Induction of Promiscuous G Protein Coupling of the Follicle-Stimulating Hormone (FSH) Receptor: A Novel Mechanism for Transducing Pleiotropic Actions of FSH Isoforms
Brian J. Arey,
Panayiotis E. Stevis,
Darlene C. Deecher,
Emily S. Shen,
Donald E. Frail,
Andrés Negro-Vilar1 and
Francisco J. López
Womens Health Research Institute Wyeth-Ayerst Research
Radnor, Pennsylvania 19087
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ABSTRACT
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Under physiological conditions, FSH is secreted
into the circulation as a complex mixture of several isoforms that vary
in the degree of glycosylation. Although it is well established that
the glycosylation of FSH is important for the serum half-life of the
hormone and coupling of the receptor to adenylate cyclase, little is
known concerning how physiologically occurring glycosylation patterns
of this hormone affect receptor signaling. In this study, we have
examined the biological activity of deglycosylated human FSH
(DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and
insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as
compared with that of purified human pituitary FSH (phFSH) using a
Chinese hamster ovarian cell line stably expressing the hFSH receptor
(3D2 cells). Differentially glycosylated forms of FSH did not bind to
the FSH receptor in the same manner as phFSH. Although all hormones
showed similar potency in competing for
[125I]phFSH binding to the hFSH receptor,
competition curves for deglycosylated and insect cell-produced FSH were
steeper. Similarly, glycosylation of FSH had a profound effect on
bioactivity of the hormone. Purified hFSH produced a sigmoidal
dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and
BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH
also elicited biphasic effects on steroidogenesis in primary cultures
of rat granulosa cells. The cellular response to BV-hFSH was dependent
on the degree of receptor-transducer activation. BV-hFSH bioactivity
was strictly inhibitory when combined with the
ED80 of phFSH. Lower concentrations of phFSH
resulted in a gradual shift from inhibition to a biphasic activity in
the presence of the ED20 of phFSH. Biphasic
responses to BV-hFSH were attributed to activation of different G
protein subtypes, since treatment of 3D2 cells with cholera toxin or
pertussis toxin differentially blocked the two phases of BV-hFSH
bioactivity. These data suggest that alternative glycosylation of FSH
leads to a functionally altered form of the hormone. Functionally
different hormones appear to convey distinct signals that are
transduced by the receptor-transduction system as either stimulatory or
inhibitory intracellular events via promiscuous,
glycosylation-dependent G protein coupling. Promiscuity in signaling of
the FSH receptor, in turn, may represent a potentially novel mechanism
for FSH action, whereby the gonad may respond in diverse ways to
complex hormonal signals such as those presented by circulating FSH
isoforms.
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INTRODUCTION
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FSH, LH, human CG (hCG), and thyroid-stimulating hormone make up
the heterodimeric glycoprotein hormone family (1). These proteins
contain a common
-subunit that is linked in a noncovalent manner
with a hormone-specific ß-subunit. Despite their similar structures
and sequence homologies, there is only marginal cross-reactivity among
the receptors (Ref. 2 and B. J. Arey, D. C. Deecher, E. S. Shen, and F.
J. López, unpublished observations). The presence of multiple
glycosylation sites on both subunits is one of the common structural
features of these hormones. FSH, for example, contains four such
glycosylation sites, two each on the
- and ß-subunits (3). The
action of FSH in the gonad involves an initial binding event with a
specific FSH receptor. The human (h) FSH receptor (hFSH-R) is a large,
integral membrane protein comprised of multiple hydrophobic regions
consistent with the seven transmembrane-spanning domains identified in
G protein-coupled receptors (4). Indeed, the FSH receptor has been
shown to be positively coupled to Gs, resulting in
activation of adenylate cyclase and cAMP production (5, 6).
In vivo, FSH is secreted into and maintained in serum as a
series of isoforms of differing isoelectric points (pI) (for review see
Refs. 7 and 8). These charge differences have been attributed to the
presence of differently glycosylated forms of the hormone (9, 10). It
is clear that the relative abundance of FSH isoforms in the circulation
is dependent on the physiological status of the subject (11, 12, 13, 14). For
example, in the rat, a shift in the pituitary content of FSH isoforms
is observed on proestrus from those of lower pI to those of higher pI
(12, 13). Because forms of FSH possessing greater pI values have a
decreased sialic acid content, this represents a shift in circulating
FSH from those species having a more complex glycosylated structure
(more acidic) and lesser bioactivity to those of higher pI and greater
bioactivity (15, 16). These observations suggest that feedback from the
ovary signals an increase in bioactive FSH before ovulation. Therefore,
the effect of a given stimulus on the gonad depends not only upon the
amount of circulating gonadotropins but perhaps, more importantly, on
the qualitative aspects of the stimulus such as the relative
distribution of diverse hormone isoforms under various physiological
conditions.
Several studies have provided evidence to indicate that FSH isoforms
exhibit different properties, which, in turn, modify their biological
activity. The data available suggest that hormone glycosylation is
important for both serum half-life (17, 18, 19) and signal transduction (7, 20, 21, 22, 23). Indeed, it has been shown that chemically deglycosylated FSH
acts as an antagonist at the FSH-R (24, 25). Because FSH binding to its
receptor involves multiple interactions between the proteins, one
possible explanation for these observations is that deglycosylation of
the hormone decreases its intrinsic activity, producing an isoform that
retains affinity for the receptor. In this respect, isoforms of FSH are
known to contain differing degrees of glycosylation and also a range of
receptor-binding characteristics (for review see Ref.8). Therefore,
the deglycosylated hormone would act as a competitive blocker of the
native hormone. Under this hypothesis then, the trophic signal to the
gonad consists of a mix of agonists and antagonists with varying
affinities and intrinsic activities that would determine gonadal
response. Alternatively, it is plausible that certain FSH isoforms may
bind to the FSH-R in a unique manner that induces ligand-specific
conformational changes to the ligand-receptor complex. Distinct
ligand-receptor complex conformations, in turn, could provide the
molecular foundation for either activation or deactivation of
alternative signaling pathways. These, therefore, may serve as a means
to provide pleiotropic responses to complex signals consisting of
multiple or varying glycosylated forms of FSH. In this study, we have
evaluated whether different glycosylated forms of hFSH are capable of
activating multiple different signal transduction pathways. For this
purpose, dose-effect relationships for differently glycosylated forms
of FSH were evaluated, not only in recombinant cell lines expressing
the hFSH-R, but also in some physiologically relevant in
vitro models of hFSH action.
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RESULTS
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The role of alternatively glycosylated forms of FSH in FSH-R
signaling was evaluated by studying the biological activity of various
preparations of FSH. For this purpose, Chinese hamster ovarian (CHO)
cells stably expressing the hFSH-R (3D2 cells) were incubated in the
presence of varying concentrations of either purified human FSH
(phFSH), chemically deglycosylated (hydrogen fluoride-treated) phFSH
(DeGly-phFSH), recombinant hFSH produced using a baculovirus expression
system (BV-hFSH), or recombinant hFSH expressed in CHO cells
(CHO-hFSH). Purified hFSH, DeGly-phFSH, BV-hFSH, and CHO-hFSH were
assayed by hFSH immunoradiometric assay (IRMA) simultaneously, and
the concentrations of stock solutions normalized to the detected
levels of immunoreactive
/ß hFSH. Purified hFSH induced a
sigmoidal dose-dependent increase in cAMP accumulation that reached
maximal levels more than 300-fold greater than control (Fig. 1
, upper left panel), consistent with the
coupling of the FSH-R to Gs (5, 6). The estimated
ED50 for phFSH in 3D2 cells was 9.55 ng/ml. Similarly,
DeGly-phFSH induced a dose-dependent elevation in cAMP accumulation
apparent at doses ranging from 110,000 ng/ml (ED50 = 9.61
ng/ml; Fig. 1
, upper right panel). However, unlike the
native hormone, DeGly-phFSH produced a biphasic (bell-shaped)
dose-response curve. Concentrations of DeGly-phFSH greater than 100
ng/ml were less efficacious in bioactivity when compared with that
elicited by this dose (Fig. 1
, upper right panel). Maximal
stimulation of cAMP accumulation was approximately 20-fold greater than
control. Thus, DeGly-phFSH is approximately 10-fold less efficacious
than the native hormone. The IC50 for DeGly-phFSH, as
estimated using a seven-parameter logistic model, was 7.62 µg/ml.

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Figure 1. Different Glycosylation Patterns of hFSH Induce
cAMP Accumulation in a Biphasic Manner
Purified human FSH (phFSH, top left, ED50=
9.55 ng/ml). Chemically deglycosylated phFSH (DeGly-phFSH, top
right, ED50 = 9.61 ng/ml and ID50= 7.62
µg/ml). Insect cell expressed hFSH (BV-hFSH, bottom
left, ED50 = 1.37 ng/ml and ID50 = 0.53
µg/ml). Mammalian cell expressed hFSH (CHO-hFSH, bottom
right, ED50 = 7.29 ng/ml). Open bars
denote cAMP accumulation in the control group. *, P
< 0.05 vs. control by ANOVA followed by the Dunnets
test on log-transformed data .
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Similar to DeGly-phFSH, a differently glycosylated [under-glycosylated
(26)] form of hFSH expressed in Hi5 insect cells (BV-hFSH) also
produced a biphasic stimulation of cAMP accumulation in 3D2 cells (Fig. 1
, lower left panel). Maximal stimulation of cAMP
accumulation by BV-hFSH was approximately 200-fold greater than control
at a dose of 100 ng/ml. Higher doses of BV-hFSH were less bioactive,
leading to a bell-shaped dose-response curve. The ED50 for
the ascending phase of the BV-hFSH bioactivity was 1.37 ng/ml, whereas
the ID50 for the descending phase of the BV-hFSH curve was
532.59 ng/ml. Moreover, in a cell-free adenylate cyclase assay using
isolated 3D2 cell membranes, phFSH induced a sigmoidal dose-dependent
stimulation in adenylate cyclase activity (Table 1
). In
contrast, increasing concentrations of BV-hFSH elicited a biphasic
effect on adenylate cyclase activity with distinct ascending and
descending phases to the dose-response relationship (Table 1
).
Supernatants of Hi5 cells that were infected with baculovirus without
the coding region of hFSH did not increase cAMP accumulation as
compared with control in the whole-cell assay (data not shown).
Furthermore, purification of BV-hFSH from Hi5 cell supernatants by ion
exchange and subsequent size exclusion chromatography revealed that a
single peak was responsible for the biphasic dose-responses induced by
crude Hi5 cell supernatants (data not shown). In contrast, FSH
expressed in a mammalian cell line (CHO cells) induced a sigmoidal
dose-dependent increase in cAMP accumulation that was nearly
indistinguishable from that observed for phFSH (ED50 = 7.29
ng/ml; Fig. 1
, lower right panel). Maximal induction of cAMP
production by CHO-hFSH was approximately 300-fold greater than that
observed in control wells.
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Table 1. Insect Cell-Expressed Human FSH (BV-hFSH)
Activates Adenylate Cyclase in a Biphasic Manner, Whereas Purified
Human FSH (phFSH) Induces a Sigmoidal Dose-Dependent Elevation of
Enzyme Activity in 3D2 Cell Membrane Preparations
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To evaluate whether altered affinities of the various FSH preparations
could account for the diverse biological activities, the ability of the
ligands to bind to the hFSH receptor was studied by radioligand-binding
assay using 3D2 cell membranes. Purified hFSH, DeGly-phFSH, and BV-hFSH
dose-dependently competed for [125I]hFSH binding to 3D2
cell membrane FSH-R (Fig. 2
). Purified hFSH competed for
[125I]hFSH binding with an ID50 = 869 ±
7 pM. Deglycosylated and BV-hFSH were slightly more potent
with an estimated ID50 of 395 ± 5 pM and
207 ± 7.8 pM, respectively. The slopes of competition
curves for DeGly-phFSH and BV-hFSH, however, were not parallel to that
of phFSH. Deglycosylated-phFSH and BV-hFSH competed with
[125I]hFSH for the hFSH-R with slope factors of 2.25 and
1.56, respectively. Purified hFSH competed for binding to the receptor
with a slope factor of 0.77. Thus, these data suggest that differently
glycosylated forms of FSH may bind to the receptor in a
hormone-specific fashion and, perhaps, elicit diverse conformations to
the ligand-receptor complex.

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Figure 2. Binding Properties of Diverse FSH Preparations to
the FSH-R Are Different
Binding of [125I]phFSH in the presence of phFSH
(open circles), BV-hFSH (solid circles),
or DeGly-phFSH (solid triangles).
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The possibility that the biphasic bioactivities of under-glycosylated
FSH may be restricted to the genetically engineered cell line (3D2)
used for these studies was ruled out by evaluating the biological
activity of phFSH and BV-hFSH in an aromatase bioassay. In this more
physiologically relevant system, incubation of rat granulosa cells with
phFSH resulted in a dose-dependent increase in estradiol secretion with
a 3-fold elevation as compared with cells cultured in the presence of
medium alone (ED50 = 0.30 ng/ml, Fig. 3
, top panel). In contrast, treatment of granulosa cells for
72 h with BV-hFSH resulted in a bell-shaped dose-response
relationship (Fig. 3
, bottom panel) similar to that observed
in 3D2 cells. BV-hFSH stimulated estradiol secretion above control at
all but the lowest dose tested (Fig. 3
, bottom panel).
Maximal stimulation of estradiol secretion was apparent at a dose of 10
ng/ml with an ED50 for the ascending portion of the BV-hFSH
dose-response curve of 0.97 ng/ml. Concentrations of BV-hFSH greater
than 10 ng/ml were less efficacious such that the highest dose of
BV-hFSH induced only a 1.4-fold increase in estradiol secretion over
control levels. The estimated ID50 for the descending phase
of the BV-hFSH dose-response curve was 295.44 ng/ml. A similar
dichotomy between phFSH and BV-hFSH in terms of the qualitative
dose-response profile on progesterone secretion was evident in an
adrenal cell line (Y1) stably expressing the hFSH-R (data not
shown).

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Figure 3. BV-hFSH in Primary Cultures of Rat Ovarian
Granulosa Cells Induces Estradiol Secretion in a Biphasic Manner
Top, Granulosa cells incubated in the presence of phFSH
for 72 h. Bottom, Granulosa cells incubated in the
presence of BV-hFSH for 72 h. Open bars denote
estradiol release by the respective control group. *,
P < 0.05 vs. respective control
group by ANOVA followed by the Dunnets test.
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To study whether the ascending and descending bioactivities of BV-hFSH
could be separated by altering the degree of receptor-transducer system
activation, 3D2 cells were incubated with the ED20,
ED50, or ED80 of phFSH (representing low,
medium, or high receptor-transducer activation, respectively). In the
presence of the ED20 of phFSH, BV-hFSH induced a
bell-shaped dose-response curve (Fig. 4
, top
panel). However, when the ED50 of phFSH was incubated in
the presence of the same doses of BV-hFSH, an intriguing difference in
biological activity was observed (Fig. 4
, middle panel). The
recombinant hormone had no effect on cAMP accumulation except at the
highest dose tested, where it inhibited cAMP accumulation induced by
the ED50 of phFSH by 40%. In contrast, when the
ED80 of phFSH (representing high receptor-transducer
activation) was incubated in the presence of varying concentrations of
BV-hFSH, the recombinant hormone was strictly inhibitory, with a
maximal inhibition of phFSH-induced cAMP accumulation of 60% at a dose
of 1.0 µg/ml (Fig. 4
, bottom panel). Thus, when the
receptor is only partially activated (low concentrations of phFSH)
BV-hFSH displays agonistic activity, and accumulation of cAMP is
observed, which is consistent with the coupling of the FSH-R to
Gs. However, when the receptor is more fully activated
(higher concentrations of phFSH), BV-hFSH becomes antagonistic,
suggesting that at high receptor-transducer activation this hormone is
either capable of uncoupling the receptor from the stimulatory
signaling pathway and/or coupling it to an inhibitory cascade.

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Figure 4. The Biphasic Activity of BV-hFSH Is Dependent on
the Degree of Receptor-Transducer Activation
Top, 3D2 cells treated with the ED20 of
phFSH (representing low receptor-transducer activation) in the presence
of varying concentrations of BV-hFSH (11000 ng/ml).
Middle, 3D2 cells treated with the ED50 of
phFSH (representing medium receptor-transducer activation) in the
presence of varying concentrations of BV-hFSH. Bottom,
3D2 cells treated with the ED80 of phFSH (representing high
receptor-transducer activation) in the presence of varying
concentrations of BV-hFSH. Open bars denote cAMP
accumulation of cells treated with medium, whereas solid barsindicate cells treated with the respective concentration of phFSH
alone. *, P < 0.05 vs. phFSH alone
by ANOVA followed by the Dunnets test on log-transformed data.
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Taken together, these data suggest that glycosylated isoforms of FSH
confer particular conformations to the receptor that may involve
subsequent postreceptor mechanisms to produce the observed biphasic
responses to De-Gly- and BV-hFSH. Therefore, the role of activation of
hFSH-R/G protein-signaling pathways in transducing the signal of
alternatively glycosylated forms of FSH was studied. 3D2 cells were
challenged with FSH in the presence or absence of either cholera toxin
(CTX) or pertussis toxin (PTX), which selectively abolish either
Gs- or Gi/Go-mediated signal
transduction mechanisms, respectively. Pretreatment of 3D2 cells with
CTX completely blocked the ascending phase of the BV-hFSH dose-response
curve when performed in the presence of the ED20 of phFSH
(Fig. 5
, top panel, solid
symbols). Low concentrations of BV-hFSH (110 ng/ml) had no
effect on cAMP accumulation as compared with cells treated with the
ED20 alone. However, higher concentrations of BV-hFSH led
to a decrease in basal cAMP accumulation (ID50 = 2.02
µg/ml). Cells not pretreated with CTX responded to BV-hFSH in a
biphasic manner in the presence of the ED20 of phFSH (Fig. 5
, top panel, open symbols). Thus, CTX
pretreatment had no effect on the descending phase of the dose-response
curve, suggesting that this portion of the bioactivity of BV-hFSH is
independent of Gs-mediated signaling.

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Figure 5. CTX and PTX Differentially Abolish the Ascending or
Descending Components of BV-hFSH Bioactivity
Top, 3D2 cells pretreated 24 h with (solid
symbols) or without (open symbols) 2 µg/ml CTX
before challenge with the ED20 of phFSH in the presence of
varying concentrations of BV-hFSH (11,000 ng/ml).
Bottom, 3D2 cells pretreated 24 h with
(solid symbols) or without (open symbols)
5 µg/ml PTX before challenge with the ED80 of phFSH in
the presence of varying concentrations of BV-hFSH. The dashed
line denotes the mean of cells treated with the
ED20 and ED80 of phFSH alone with or without
toxins for the top and bottom panels,
respectively. *, P < 0.05 vs. phFSH
alone by ANOVA followed by the Dunnets test on log-transformed
data.
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Pretreatment of 3D2 cells with PTX completely blocked the inhibitory
effect of BV-hFSH in the presence of the ED80 of phFSH
(Fig. 5
, bottom panel, solid symbols). In cells
not treated with PTX, BV-hFSH was inhibitory to cAMP accumulation
induced by the ED80 of phFSH (Fig. 5
, bottom
panel, open symbols). As a confirmation of these
observations, we also tested the effect of PTX on BV-hFSH and phFSH
bioactivity in the absence of additional phFSH. As observed in Fig. 6
, in the absence of PTX (solid symbols),
phFSH (top left panel) produced a sigmoidal dose-dependent
increase in cAMP accumulation, while BV-hFSH (top right
panel) induced its typical biphasic dose-response relationship.
However, PTX pretreatment completely abolished the descending phase of
BV-hFSH bioactivity. In the presence of PTX (Fig. 6
, open
symbols), BV-hFSH (bottom right panel) induced a
sigmoidal dose-dependent increase in cAMP accumulation that was nearly
identical to that of phFSH (bottom left panel).
Interestingly, PTX treatment decreased the efficacy of both phFSH and
BV-hFSH, as well as forskolin (data not shown), to increase cAMP levels
approximately 6-fold when compared with control cells. Taken together,
these data suggest that the biphasic activity of BV-hFSH is mediated
through recruitment of differing G proteins.

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Figure 6. PTX Abolishes the Descending Phase of BV-hFSH
Bioactivity
Top left, 3D2 cells pretreated in the absence of PTX
before challenge with varying concentrations of phFSH (0.11,000
ng/ml). Top right, 3D2 cells pretreated in the absence
of PTX before challenge with varying concentrations of BV-hFSH
(0.110,000 ng/ml). Bottom left, 3D2 cells pretreated
24 h with 5 µg/ml PTX before challenge with varying
concentrations of phFSH. Bottom right, 3D2 cells
pretreated 24 h with 5 µg/ml PTX before challenge with varying
concentrations of BV-hFSH. *, P < 0.05
vs. the control group by ANOVA followed by the Dunnets
test on log-transformed data.
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DISCUSSION
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During recent years it has become apparent that the actions of FSH
on the gonad represent a multifaceted phenomenon. Rather than a classic
ligand-receptor interaction in which a single ligand docks onto a
specific receptor, the FSH-R is exposed to a multitude of FSH isoforms
with varying characteristics that are present in different proportions
depending on the physiological status (8). Therefore, integration
of complex signals represents a challenge to the target organ in
general and to the receptor in particular. Such a richness of signals
complicates our understanding of how these inputs are transduced. Many
studies, in partial explanation of the relative roles of different FSH
isoforms, have implicated differences in binding characteristics and
serum half-life as the major properties of diverse FSH isoforms (8).
However, studies aimed at evaluating whether the signal transduction
system has sufficient versatility to respond to diverse FSH signals are
scarce (20, 21). In our current studies, we have evaluated the role of
glycosylation in activation of downstream signaling components by the
FSH-R. Our data demonstrate a glycosylation-dependent coupling of the
ligand-receptor complex with multiple signaling pathways, which
provides evidence for the existence of a versatile coupling of the
FSH-R in conveying FSH signals to the gonad.
Many investigators have shown the importance of secondary protein
processing for bioactivity in several homeostatic systems (7, 24, 25).
Glycosylation patterns found in FSH and the FSH-R are likely to play a
significant role in ligand binding and possibly G protein coupling.
Under physiological conditions, FSH is found in serum of many species
as a myriad of different isoforms with differing pI values (7). The
differences in pI have been shown to be primarily due to
secretion of alternatively glycosylated forms of the hormone (10, 27). Therefore, the observed biological effects of FSH could be the
result of a highly complex interaction between many different factors.
For example, FSH bioactivity could depend upon the number of receptors
present on the cell surface, the concentration of FSH in the serum, as
well as the ratio of the isoforms of the hormone secreted. However,
these factors would transduce signals solely as positive inputs to the
target organ, since the FSH-R is thought to be coupled to a stimulatory
G protein pathway (5, 6). An alternative mechanism to provide
plasticity to the responsiveness of the target organ could conceivably
involve coupling of the FSH-R with inhibitory transduction pathways
(i.e. G proteins different than Gs). If such a
mechanism operates within the FSH-R-transducer system, then the ability
of the FSH-R to couple to different G proteins provides a means to
respond in a pleiotropic manner to a complex ligand, a mix of multiple
isoforms of FSH.
In our studies, we have taken advantage of chemically deglycosylated
hFSH and the altered secondary processing of newly synthesized proteins
in insect cells (26) to produce alternatively glycosylated forms of
hFSH. Studies of the expression of other proteins have shown that
insect cells have the ability to produce small truncated sugar
side chains in place of the more complex oligosaccharide structures
produced by cells of higher organisms (26). Using this paradigm, we
demonstrate that such alterations in glycosylation of FSH can have
profound effects on its biological activity. Whereas phFSH and
mammalian cell-expressed hFSH (CHO-hFSH) induced sigmoidal stimulation
in cAMP accumulation in 3D2 and granulosa cells, DeGly-phFSH and
BV-hFSH induced bell-shaped dose-response curves. Furthermore, the
differences in bioactivity between phFSH and alternatively glycosylated
FSH is evident at the membrane level, since adenylate cyclase activity
in a cell-free paradigm revealed similar differences in bioactivity
between phFSH and BV-hFSH. This may be due to an altered
receptor-ligand conformation, since we have also shown that
deglycosylation of the hormone had a notable effect on receptor
binding. Both BV-hFSH and DeGly-phFSH competed for the hFSH-R with
similar slope factors to each other, but different from that of phFSH,
as has been described by others (28). It has been proposed that
isoforms of hFSH or hLH could act in both a competitive or
noncompetitive manner with native hormone for interaction with the
receptor. In fact, chemically deglycosylated hFSH can antagonize the
effects of native FSH (24, 25). Similarly, deglycosylated hCG acts as a
noncompetitive antagonist (with equimolar affinity) to native LH,
suggesting that these two ligands do not share the same binding site(s)
(29). We have observed that, depending on the degree of
receptor-transducer system activation, the underglycosylated BV-hFSH
could behave either in a strictly inhibitory or stimulatory/inhibitory
manner simultaneously. Thus, our data have extended earlier
observations to clearly demonstrate that alternatively glycosylated
hFSH is not a true antagonist of the hFSH-R, but actually an analog
with partial agonistic activity capable of inducing the FSH-R to
activate other signaling pathways than those already established for
this receptor.
We hypothesized that the mechanism for the dual bioactivity of these
hormones is related to the ability of the ligands to stabilize certain
receptor conformations that permit interaction with multiple G
proteins. This was evaluated by the use of two ADP-ribosylating toxins,
PTX and CTX. These two toxins block either Gs- or
Gi/Go-activated signaling pathways,
respectively. Treatment of 3D2 cells with CTX specifically blocked the
ascending phase of BV-hFSH bioactivity, but did not abolish the
descending phase. In contrast, PTX completely and selectively blocked
the descending phase of BV-hFSH bioactivity. The two toxins, therefore,
differentially affected BV-hFSH-induced responses in 3D2 cells.
Interestingly, PTX treatment also altered the efficacy of both phFSH-
and BV-hFSH-induced cAMP accumulation. These observations imply that a
PTX-sensitive mechanism participates in maintenance of a fully
responsive transduction system. The reason(s) for these findings is not
readily apparent; however, two possible mechanisms could be invoked.
First, it is possible that either multiple PTX-sensitive G proteins
(e.g. Go) or another PTX-sensitive pathway
confers full responsiveness to the system. Alternatively, PTX-dependent
chronic activation of adenylate cyclase (due to the removal of tonic
Gi-dependent inhibitory inputs) could lead to
densensitization of the enzyme. The latter hypothesis is supported by
the observation that PTX treatment reduced forskolin responsiveness in
terms of cAMP production (data not shown); however, this observation
does not completely refute the first mechanism. Experiments are
currently underway in our laboratory to address these possibilities in
an attempt to discern whether other signaling pathways are involved in
maintaining the gain of the FSH/FSH-R transduction system.
Our data provide strong evidence that the FSH-R is capable of
activating alternate signaling cascades other than those activated
through Gs. Moreover, the ability of the FSH-R to associate
with alternative signaling molecules was dependent upon the degree of
receptor-transducer system activation. At low levels of activation,
BV-hFSH was capable of inducing both an ascending (stimulatory) or
descending (reduced activity) bioactivity profile. At a midrange of
receptor-transducer activation, addition of BV-hFSH did not increase
cAMP accumulation over levels observed with phFSH alone. Furthermore,
an inhibitory component was only identifiable at high concentrations of
BV-hFSH. Similarly, at high receptor-transducer activation, BV-hFSH was
inhibitory at all doses tested. Because in these studies cells were
exposed to a mix of differently glycosylated forms of FSH, these
conditions could resemble what occurs in vivo,
i.e. circulating FSH isoforms with different glycosylation
patterns. Taken together with the fact that biphasic responses were
also evident in primary granulosa cells, these data suggest that this
phenomenon may occur under physiological conditions. It is apparent
from our data that high ratios of fully to incompletely glycosylated
hormone result in positive intracellular signals, whereas lower ratios
convey inhibition. Overall, it is tempting to speculate that, while the
FSH receptor has an affinity for other G proteins, it has a higher
affinity for Gs. Therefore, our data would define the FSH-R
as a preferentially Gs-coupled receptor, but with capacity
to associate with other (Gi/Go) proteins as
well. This so-called promiscuity in receptor-signal transducer coupling
is well documented for catecholamine and adenosine receptors
(30, 31, 32). However, unlike the catecholamine and adenosine receptors in
which different ligands induce promiscuity, the ability of the FSH-R to
couple to multiple G protein signaling pathways appears to be dependent
on different physiological statuses (glycosylation) of the same ligand
(i.e. the FSH molecule). Promiscuity of the FSH-R for
coupling would be a physiologically relevant event by empowering the
signal transduction system to respond in a positive or negative
fashion, depending on the prevailing gonadotropic stimulus interacting
with the system.
In conclusion, these data provide evidence that the FSH-R is capable of
coupling with more than one G protein subtype and that its association
with other subtypes is dependent on the glycosylation pattern of the
ligand bound and the degree of receptor-transducer activation. Perhaps,
more importantly, our data provide some basis for a physiological role
of alternatively glycosylated isoforms of circulating FSH. That is,
depending on the prevailing physiological status of the subject, the
ovary may be presented with and respond to differing pleiotropic
signals from the pituitary that are sensed by the FSH-R and perceived
as activation of alternative signaling pathways.
 |
MATERIALS AND METHODS
|
---|
Isolation and Expression of hFSH cDNA in Hi5 Insect Cells
The cDNAs for FSH
and ß were isolated from human pituitary
poly-A+ RNA (Clontech, Palo Alto, CA) by RT-PCR using
gene-specific primers: FSH
1,
5'CGCGGATCCGCCATGGATTACTACAGAAAATATGC3'; FSH
2,
5'CGCAAGCTTAGCAGTCATCAAGACAGCAC3'; FSHß1,
5'CGCGGATCCCA-GGATGAAGACACTCCAG3'; FSHß2, 5'CGCAAGCT
TCAGGACAAGGGTATGTGGC3'. The cDNA fragments were cloned into pCRII
(Invitrogen, San Diego, CA) and sequenced to verify identity and
fidelity of the cloned fragments. The fragments included restriction
digestion sites (BamHI-hFSH-HindIII) for use in
subcloning into the corresponding sites of the baculovirus transfer
vector, pBluBacIII (Invitrogen). The resulting transfer vectors
containing hFSH
and hFSHß were cotransfected into Sf9 insect cells
with linearized AcNPV (Baculogold, Pharmingen, San Diego, CA) to
generate recombinant virus. The latter was purified by successive
rounds of plaque purification. A high titer viral stock was generated
by two rounds of infection with the purified viral stock. For
expression experiments, the high titer viral stock was used to infect
Hi5 cells (Invitrogen), cultured in serum-free medium using a
multiplicity of infection of 5 for each recombinant virus. The culture
supernatant was collected at 96 h postinfection and analyzed for
production of immunoreactive hFSH
/ß dimer by a hFSH IRMA and by
Western blot using antibodies specific for FSH
or FSHß. The
expression level of intact hFSH
/ß dimer ranged from 1 to 5
mg/liter.
Deglycosylation of phFSH
Deglycosylated phFSH was obtained from Dr. P. M. Sluss
(Massachusetts General Hospital, Charlestown, MA). Purified hFSH (2 mg,
Cortex Biochem, San Leandro, CA) was deglycosylated by a 60-min
exposure to hydrogen fluoride gas at room temperature. After exposure
to hydrogen fluoride, the deglycosylated (all but the N-linked sugar)
hormone was separated from all other carbohydrates by gel filtration
chromatography. The mass of the purified material was determined by
amino acid analysis and found to be consistent with acid hydrolysis of
phFSH. The deglycosylated phFSH had an apparent molecular mass of 29
kDa, with no larger forms of the protein detectable by SDS-PAGE
analysis. Dubois assay of phFSH revealed no detectable carbohydrate
beyond the N-linked sugar of a 20-µg aliquot. The remaining material
was lyophilized and reconstituted in 0.1 M acetic acid
before use in bioactivity studies.
Primary Culture of Granulosa Cells and Aromatase Bioassay
All procedures using animals were approved by the Radnor Animal
Care and Use Committee.Twenty one-day-old immature female
Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were
housed under controlled light (12-h light, 12-h dark) and temperature
(25 C) conditions. Food and water were available ad libitum.
Animals were treated by single daily injections of 100 µg/kg
diethylstilbestrol (DES) in olive oil for 3 days. On the fourth
day, animals were euthanized by rapid CO2 asphyxiation, and
the ovaries were removed. Ovaries were washed three times in 50 ml of
sterile HEPES-buffered saline (pH 7.4). Granulosa cells were harvested
by incubating ovaries in a serum-free hypertonic medium consisting of
McCoys 5A medium (GIBCO Life Sciences, Grand Island, NY) supplemented
with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite
(ITS, Sigma Chemical Co, St Louis, MO), 146 µg/ml
L-glutamine, 100 nM testosterone, 100
nM DES, and 100 U/ml penicillin/10 mg/ml streptomycin/250
ng/ml amphotericin B (antibiotic/antimycotic, GIBCO) containing
0.5 M sucrose (Sigma) and 0.1 mM EGTA (Sigma).
Ovaries were then incubated for 45 min at 37 C in a humidified
incubator gassed with 95% air/5% CO2. Subsequently,
ovaries were washed three times with 10 ml isotonic medium (hypertonic
medium without sucrose and EGTA) and incubated for an additional 45 min
in isotonic medium at 37 C. Granulosa cells were harvested by puncture
of swollen follicles using a 23 gauge needle. Isolated granulosa cells
were placed in a 50-ml centrifuge tube and washed two times by the
addition of 50 ml serum-free McCoys 5A medium followed by
centrifugation at 700 x g for 5 min. The final cell
pellet was resuspended by gentle trituration in 25 ml serum-free
isotonic medium. Cell number was determined using a hemocytometer, and
viability was estimated by trypan blue exclusion. Cells were plated
into 24-well Nunc (Naperville, IL) tissue culture plates at 100,000
viable cells per well.
The aromatase bioassay was performed according to the method of Hsueh
et al. (33). Briefly, cells were challenged with test
substances in isotonic McCoys 5A medium supplemented with 0.1% BSA
(Fraction V, Sigma), ITS, testosterone, DES, glutamine, and
antibiotic/antimycotic mix in a total incubation volume of 500 µl.
The cells were incubated for 72 h at 37 C with the test
substances. At the end of the challenge period, the medium was assayed
for estradiol concentration by RIA.
CHO Cell Line and cAMP Accumulation Assay
A CHO cell line expressing the hFSH-R was used to study effects
of FSH on receptor activation (kindly provided by Dr. Kerry Koller,
Affymax Inc., Palo Alto, CA). CHO cells were stably transfected with
the cDNA for the hFSH-R, which was cloned by RT-PCR from human ovarian
RNA. One clone (3D2) was found to express the hFSH-R and to respond to
phFSH with a dose-dependent stimulation in cAMP production. 3D2 cells
were maintained at 37 C in 1:1 DMEM/F12 medium supplemented with 10%
FBS (GIBCO), 146 µg/ml L-glutamine, and 100 U/ml
penicillin/10 µg/ml streptomycin. The cells were plated 1 day before
each experiment into 24-well or 96-well Nunc tissue culture plates at
200,000 or 30,000 cells per well, respectively.
FSH activation of the FSH-R was studied by monitoring cAMP
accumulation. Cells were washed twice with Optimem (GIBCO)/0.1% BSA.
After the second wash, cells were preincubated in either 500 µl
(24-well format) or 100 µl (96-well format) Optimem/0.1% BSA for 30
min at 37 C. The medium was removed from the wells, and the cells were
challenged for 30 min at 37 C in Optimem/0.1% BSA containing test
substances in a total incubation volume of 250 µl or 50 µl for the
24-well and 96-well formats, respectively. Experiments were terminated
by the addition of an equal volume of 0.2 N HCl, and cAMP
accumulation was measured by RIA.
Adenylate Cyclase Activity Assay
Adenylate cyclase assays were performed on isolated 3D2
cell membranes. 3D2 cells were grown to 90% confluency on 15-cm Nunc
tissue culture dishes in growth medium. The medium was removed and
cells were scraped from the plate into 30 ml FSH-binding buffer
(10 mM Tris-HCl, 1 mM MgCl2, 1
mM CaCl2, 0.1% BSA, and 0.025% sodium azide,
pH 7.2), and the cells were homogenized. The homogenate was centrifuged
at 15,000 x g for 10 min, and the pellet was
resuspended in binding buffer and centrifuged again. The supernatant
was discarded and the pellet resuspended to 100150 µg/ml protein in
binding buffer. At the start of the assay, 3D2 membranes were pelleted
as above and resuspended in a volume of membrane buffer (50
mM Tris-HCl, pH 7.2, 10 mM MgCl2,
and 2 mM EGTA) to give 2.5 mg membrane protein/ml. Assays
were performed in 96-well plates (Nunc). The following additions were
made to each well in order: 20 µl 0.25% BSA (Sigma), 20 µl of each
concentration of hormone in 0.25% BSA, 20 µl of a solution
containing 2,500 U/ml phosphocreatine kinase (Sigma), 50 mM
creatine phosphate (Sigma) and 0.5% BSA, 40 µl of buffer containing
450 mM Tris-HCl, pH 7.4, 40 mM
MgCl2 and 5 mM isobutylmethylxanthine (Sigma),
10 µl 2 mM GTP (Sigma), and 50 µl 4 mM ATP
(Sigma). Each hormone concentration was assayed in quadruplicate. The
plates were incubated for 10 min at 37 C. After the incubation, the
content of each well was rapidly transferred to a 1.5-ml centrifuge
tube and spun at 12,000 x g for 5 min at room
temperature. The supernatants were placed into fresh tubes and stored
at -20 C until assayed for cAMP by RIA.
Radioligand-Binding Assay
Binding assays were performed using the same 3D2 cell membrane
preparations used in the adenylate cyclase assays. To perform the
binding assay, 100 µl/well (100 µg membrane protein) of the 3D2
membrane homogenate were added to a 96-well microtiter plate followed
by the addition of 50 µl of either binding buffer (total binding),
phFSH, DeGly-phFSH, or BV-hFSH at varying concentrations. Nonspecific
binding was determined in the presence of 1 µM phFSH.
Reactions were initiated by the addition of 50 µl
[125I]phFSH (50 pM; 55,000 cpm, 35004500
Ci/mmol; NEN, Boston, MA) in binding buffer, for a final reaction
volume of 200 µl. Plates were incubated on an orbital shaker for
2 h at 25 C.
The binding assay was terminated by harvesting the cell membranes using
a 96-well vacuum harvester (Skatron Instruments, Inc, Sterling, VA)
onto presoaked (30 min in 50 mM Tris/1% BSA, pH 7.2)
Skatron Blue mat 11740 glass fiber filters. Harvesting was completed by
washing unbound radioactivity from the mats with five cycles of 3.5 ml
of 50 mM Tris-HCl (4 C). Filters were individually punched
out and the bound radioligand was determined by counting single disks
for 1 min in a
-counter (ICN Biomedical, Costa Mesa, CA).
RIAs
Estradiol levels in medium samples from the aromatase bioassay
were measured using a commercially available Coat-a-Count kit with
modifications (Diagnostic Products Corp., Los Angeles, CA). Medium
samples were preincubated in the presence of assay buffer (100 µl
total volume) in antibody-coated tubes for 1 h at 37 C and after
the addition of [125I]estradiol (1 ml), tubes were
incubated for 2 h at room temperature. The assay was terminated by
draining the tubes, and bound radioactivity was counted in an ICN
-counter for 1 min. This assay has a sensitivity of 0.25 pg/tube.
Intra- and interassay variability is 4.3% and 6.8%, respectively.
Cyclic AMP accumulation in the 3D2 cells was measured using a
commercially available double-antibody RIA kit with some modifications
(Amersham, Arlington Heights, IL). Medium samples were incubated in the
presence of tracer and primary antibodies for 1 h at room
temperature. Secondary antibodies were added, and the tubes were
incubated for 10 min at room temperature and centrifuged at 1000
x g for 15 min. The supernatant was drained and the pellets
counted in an ICN
-counter for 1 min. This assay has a sensitivity
of 2 fmol/tube. The intra- and interassay variation for this assay is
approximately 6.7% and 10.8%, respectively.
For the adenylate cyclase activity assay, an acetylation step was
performed on samples and standards before assay for cAMP as above
(Amersham). Using this protocol, the assay did not detect ATP at the
concentrations used in the adenylate cyclase activity reactions. This
assay has a sensitivity of 0.25 fmol/tube. The intra- and interassay
variation for the acetylated protocol is approximately 4.8 and 6.6%,
respectively.
To normalize for FSH concentration of phFSH, CHO-hFSH, and
DeGly-phFSH, as well as crude and purified Hi5 cell supernatants,
hormones were assayed using a hFSH IRMA. This assay was performed using
commercially available reagents (Diagnostic Products Corp.). Aliquots
of the hormones were serially diluted in assay buffer and assayed as
5-µl aliquots in 95 µl assay buffer in tubes coated with primary
hFSH antibodies. One milliliter of 125I-labeled secondary
hFSH antibodies was added to the tubes, and they were incubated on an
orbital shaker at room temperature for 1 h. Thereafter, the tube
content was discarded, and the tubes were washed twice by the addition
of 500 µl assay wash buffer per wash and then counted in an ICN
-counter for 1 min. This assay has a sensitivity of 0.019 mIU/tube.
The intra- and interassay variation is approximately 2.4% and 4.0%,
respectively.
Statistical Analysis
Statistical analyses were performed for bioassay data
using the SigmaStat software package (Jandel Scientific, San Raphael,
CA). Differences between treatment groups were analyzed by ANOVA.
Differences vs. the control group were analyzed after a
significant ANOVA by the Dunnets test. In some instances, data were
found to be skewed from normality or to have heterogeneous variance. In
such cases, log transformation of the data was performed. Differences
between treatment groups were considered significant if
P < 0.05.
Sigmoidal dose-response curves were fitted and ED50s
determined mathematically using a four-parameter logistic equation and
the SigmaPlot software (Jandel Scientific). Bell-shaped dose-response
curves were fitted and ED50s and ID50s
determined mathematically using a seven-parameter logistic equation as
described by Rovati et al. (34) with some modifications:
{((a - d1)/(1 + (x/c1)b1) +
d1) - ((a - d2)/(1 +
(x/c2)b2) + d2)}; where a =
asymptotic maximum, b1 = ascending slope factor,
c1 = ED50 of ascending portion of the curve,
d1 = asymptotic minimum for the ascending portion of the
curve, b2 = slope factor of the descending portion of the
curve, c2 = ID50 of descending portion of the
curve, and d2 = asymptotic minimum for the descending
portion of the curve.
Data from radioligand binding studies were analyzed using the JMP
software package (SAS Inc, Cary, NC). Square-root transformation of the
data was performed in conjunction with a Huber weighting procedure. A
four-parameter logistic equation was used to fit competition curves and
calculate ID50s from the transformed, weighted data.
 |
ACKNOWLEDGMENTS
|
---|
The authors gratefully acknowledge the technical assistance of
Karen Allegretto, Jennifer Herrlinger, and Christopher Bove of the WHRI
RIA Core Facility who performed the RIAs. We also offer our sincere
gratitude to Debra McMahon of the WHRI Cell Culture Core Facility for
maintaining the 3D2 cell line. We are indebted to the Wyeth-Ayerst
Molecular Biology Core Facility as well for production of CHO-hFSH and
purification of BV-hFSH, to Affymax Inc. for supplying the 3D2 cell
line, and to Dr. Krish Ghosh for development of JMP scripts used for
the analysis of radioligand-binding data. The authors thank Bela
Kanyicska for his invaluable discussions concerning G protein-coupled
receptors. F. López-Acosta dedicates this manuscript in loving
memory of his father.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Francisco J. López M.D., Ph.D., Womens Health Research Institute, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087.
1 Current Address: Ligand Pharmaceuticals, Inc., 10255 Science Center
Drive, San Diego, California 92121. 
Received for publication December 4, 1996.
Revision received February 6, 1997.
Accepted for publication February 18, 1997.
 |
REFERENCES
|
---|
-
Boothby M, Ruddon RW, Anderson C, McWilliams D, Boime I 1981 A single gonadotropin alpha-subunit gene in normal tissue and
tumor-derived cell lines. J Biol Chem 256:51215127[Abstract]
-
Kammerman S, Canfield RE, Kolena J, Channing CP 1972 The
binding of iodinated hCG to porcine granulosa cells. Endocrinology 91:6574[Medline]
-
Sairam MR 1981 Primary structure of the ovine pituitary
follitropin alpha-subunit. Biochem J 197:535539[Medline]
-
Reichert Jr LE 1994 The functional relationship between FSH
and its receptor as studied by synthetic peptide strategies. Mol Cell
Endocrinol 100:2127[CrossRef][Medline]
-
Rao AJ, Ramachandran J 1975 Cyclic AMP production in isolated
rat seminiferous tubule cell preparations: a potential in
vitro assay for follicle stimulating hormone. Life Sci 17:411416[CrossRef][Medline]
-
Dufau ML, Catt KJ 1978 Gonadotropin receptors and regulation
of steroidogenesis in the ovary. Vitam Horm 36:462585
-
Dahl K, Stone MP 1992 FSH isoforms, radioimmunoassays,
bioassays and their significance. J Androl 13:1122[Abstract/Free Full Text]
-
Ulloa-Aguirre A, Midgley AR, Beitins IZ, Padmanabhan V 1995 Follicle stimulating hormone isohormones-characterization and
physiological relevance. Endocr Rev 16:765787[Medline]
-
Reichert Jr LE 1971 Electrophoretic properties of pituitary
gonadotropins as studied by electrofocusing. Endocrinology 88:10291044[Medline]
-
Chappell SC, Coutifaris C, Jacobs SJ 1982 Studies on the
microheterogeneity of follicle-stimulating hormone present within the
anterior pituitary gland of ovariectomized hamsters. Endocrinology 110:847854[Medline]
-
Chappel SC, Bethea CL, Spies HG 1984 Existence of multiple
forms of follicle-stimulating hormone within anterior pituitaries of
cyngomolus monkeys. J Med Primatol 14:177194
-
Ulloa-Aguirre A, Espinoza R, Damian-Matsumura P, Larrea F,
Flores A, Morales L, Dominguez R 1988 Studies on the microheterogeneity
of anterior pituitary follicle-stimulating hormone in the female rat:
isoelectricfocusing throughout the estrous cycle. Biol Reprod 38:7078[Abstract]
-
Ulloa-Aguirre A, Damian-Matsumura P, Espinoza R, Dominguez R,
Morales L, Flores A 1990 Effects of neonatal androgenization on the
chromatofocusing pattern of anterior pituitary FSH in the female rat. J
Endocrinol 126:323332[Abstract]
-
Galle PC, Ulloa-Aguirre A, Chappell SC 1983 Effects of
oestradiol, phenobarbitone and luteinizing hormone releasing hormone
upon the isoelectric profile of pituitary follicle-stimulating hormone
in ovariectomized hamsters. J Endocrinol 99:3139[Abstract]
-
Chappel SC, Ulloa-Aguirre A, Ramaley J 1983 Sexual maturation
in female rats: time-related changes in the isoelectric focusing
pattern of anterior pituitary follicle-stimulating hormone. Biol Reprod 28:196205[Abstract]
-
Ulloa-Aguirre A, Mejia JJ, Dominguez R, Guevara-Aguirre J,
Diaz-Sanchez V, Larrea F 1986 Microheterogeneity of anterior pituitary
FSH in the male rat: isoelectric focusing pattern throughout sexual
maturation. J Endocrinol 110:539549[Abstract]
-
Morell AG, Gregoriadis G, Scheinberg IH 1971 The role of
sialic acid in determining the survival of glycoproteins in the
circulation. J Biol Chem 246:14611467[Abstract/Free Full Text]
-
Blum WFP, Gupta D 1985 Heterogeneity of rat FSH by
chromatofocusing: studies on serum FSH, hormone release in
vitro and metabolic clearance rates of its various forms. J
Endocrinol 105:2937[Abstract]
-
Wide L 1986 The regulation of metabolic clearance rate of
human FSH in mice by variation of the molecular structure of the
hormone. Acta Endocrinol (Copenh) 112:336344[Medline]
-
Sairam MR, Bhargavi GN 1985 A role of glycosylation of the
subunit in transduction of biological signal in glycoprotein hormones.
Science 229:6567[Medline]
-
Padmanabhan V, Sairam MR, Hassing JM, Brown MB, Ridings JW,
Beitins IZ 1991 Follicle-stimulating hormone signal transduction: role
of carbohydrate in aromatase induction in immature rat sertoli cells.
Mol Cell Endocrinol 79: 112119
-
Stanton PG, Robertson DM, Burgon PG, Schmauk-White B, Hearn
MTW 1992 Isolation and physicochemical characterization of human
follicle-stimulating hormone isoforms. Endocrinology 130:28202832[Abstract]
-
Burgon P, Robertson D, Stanton PG, Hearn M 1993 Immunological
activities of highly purified isoforms of human FSH correlate with
in vitro bioactivities. J Endocrinol 139:511518[Abstract]
-
Sairam MR, Manjunath P 1982 Studies on pituitary
follitropin. XII. Enhanced thermal stability induced by chemical
deglycosylation. Mol Cell Endocrinol 28:151159[CrossRef][Medline]
-
Sairam MR, Manjunath P 1982 Studies on pituitary follitropin.
XI. Induction of hormonal antagonistic activity by chemical
deglycosylation. Mol Cell Endocrinol 28:139150[CrossRef][Medline]
-
Kuroda K, Geyer H, Geyer R, Doefler W, Klenk H-D 1990 The
oligosaccharides of influenza virus hemagglutinin expressed in insect
cells by a baculovirus vector. Virology 174:418429[Medline]
-
Chappel SC, Ulloa-Aguirre A, Coutifaris C 1983 Biosynthesis
and secretion of follicle-stimulating hormone. Endocr Rev 4:179211[Medline]
-
Manjunath P, Sairam MR, Sairam J 1982 Studies on pituitary
follitropin. X. Biochemical, receptor binding and immunological
properties of deglycosylated ovine hormone. Mol Cell Endocrinol 28:125138[CrossRef][Medline]
-
Dunkel L, Jia X-C, Nishimori K, Boime I, Hsueh AJW 1993 Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG
stimulation of 3',5'-cyclic adenosine monophosphate accumulation
through a non-competitive interaction with recombinant human
luteinizing hormone receptors. Endocrinology 132:763769[Abstract]
-
Munshi R, Linden J 1989 Co-purification of A1 adenosine
receptors and guanine-nucleotide binding proteins from bovine brain.
J Biol Chem 264:1485314859[Abstract/Free Full Text]
-
Munshi R, Pang I-H, Sternweis PC, Linden J 1991 A1
adenosine receptors of bovine brain couple to guanine
nucleotide-binding proteins Gi1, Gi2, and
Go. J Biol Chem 266:2228522289[Abstract/Free Full Text]
-
Kimura K, White BH, Sidhu A 1995 Coupling of human D-1
dopamine receptors to different guanine nucleotide binding proteins:
evidence that D-1 dopamine receptors can couple to both Gs and Go.
J Biol Chem 270:1467214678[Abstract/Free Full Text]
-
Hsueh AJ, Bicsak TA, Jia X-C, Dahl KD, Fauser BCJM, Galway AB,
Czwkala N, Pavlou SN, Pakoff H, Keene J, Boime I 1989 Granulosa cells
as hormone targets: The role of biologically active
follicle-stimulating hormone in reproduction. Recent Prog Horm Res 45:209277[Medline]
-
Rovati GE, Nicosia S 1994 Lower efficacy: interaction with an
inhibitory receptor or partial agonism? Trends Pharmacol Sci 15:140144[CrossRef][Medline]