(Received for publication, November 7, 1996, and in revised form, January 10, 1997)
From the Department of Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 and the § Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317
Disrupting disulfide loops in the human chorionic
gonadotropin subunit (CG
) inhibits combination with the
subunit. Because the bioactivity requires a heterodimer, studies on the
role of disulfide bonds on receptor binding/signal transduction have
previously been precluded. To address this problem, we bypassed the
assembly step and genetically fused CG
subunits bearing paired
cysteine mutations to a wild-type
(WT
) subunit. The changes
altered secretion of the single-chain mutants which parallel that seen for the CG
monomeric subunit. Despite conformational changes in CG
disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to
the lutropin/CG receptor and activated adenylate cyclase in
vitro. The data show that the structural requirements for
secretion and bioactivity are not the same. The results also suggest
that the extensive native subunit interactions determined by the
cystine bonds are not required for signal transduction. Moreover, these studies demonstrate that the single-chain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.
Mutational and structural studies have revealed that small
clusters of amino acids rather than large structural motifs often contribute to most of the energy involved in protein-protein
interactions such as hormone binding to its receptor (for review, see
Refs. 1-3). However, it is not clear how the conformation of a peptide ligand contributes to the coupling of binding to signal transduction. This is especially an issue for the function of hormone-receptor complexes involving multisubunit ligands. A convenient model for studying the tertiary and quaternary determinants in signaling is the
glycoprotein hormone family which include human chorionic gonadotropin
(hCG),1 lutropin (LH), follitropin (FSH),
and thyrotropin. Each is a non-covalent heterodimer composed of a
common and unique
subunit which allows recognition of the
corresponding G-protein-coupled receptor (for review, see Ref. 4).
Recent crystallographic studies of hCG revealed a significant
structural similarity to several growth factor families,
e.g. transforming growth factor , which contain a cystine
knot motif composed of three pairs of bridged cysteine residues (5-8).
Each hCG subunit contains a cystine knot configuring three
disulfide-bonded loops which are the major structural motifs (7, 8).
Based on the crystallographic studies of hCG (7, 8), the disulfide bonds in the CG
subunit are at positions 9-57, 34-88, 38-90, 23-72, 93-100, and 26-110 (Fig. 1A). The
folding intermediates associated with the ordered formation of these
bridges to acquire an assembly-competent form is well documented (9,
10). Current models of hCG action presume that the conformation of the
dimer and the highly interactive contacts between the two subunits are critical for function (11-14). One method to examine the functional role of the structural motifs in the glycoprotein hormones is to assess
the biologic activity of variants containing mutated cysteine residues.
However, breaking single disulfide bonds of the
subunit inhibits
secretion and assembly with the
subunit, and as a result dimer
recovery is dramatically reduced (Ref. 15 and Table I). Since only the
heterodimer binds to the receptor, it is virtually impossible to
examine the bioactivity of these variants. Recently, a single gene
encoding a protein containing CG
and
subunit was constructed
(16, 17). The tethered hormone exhibited secretion kinetics and
bioactivity similar to that of the non-covalent heterodimer. Here we
compile into a single protein subunits that cannot combine efficiently,
namely the
subunit and CG
with cysteine to alanine mutations.
Because this tethered construction by-passes the assembly step, we can
examine the role of the disulfide bonds on the biological activity of
hCG. The data show that they are primarily required for assembly with
the
subunit and secretion of the heterodimer. The extensive native subunit interaction in the dimer which is altered by these mutations is
not critical for receptor binding and signal transduction.
|
A KpnI-XhoI
fragment (2.7 kilobases; Fig. 1B) containing single-chain
hCG (CG) in vector pM2HA (16) was subcloned into
pBluescript II KS (Stratagene). Construction of cysteine (Cys) to
alanine (Ala) mutants in the CG
subunit gene (CG
Cys) was
described (15). The ApaI fragment containing the first 135 amino acids of CG
sequence in CG
was exchanged for the
ApaI fragment in CG
Cys. The new
KpnI-XhoI fragment was inserted into
pM2AH and rechecked by restriction enzyme analysis. The
mutations were confirmed using Taq DyeDeoxy Terminator Cycle
Sequencing Kit and an ABI prism DNA Sequencer (Perkin Elmer).
Clones of
transfected Chinese hamster ovary (CHO) cells were maintained as
described (15, 16). We used polyclonal antiserum for precipitation
and Western blots since WT
subunit is tethered to the mutated CG
sequence, and the immunoreactivity of all variants to the antiserum
should be similar. Pulse-chase experiments were performed in 12-well
dishes (300,000 cells/well) (15). Precipitates were resolved on SDS
gels and were quantitated with a PhosphorImager (Molecular Dynamics) or
a laser densitometer (LKB).
Condition medium obtained from cells
grown in serum-free F12 was concentrated (using Amicon Centriprep 10 concentrators), the concentrate was diluted (1:15) in
phosphate-buffered saline, and the volume reduced again. The mutants
were quantitated using our polyclonal antiserum and the double
antibody RIA (Diagnostic Products Corp.). A human kidney-derived cell
line (293), stably transfected with the cDNA-encoding human LH/CG
receptor, was used for receptor binding and cAMP production (18).
To assess the
intracellular effect of the disulfide loops in the CG domain, we
examined the secretion of tethered hCG variants in which both cysteines
of a proposed pair were mutated. Transfected CHO cells stably
expressing the Cys mutants were metabolically labeled and subjected to
SDS-polyacrylamide gel electrophoresis analysis under reduced
conditions. The electrophoretic migration of the secreted proteins is
slower than the corresponding intracellular species (Fig.
2A) which reflects both the processing of the
N-linked and addition of the O-linked
oligosaccharides to the CG
subunit just prior to secretion (15). The
experiment reveals that the secretion kinetics and extent of recovery
of the mutants from the media are variable. For example, the 34-88
mutant is not detected in the media after 4 h of labeling
(lane 7). Pulse-chase analysis shows that the unmodified
single-chain hCG (CG
) is secreted efficiently
(t1/2 = 85 min; recovery = 75%; Table
I) which is comparable to that of the heterodimer (15).
As previously observed for the monomeric CG
subunit (15), release of
the 26-110 mutant is accelerated (t1/2 = 60 min,
recovery 90%). In contrast, mutations in the cystine knot
(i.e. 9-57, 34-88, and 38-90) resulted in variants that
were secreted slower and less efficiently. This is especially evident
for the 34-88 mutant where the t1/2 is 350 min and
25% of the protein is recovered. The reduced recovery suggests that
the mutants were altered resulting in degradation of a significant
fraction of the pool. This is substantiated by pulse-chase analyses
that show in contrast to CG
, where most of the variant is
secreted, 75% of the 34-88 mutant that accumulates in the lysate does
not exit the cell and is no longer detected after 24 h (Fig.
2B). Thus, the disulfide bonds which comprise the core of
the subunit, namely 9-57, 34-88, and to a lesser extent 38-90, are
essential for efficient secretion of the CG single chain. The secretion
kinetics generally parallel those seen for CG
monomer (Table I),
which implies that these mutation-induced alterations in intracellular
behavior are not the result of the single-chain construction.
Conformational Changes of the Cys Analogs
If the conformation
is altered by breaking the disulfide bonds, we should detect
differences in electrophoretic mobility and immunoreactivity on SDS
gels. To test this prediction, Western blots were performed under
non-reduced conditions using a monoclonal antibody directed against subunit epitopes that are exposed primarily in the heterodimer
(designated A407; Ref. 19). The antibody recognized the CG
,
suggesting that dimer-like structure is preserved despite the tethering
(Fig. 3A, lanes 1 versus 6). The
mutants lacking the bonds that form late in the folding sequence of
CG
monomer, i.e. 23-72, 26-110, and 93-100 (9, 10),
were immunoreactive (Fig. 3A, lanes 2,
7, and 8). The recognition of the 23-72 and
26-110 Cys variants by the dimer-specific antibodies presumably
reflects the limited assembly of such mutated CG
monomers with the
subunit (Table I and Ref. 15). It is unclear why the 93-100 Cys
mutant is detected because no discernible assembly is seen with monomer
CG
subunit bearing this mutation (Table I and Ref. 15), but this
single-chain mutant may be partially configured to a heterodimeric
form. Of significance, analogs lacking bonds of the cystine knot which
are the first to form in CG
(9, 10) display little or no
signal (Fig. 3A, lanes 3-5). Comparable results
were obtained with B109 (data not shown), a monoclonal antibody raised
against a CG
epitope specific to the heterodimer (12). When the same
blot was reprobed with polyclonal
antiserum (Fig. 3B),
all analogs and free
monomer were detected. This indicates there
was sufficient protein in the blot and illustrates the
discrimination between the conformation of free and dimerized subunit
by the monoclonal antibody (lane 1).
As previously reported, the CG migrated slightly faster
than the heterodimer under non-reduced conditions (Fig. 3, lanes 1 and 6, and Ref. 16). In addition, the electrophoretic
migration of each analog differs compared with CG
further
demonstrating conformational changes induced by disrupting the
disulfide bridges. We also observed slower migrating species on the
blots and that this heterogeneity for the CG
and the Cys mutants
is variable; it is most pronounced for mutants 38-90, 9-57
(panel B, lanes 3 and 4), and, for the
34-88 mutant, the higher molecular weight protein represents a
considerable fraction of the total (lane 5). The high
molecular weight forms of CG
are non-covalent since heating
(95 °C, 5 min) under non-reduced conditions converts them to the
expected size of the single chain (data not shown). The larger forms of
the Cys variants apparently consist mainly of covalent polymers since
they are resistant to heat (95 °C, 5 min) but dissociate when boiled
in the presence of
-mercaptoethanol (data not shown). Aggregate
formation, often associated with misfolded forms of a protein, was
observed when Cys mutants of CG
subunit monomers were analyzed under
non-reduced conditions (10) and during purification of native urinary
hCG (20, 21). Taken together, the data imply that bonds of the cystine
knot motif are the scaffold for the structure, and the cysteine
mutations induce conformational changes in the single chains. In
contrast to the inability of such CG
subunits to efficiently combine
and thus interact with the
subunit, the mutated
domains in the single chain are nevertheless tethered to an
domain and secreted. The improved recovery (Table I) now permits analysis of the effects of
disrupting the disulfide-bonded loops on biological activity.
Binding of
single-chain variants to the human LH/CG receptor stably expressed in
293 cells was examined. We unexpectedly found that all CG variants bind
to the receptor including those mutants that as subunits assemble
poorly (i.e. 34-88, 9-57, 93-100, and 38-90). The
single-chain mutants, except analogs 38-90 and 34-88, displayed
similar dose-response curves to the purified heterodimer (CR129)2 and CG (Fig.
4A); the affinity of mutants 38-90 and
34-88 was apparently reduced by 7- and 10-fold, respectively (Table
II). We cannot exclude the possibility that these
changes are due to the observed aggregation (see above; Fig.
3B, lanes 3 and 5); purification is
needed to address this point. That the binding affinities observed were
not due to an altered specificity induced by the single-chain
configuration, we tested the action of single-chain FSH (22). As
expected, this protein did not displace the bound hCG (Fig.
4A). The signal-transducing responses of the hCG variants were also assessed by quantifying adenylate cyclase activation. All
these mutants increased cAMP levels, and the signal transduction paralleled binding efficiency, resulting in similar coupling ratios (Fig. 4B and Table II). These data show that bioactivity was
preserved and mutations in the CG
monomer which block assembly with
the
subunit are without effect on the efficiency of signal
transduction when expressed as single chains.
|
Structural analyses of hCG revealed that the overall shape of the
glycoprotein hormones is elongated rather than globular with the
disulfide-bonded loops primarily surface-exposed (7, 8). However,
despite their relatively large structures, conformational changes
induced by deleting each of the disulfide bonds and altering any of the
loops in the CG domain does not abolish binding/signal transduction
nor does it significantly affect coupling. These results were not
anticipated since it is well accepted that the intracellular/extracellular behavior of the glycoprotein hormones is
conformationally sensitive (11-14). Mutations inhibiting heterodimer formation and eliminating dimer-specific epitopes in the tethered analogs did not significantly affect the bioactivity of the
single-chain mutants. The glycoprotein hormone-specific quaternary
structure, i.e. the heterodimer, signals key functional
intracellular events such as efficient secretion and hormone-specific
processing of the subunit N-linked oligosaccharides (23).
Our data imply that analogs with different conformations can bind to
the receptor and activate adenylate cyclase. Alternatively, the hormone
could exist in a series of resonant conformers, and the receptor favors only the binding-competent species. We consider this less likely because for Cys knot mutants no signal indicative of the native form
was seen with monoclonal antibody. Therefore, we would conclude that
the tertiary and quaternary features created by single disulfide loops
in the
subunit and the tight interaction between subunits are
essential for the normal intracellular behavior of the gonadotropins, but not for the coupling of receptor binding/signal transduction in vitro.
The bioactivity of Cys analogs can be explained by several models.
(a) As previously proposed for numerous protein-protein interactions (reviewed in Refs. 1-3) including the gonadotropins and
their receptors (24-28), the contact sites at the hormone receptor interface are likely established by small clusters of amino acid residues. These determinants for bioactivity could not be disrupted by
the conformational changes induced by the Cys mutations; (b) hCG may contain redundant binding/signaling determinants, eliminating one is compensated by others in the intact loops; (c)
binding of the complex to the receptor results in conformational
changes of the CG subunit that restores activity of the Cys mutated
analogs.
The 91-110-amino acid stretch of CG envelops the
subunit and
includes domains for bioactivity such as the "determinant loop"
(i.e. residues 93-100; Ref. 29). Because the single-chain 26-110 or 93-100 mutants were as active as CG
, a major role for
this sequence is apparently to stabilize the noncovalent heterodimer (7, 8). Thus, the primary and/or secondary structure of loci within
each of these individual loops rather than their tertiary structure are
likely determinants for functional receptor recognition.
By-passing the assembly step with the single-chain approach enabled us to expand the spectrum of analogs for structure-function analysis. Similarly, mutations in other multisubunit hormones and growth factors of the cystine knot superfamily which result in inefficient assembly, secretion, and loss of bioactivity (6 and references therein) can be examined.
We thank Dr. Steven Birken for providing monoclonal antibodies and his helpful suggestions and Susan Carnes for excellent assistance in preparing the manuscript. We thank Drs. M. Muyan, D. Ornitz, and D. Towler for reading the manuscript and Takashi Hiro'oka and Burkhard Hirsch for FSH control.