,
surprisingly, despite the reduced binding affinity, this analog can
activate LHR to the same extent as YhCG1 when added exogenously or when
covalently linked to the receptor, indicating that free N- and
C-termini of hCGß and the -subunit, respectively, are not absolute
requirements for receptor binding and activation. These results also
suggest that binding of YhCG3 to LHR results, perhaps, in a more active
conformation of the hormone and/or receptor than that produced by
binding of YhCG1 to LHR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of YhCG3 and YHR3
To generate YhCG3, the nucleotide sequence encoding the
full-length -subunit, including the signal sequence, was fused
in-frame via the CTP to the 5'-terminus of full-length hCGß
containing the entire coding sequence of the mature protein. The
-subunit containing the CTP sequence was generated by PCR
amplification of a previously constructed clone, YR, in which the
-subunit was covalently linked to LHR via the CTP (20). The
sequences of the 5'- and 3'-primers, used in generating the -subunit
sequence were as follows: 5'-CTACGAGGTACCCAGTAACCGCCCTGA-3', containing
a KpnI restriction enzyme site and 15 nucleotides
corresponding to the 5'-end of the -subunit complementary DNA
(cDNA), and 5'-CGAGCTAGCTTGTGGGAGGATCGG-3', containing the sequence
corresponding to the last five codons of the CTP and a NheI
restriction enzyme site. The hCG ß-subunit without the signal
sequence was also generated by PCR amplification of hCGß cDNA using
the following two primers: 5'-CGAGCTAGCTCCAAGGAGCCGCTTCGG-3' containing
a NheI restriction enzyme site and the sequence
corresponding to the first six codons of the mature protein, and
5'-CGAGGATCCTTATTGTGGGAGGATCGG-3' containing the sequence corresponding
to the last five codons of hCGß followed by a stop codon and a
BamHI restriction enzyme site. The -subunit and the
hCGß sequences were ligated to each other and to the
KpnI/BamHI-digested expression vector pcDNA3 in a
single ligation reaction. To generate YHR3, a previously constructed
clone, YßR containing the hCGß sequence covalently linked to LHR
via the CTP and factor Xa protease recognition sequence (20), was used
as the template to generate the hCGß-LHR sequence. The 5'- and
3'-primers used were as follows: 5'-CGAGCTAGCTCCAAGGAGCCGCTTCGG-3'
containing a NheI restriction enzyme site and the sequence
corresponding to the first six codons of the mature protein, and
5'-ACGAGGATCCTTAGTGAGTTAACGCTCT-3' containing the sequence
corresponding to the last five codons of LHR followed by a stop codon
and a BamHI restriction enzyme site. This hCGß-LHR
sequence was ligated to the -subunit sequence used in the
construction of YhCG3 to generate YHR3, which was cloned into
KpnI/BamHI-digested pcDNA3 for subsequent
expression. Both clones were completely sequenced.
Transfection and selection of stable cell lines
Transfection of CHO K1 and HEK 293 cells was performed using
Lipofectamine as the transfection agent. YhCG1 and YhCG3 cDNAs as well
as the empty pcDNA3 vector DNA were transfected into CHO K1 cells, and
stable cell lines were selected by growth in DMEM-Ham’s F-12 medium
(1:1) supplemented with 10% (vol/vol) FBS, 50 U/ml penicillin, 50
µg/ml streptomycin, 0.125 µg/ml Amphotericin, and 600
µg/ml geneticin. YHR and YHR3 cDNAs were transfected into HEK 293
cells that were maintained in DMEM supplemented with 10% (vol/vol)
horse serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.125
µg/ml Amphotericin. Colonies resistant to 700 µg/ml
geneticin were isolated using cloning cylinders. A clonal line each of
YHR and YHR3 was chosen for further characterization.
Analysis of hormone expression
The expression medium from confluent flasks of YhCG1- and
YhCG3-expressing CHO cell lines were collected and concentrated with
Biomax-10 columns. The amount of yoked hormones in the medium was
quantified by a solid phase, two-site immunoradiometric assay
(TANDEM-RHCG) specific for hCG molecules containing both subunits or a
hCG RIA capable of detecting both free ß-subunit as well as
heterodimer.
In vitro bioassays
Competitive binding experiments were performed with a stable
cell line of HEK 293 cells expressing LHR (7). Cells in 12-well tissue
culture plates were incubated with 50 pM
[125I]hCG and various dilutions of unlabeled
hCG or yoked hormones. Nonspecific binding was measured in the presence
of 1 µg/ml hCG. For determination of intracellular levels of cAMP,
LHR-expressing HEK 293 cells in 12-well tissue culture plates were
incubated with various concentrations of hCG or yoked hCG for 30 min at
37 C in Waymouth’s MB752/1 medium containing 0.8 mM
isobutylmethylxanthine. The incubation medium was removed, and the
cells were lysed in 100% ethanol at -20 C overnight. The lysed cells
were pelleted by centrifugation, and the ethanol supernatants were
collected, dried, and dissolved in the assay buffer of the
[125I]cAMP RIA kit that was used to determine
cAMP concentrations. Binding and cAMP determinations were performed in
a similar manner with YHR- and YHR 3-expressing cell lines.
Western blot analysis
Membrane fractions were isolated as described previously (7),
and their protein concentrations were determined by the bicinchoninic
acid assay. Membrane fractions were resolved on 10% SDS-polyacrylamide
gels under reducing conditions and transferred to Immobilon P. Blots
were incubated for 1 h at room temperature with rabbit anti-hCGß
CTP antiserum raised against amino acid residues 109–145 of hCGß.
The blots were developed using the ECL detection system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit was linked to the N-terminus of
hCGß via the CTP linker containing amino acid residues 116–145 of
hCGß, with the replacement of Ser132 by Thr
(Fig. 1
) (7). Compared with YhCG1, in which
the C-terminus of hCGß was linked to the N-terminus of the
-subunit, this new analog contains two CTP sequences, one present
naturally at the C-terminus of the ß-subunit and the other that was
introduced as a linker between the two subunits. YhCG1 and YhCG3 were
transfected into CHO cells, and stable CHO cell lines expressing the
two hormones were established. Conditioned media from these cell lines
and from a mock-transfected cell line were concentrated, and the levels
of YhCG1 and YhCG3 were determined by two independent RIA assays. For
both YhCG1 and YhCG3, the solid phase, two-site assay (which recognizes
only the hCG heterodimer) gave values that were 1.5- to 2-fold higher
than those obtained with the RIA (which recognizes both free ß as
well as heterodimer), indicating that both analogs were recognized with
similar affinities by antibodies used in the two assays. When the
standards supplied with the RIA kit were measured as unknowns in the
solid phase, two-site assay, the values obtained were approximately
1.5-fold higher than expected. Therefore, it appears that the
difference in the measured values of YhCG1 and YhCG3 by the two assays
is a reflection of the differences in the absolute concentrations of
the standards supplied with the two kits. The concentrations of YhCG1
and YhCG3 that were determined by the solid phase, two-site assay were
used in the bioassays. No detectable hCG was measured in medium from
mock-transfected cells.
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and Table 1). However, despite the lower binding
affinity, YhCG3 was able to stimulate intracellular cAMP with an
EC50 only 3- and 6-fold higher than those of hCG
and YhCG1, respectively (Fig. 2B and Table 1). Furthermore, the cAMP
levels achieved with the maximal dose (500 ng/ml) were not
significantly different for the three ligands. Treatment of cells with
a volume of concentrated conditioned medium from mock-transfected cells
corresponding to the highest volume used in the YhCG1 and YhCG3 assays
did not yield increased intracellular cAMP levels over the basal value
or compete with radiolabeled hCG for receptor binding (data not
shown).
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). YHR3 was transfected
into HEK 293 cells, and a stable clonal cell line was established; its
binding and signaling characteristics were then compared with YHR (Fig. 1), which we had previously characterized (7). Western blot analysis
with membrane fractions from both YHR- and YHR3-expressing cells showed
a specific band with an apparent molecular mass of 137 kDa (Fig. 3). However, in the clonal line chosen, YHR3
was expressed at a significantly lower level than YHR. The basal level
of intracellular cAMP in YHR3-expressing cells was elevated 19-fold
compared with that in LHR-expressing cells (Fig. 4), indicating that, as previously observed
with YHR (7), the YhCG3-LHR complex is functional. In contrast to the
ligand-mediated 14-fold increase in cAMP in LHR-expressing cells, no
further increase in cAMP was observed upon addition of a saturating
dose of hCG with either YHR- or YHR3-expressing cell lines, indicating
that the receptor is fully occupied by the covalently linked yoked
hormone. This result is consistent with the lack of binding observed
with radiolabeled hCG and cells expressing either YHR or YHR3 (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-CTP-ß-C binds LHR with a
reduced affinity compared with YhCG1, containing the tandem arrangement
of N-ß--C. However, it is able to activate the receptor to the
same extent as YhCG1, albeit with a somewhat higher
EC50. In addition, YhCG3 can be covalently linked
to LHR to generate a hormone-receptor complex that is constitutively
active, resulting in elevated levels of basal cAMP in transfected cells
and providing further evidence that YhCG3 is functional.
Various studies have shown that although progressive deletion of amino
acid residues from the C-terminus of the -subunit failed to abolish
holoprotein formation, the C-terminal deletion mutants progressively
exhibited a loss of receptor binding effectiveness (13, 14, 15, 17, 18).
Previous reports have also implicated the N-terminal region of hCGß
in receptor binding (8, 9, 10, 11). However, none of these studies focused on
the necessity of a free -carboxyl group on the -subunit or a free
-amino group on hCGß. Our findings with YhCG3 demonstrate that
neither of these free termini is required for a functional single chain
gonadotropin.
The 30-fold reduction in binding affinity of YhCG3 compared with YhCG1
for LHR suggests that the conformation of YhCG3 may be different from
that of YhCG1. From the crystal structure of deglycosylated hCG (21, 22), it is clear that the N-terminal regions of the two subunits are in
relatively close proximity to each other. Moreover, the most C-terminal
amino acid residues resolved in the hCG crystal structure,
i.e. Tyr89 in and
Asp111 in ß, are located on the opposite side
of the holoprotein from the two N-termini. Molecular modeling suggested
that the complete CTP as well as a shorter version could loop around
the molecule in YhCG1 without disrupting the conformations and
intersubunit interactions of and ß (1). Conversely, it seems
reasonable that the CTP could serve in a similar fashion by linking the
C-terminus to the ß N-terminus to form YhCG3. The CTP has many
attributes to serve as an innocuous linker. For example, it is quite
hydrophilic and appears to be a random glycosylated polypeptide chain,
perhaps with a few ß turns (23, 24). Yet, others have found that
functional single chain gonadotropins of the form, N-ß--C, can be
prepared with no intervening CTP, e.g. in LHß (4), FSHß,
and hCGß devoid of its CTP (3), or with a few repeats of Ser-Gly (6).
These findings and the data presented in this study suggest flexibility
in the gonadotropin structures, particularly in the regions of the
chain termini, and also bolster the argument that the native
heterodimeric structure of hCG is not required for biological activity
(25, 26).
The observation that despite the low receptor binding affinity YhCG3 can activate LHR almost as well as YhCG1 suggests that perhaps binding of YhCG3 to LHR results in a conformation of the complex that is more effective than that of YhCG1and LHR in coupling to second messenger signaling systems. Additional support for this argument is provided by YHR3, which, although expressed at a lower density than YHR in HEK 293 cells, is able to stimulate basal cAMP to levels higher than that produced by YHR. The low binding affinity exhibited by YhCG3 can apparently be overcome when the hormone is covalently linked to the receptor. A similar observation was made by Boime, Hsueh, and colleagues (2) when they compared the biological activity of single chain hCG (similar to YhCG1) and that of heterodimeric hCG expressed in CHO cells. They noted that although the binding affinity of the single chain hormone was 2- to 3-fold less than that of hCG, the EC50 for cAMP stimulation was 3-fold greater than that for hCG.
A construct similar to the one reported herein, but lacking the natural
CTP on hCGß was found to bind to LHR, but with an affinity some
50-fold less than that of hCG (Boime, I., and D. Ben-Menahem, personal
communication). Interestingly, the absence of a CTP linker between
and hCGß renders the single chain protein inactive in binding to
receptor (27).
In summary, our data with the single chain hCG analogs demonstrate that
neither a free hCGß N-terminus nor a free -subunit C-terminus is
essential to produce a functional hormone. These analogs can activate
LHR either when added exogenously or when covalently linked.
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Acknowledgments |
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Footnotes |
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Received August 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chorionic
gonadotropin ß subunits in tandem. Proc Natl Acad Sci USA 92:2041–2045 subunit and the follicle-stimulating
hormone ß subunit. J Biol Chem 271:10445–10448-subunits. J
Biol Chem 254:6010–6015 results in
the loss of cAMP inducibility. J Biol Chem 266:17741–17743-subunit: contributions to
receptor binding and signaling in human chorionic gonadotropin. Mol
Endocrinol 6:914–919 subunit play common and different
roles in human choriogonadotropin and follitropin. J Biol Chem 268:13034–13042-subunit are crucial for receptor-binding and hormone action of
follicle-stimulating hormone (FSH) and play hormone-specific roles in
FSH and human chorionic gonadotropin. Endocrinology 136:2984–2953[Abstract]
subunit-LH/CG receptor. 79th Annual Meeting of
The Endocrine Society, Minneapolis, MN, 1997, p 383 (Abstract)
subunit is critical for intracellular behavior but
not for in vitro biological activity. J Biol Chem 272:18098–18103, and
ß domains in a single chain affects the heterodimeric interaction of
the subunits. 30th Annual Meeting of the Society for the Study of
Reproduction, Pullman, WA, 1999, p 274 (Abstract)
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