Derivation of Functional Antagonists Using N-Terminal Extracellular Domain of Gonadotropin and Thyrotropin Receptors
Yutaka Osuga,
Masataka Kudo,
Antti Kaipia,
Brian Kobilka and
Aaron J. W. Hsueh
Division of Reproductive Biology (Y.O., M.K., A.K., A.J.W.H.),
Department of Gynecology and Obstetrics and Howard Hughes Medical
Institute (B.K.), Stanford University Medical School, Stanford,
California 94305
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ABSTRACT
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Receptors for the glycoprotein hormones, LH/CG,
FSH, and TSH, are a unique subclass of the seven-transmembrane, G
protein-coupled proteins with a large N-terminal extracellular (ecto-)
domain. Although ecto-domains of gonadotropin receptors confer ligand
binding, expression of soluble binding proteins has been difficult. We
fused the ecto-domains of LH or FSH receptors to the
single-transmembrane domain of CD8 and found that hybrid proteins
anchored on the cell surface retained high-affinity ligand binding.
Inclusion of a junctional thrombin cleavage site in the hybrids allowed
generation of soluble receptor fragments that interfered with
gonadotropin binding to their receptors and blocked cAMP production
stimulated by gonadotropins. Cross-linking analyses confirmed the
formation of high molecular weight complexes between receptor
ecto-domains and their specific ligands. A similar approach also
generated a soluble TSH receptor fragment capable of blocking
TSH-induced signal transduction. When administered to rats, the soluble
FSH receptor fragment retarded testis growth and induced testis cell
apoptosis. These findings demonstrate the feasibility of generating
ligand-binding regions of glycoprotein hormone receptors to selectively
neutralize actions of gonadotropins and TSH, thus allowing future
design of novel contraceptives and management of different gonadal and
thyroid dysfunction. The present study represents the first successful
derivation of soluble, ligand-binding domains from glycoprotein hormone
receptors as functional antagonists. Similar approaches could allow
generation of ecto-domains of related receptors to neutralize actions
of ligands or receptor antibodies and to facilitate
structural-functional analysis.
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INTRODUCTION
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Gonadotropins stimulate gonadal development following activation
of specific target cell receptors. Gonadotropin receptors belong to the
large family of G protein-coupled receptors characterized functionally
by their interaction with guanine nucleotide-binding proteins and
structurally by their seven hydrophobic,
-helical transmembrane
domains. Members of this superfamily are functionally diverse and
include receptors ranging from the cAMP receptor in slime mold to
mammalian neurotransmitter and glycoprotein hormone receptors (1).
The glycoprotein hormone receptor subfamily, including receptors for
LH/CG, FSH, TSH, diverges structurally from other G protein-coupled
receptors in having a large N-terminal extracellular (ecto-) domain
required for interaction with the large glycoprotein hormones (2, 3, 4).
These ecto-domains of glycoprotein hormone receptors confer ligand
specificity and are homologous to those of the leucine-rich repeat
protein family, including the ribonuclease inhibitor (5). In addition
to gonadotropin and TSH receptors, several other G protein-coupled
receptors also have large ecto-domains. Included are those in the
secretin/VIP/calcitonin/CRF/PTH receptor family (6, 7, 8), the
metabotropic glutamate receptor (9), and parathyroid calcium
ion-sensing receptors (10). For the PTH/PTH-related peptide receptor,
the ecto-domain is also important for ligand binding based on studies
using chimeric receptors (11).
Many receptors for hormones of the hematopoietin family have soluble
isoforms derived either from alternative mRNA splicing (receptors for
granulocyte colony-stimulating factor, interleukin-4,
leukemia-inhibiting factor, etc.) or from proteolytic cleavage of the
extracellular region of the receptor (receptors for interleukin-1,
tumor necrosis factor, etc.) (reviewed in 12 . Furthermore,
isolation of circulating GH-binding proteins indicated that they are
truncated forms of tissue receptors with deletion of the transmembrane
region (13). Among these truncated receptors, some of them act as
functional antagonists. In contrast to soluble fragments derived from
these receptors with a single transmembrane domain, generation of the
ecto-domains of seven-transmembrane, G protein-coupled receptors as
soluble binding proteins has been difficult. Although the large
ecto-domains of gonadotropin receptors confer ligand binding, truncated
gonadotropin receptors are trapped intracellularly (14, 15).
Using an anchored fusion receptor approach followed by proteolytic
cleavage, we have generated ligand-binding regions of gonadotropin and
TSH receptors as soluble binding proteins to neutralize the action of
specific glycoprotein hormones. This approach could allow structural
analysis of the ligand binding region of glycoprotein hormone receptors
as well as future design of contraceptives and novel treatment
protocols for gonadal and thyroid dysfunction associated with aberrant
secretion of glycoprotein hormones.
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RESULTS
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Anchoring of Ligand Binding Regions of Gonadotropin Receptors and
Derivation of Soluble Receptor Fragments
Using PCR-based mutagenesis, we fused the ecto-domains of human
FSH and LH receptors to the single-transmembrane domain of CD8 through
a thrombin cleavage site and named them as FtCD8 and LtCD8,
respectively (Fig. 1A
). Human 293 cells
transfected with FtCD8 or LtCD8 showed a high-affinity cell surface
binding to their specific ligand with expression levels comparable to
those of the wild type receptors (Fig. 1B
). For anchored FtCD8, the
binding affinity to radiolabeled FSH was 3-fold higher than that of the
wild type receptor, whereas anchored LtCD8 showed a binding affinity
comparable to that of the wild type receptor (Kd values:
FtCD8, 0.31 nM; wild type FSH receptor, 1.03
nM; LtCD8, 0.61 nM; wild type LH receptor, 0.28
nM). The expression levels of anchored chimeric receptors
are about 50% of that found for the wild type receptors. These results
demonstrated that the ecto-domains of gonadotropin receptors can be
expressed on the cell surface independent of the seven-transmembrane
region and still retain their ligand-binding ability.

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Figure 1. Ecto-domains of FSH and LH Receptors Anchored
on the Plasma Membrane Retain High-Affinity Binding to Specific
Ligands: Derivation of Hormone-Specific Binding Proteins following
Thrombin Cleavage
A, Diagram of wild type and anchored receptors, FtCD8 and LtCD8. The
ligand-binding ecto-domains of human gonadotropin receptors were fused
to the single transmembrane region of CD8 through a thrombin cleavage
site. B, Binding kinetics of wild type FSH receptor and FtCD8, as well
as wild type LH receptor and LtCD8. After transfection with cDNAs
encoding chimeric or wild type receptors, 293 cells were incubated with
labeled ligands (FSH or hCG) to determine equilibrium binding constants
using Scatchard plot analysis. C, Cross-linking of labeled ligands to
solubilized ecto-domains of anchored receptors following thrombin
cleavage. Cells expressing wild type or anchored receptors were treated
with 10 IU/ml -thrombin before incubation of conditioned media with labeled ligands followed by
cross-linking with disuccinimidyl suberate. Thrombin treatment was
omitted for control groups whereas some groups were incubated with
excess nonlabeled ligands to demonstrate binding specificity. Migration
patterns of molecular mass markers and labeled ligands are shown. D,
Competition of complex formation between solubilized ecto-domains of
gonadotropin receptors and their specific labeled ligands by increasing
doses of nonlabeled gonadotropins. wt FSHR, Wild type FSH receptor; wt
LHR: wild type LH receptor.
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To solubilize ligand-binding regions of these anchored receptors, we
treated cells expressing FtCD8 or LtCD8 with
-thrombin to release
gonadotropin receptor ecto-domains. Conditioned medium was incubated
with labeled ligands followed by cross-linking with disuccinimidyl
suberate. As shown in Fig. 1C
, labeled FSH migrated as a 45 kDa band
after gel electrophoresis, whereas the conditioned media from
FtCD8-expressing cells pretreated with thrombin showed a higher
molecular mass band (90 kDa), indicating the formation of complexes
between labeled FSH and the ecto-domain of FSH receptor. Complex
formation was inhibited with the inclusion of excess nonlabeled FSH and
was not found in cells expressing wild type receptors or without
thrombin pretreatment. Likewise, thrombin pretreatment allowed the
formation of a 105 kDa complex between labeled human (h) CG and the
ecto-domain of LH receptor (Fig. 1C
). The deduced large size of the
ecto-domains of both gonadotropin receptors is consistent with our
studies using N-glycanase treatment, suggesting that these
molecules are fully glycosylated (data not shown).
To demonstrate that the solubilized receptor fragments still retained
high-affinity binding to their specific ligand, competition analyses
were performed. As shown in Fig. 1D
, addition of increasing amounts of
nonlabeled ligand competed for complex formation in a dose-dependent
manner, and the kDa values for soluble FSH and LH receptor ecto-domains
were estimated to be 200 pM and 2 nM,
respectively. These results demonstrated that high-affinity
ligand-binding domains of gonadotropin receptors could be generated
using the anchored receptor approach. The soluble receptor
fragments were named FBP (FSH-binding protein) and LBP (LH/hCG-binding
protein), respectively.
Receptor Fragments Blocked Gonadotropin Receptor Ligand
Signaling
We used solubilized ligand-binding regions of FSH and LH
receptors to block gonadotropin actions in vitro. In a
radioligand binding assay, 293 cells expressing wild type FSH receptors
were incubated with labeled FSH in the presence or absence of
increasing concentrations of FBP or LBP. As shown in Fig. 2A
, addition of FBP prevented binding of
labeled FSH to the wild type receptors in a dose-dependent manner,
reaching a level comparable to nonspecific binding. In contrast,
inclusion of LBP was ineffective. Furthermore, inclusion of the same
preparations of receptor fragments in the hCG binding assay using wild
type LH receptors led to a dose-dependent inhibition of binding by LBP
but not by FBP (Fig. 2B
). Thus, FBP and LBP specifically prevented the
binding of their respective ligands. We further evaluated the ability
of these binding proteins to interfere with signal transduction induced
by gonadotropins. 293 cells expressing wild type FSH or LH receptors
were incubated with increasing concentrations of FSH or hCG for 3
h at 37 C to stimulate cAMP production. As shown in Fig. 2C
, addition
of FBP completely blocked cAMP production induced by FSH in FSH
receptor-expressing cells whereas LBP was ineffective. Conversely,
addition of LBP inhibited cAMP stimulation by hCG in LH
receptor-expressing cells whereas FBP was ineffective (Fig. 2D
). Thus,
these binding proteins are capable of selective blockage of
gonadotropin-induced signal transduction in vitro.

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Figure 2. Competition for Receptor Binding and
Neutralization of Gonadotropin Actions in Vitro by Soluble
Ecto-domains of FSH and LH Receptors
A, Competition of labeled FSH binding to FSH receptors by soluble
ecto-domain of FSH receptor (FBP) but not LH receptor (LBP). Cells
expressing human FSH receptors were incubated with labeled FSH with or
without increasing amounts of FBP or LBP. B, Competition of labeled hCG
binding to LH receptors by LBP but not FBP. Cells expressing LH
receptors were incubated with labeled hCG with or without LBP or FBP.
C, Antagonism of FSH stimulation of cAMP production by FBP but not LBP.
Cells expressing FSH receptors were incubated with increasing amounts
of FSH in the presence or absence of FBP or LBP (107 cells
equivalent per well) for 3 h at 37 C before cAMP determination by
RIA. Concentration of binding proteins was determined based on their
ability to inhibit the binding of respective labeled ligands to wild
type receptors. D, Antagonism of hCG stimulation of cAMP production by
LBP but not FBP. Cells expressing human LH receptors were incubated
with hCG with or without LBP or FBP.
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Solubilized TSH Receptor Fragment Interfered with TSH Induction of
cAMP Production
We used the same anchored receptor approach to generate the
soluble ecto-domain of TSH receptor and named it TBP. Although the
exact binding affinity of TBP to TSH could not be adequately evaluated
due to high nonspecific binding of the tracer (4), ligand cross-linking
analysis indicated that soluble TBP released from cells transfected
with a plasmid encoding anchored ecto-domain of human TSH receptor
(TtCD8) formed high molecular mass (105 kDa) complexes with labeled TSH
(Fig. 3A
). Complex formation was blocked
following the addition of excess unlabeled TSH. Furthermore, TSH
stimulation of cAMP production by cells expressing wild type TSH
receptors was substantially blocked by cotreatment with TBP but not
with LBP coaddition (Fig. 3B
), whereas the same TBP preparation was
ineffective in interfering with the hCG stimulation of cAMP in LH
receptor-expressing cells (Fig. 3C
).

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Figure 3. Ecto-domain of Human TSH Receptor Is a
Functional Antagonist
To generate TSH-binding proteins, 293 cells expressing TtCD8 were
treated with thrombin as described for anchored gonadotropin receptors.
A, Cross-linking analysis. Conditioned media from cells expressing
TtCD8 were incubated with labeled bovine TSH (10,000 cpm/tube) followed
by cross-linking with disuccinimidyl suberate. SDS-PAGE analysis
indicated the presence of a high molecular mass complex of 97 kDa. In
contrast, incubation with excess nonlabeled TSH (1 µg/tube)
completely blocked complex formation. Migration patterns of molecular
mass markers are shown. B, Neutralization of TSH stimulation of cAMP by
TBP. Cells expressing wild type TSH receptors were incubated with
increasing amounts of TSH in the presence or absence of TBP or LBP
(107 cells equivalent per well) for 3 h at 37 C before
determination of total cAMP production using RIA. C, Antagonism of hCG
stimulation of cAMP production by LBP but not by TBP. Cells expressing
human LH receptors were incubated with hCG with or without LBP or
TBP.
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FSH Receptor Fragment Suppressed Testis Function in Rats
To test whether FBP could be used as a functional antagonist
in vivo, we scaled up the production of FBP using a
baculovirus expression system. Experimental data indicated that FBP
produced by FtCD8-expressing insect cells retained its ability to
inhibit the binding of labeled FSH to wild type FSH receptors in
vitro. We administered conditioned media from insect cells
containing FBP into immature male rats. Treatment with FBP every 6
h for 2 days attenuated testis growth by 33% (untreated, 192 ±
28 mg; FBP-treated, 128 ± 30 mg; n = 14). Analysis of testis
DNA fragmentation using a 3'-end-labeling method followed by gel
electrophoresis further indicated major increases in testis cell
apoptosis in the FBP-treated group, as evidenced by the appearance of
internucleosomal DNA fragmentation (Fig. 4A
). In rats treated with a GnRH
antagonist to suppress both LH and FSH secretion, comparable decreases
in testis weight (140 ± 18 mg) and increases in apoptotic DNA
fragmentation were found. In contrast, no retardation of testis growth
(201 ± 26 mg) or alteration of testis cell apoptosis was seen in
rats treated with conditioned media of insect cells infected with the
wild type baculovirus. In situ apoptosis analysis further
indicated that DNA fragmentation was restricted to primary
spermatocytes in rats treated with FBP or the GnRH antagonist but not
found in untreated rats (Fig. 4B
, arrowhead). These results
suggested that FBP is capable of neutralizing the action of endogenous
FSH, which is essential for testis germ cell survival (16, 17).

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Figure 4. Induction of Testis Cell Apoptosis following
FBP Administration in Vivo
For expression of FtCD8 in insect cells, recombinant baculovirus was
prepared using the Bac-to-Bac baculovirus expression system, and SF9
cells were infected with the recombinant viruses. After a 72-h culture
at 27 C in media containing 5% serum and thrombin, cells were
centrifuged and conditioned media concentrated before filtration with
0.2-µm filters. Immature male rats (21 days of age) were injected
subcutaneously (every 6 h) with conditioned media of SF9 cells
(108 cells equivalent per injection) containing soluble
FBP. Two days later, testis weight was determined and testis cell
apoptosis quantitated using a 3'end-labeling method. Some animals were
treated with a potent GnRH antagonist (ANT; 50 µg/rat) to suppress
pituitary release of LH and FSH whereas others were treated with
conditioned media of SF9 cells infected with wild type baculovirus.
In situ analysis of specific cell types undergoing
apoptosis was performed using the Apotag kit. A, Pattern of apoptotic
DNA fragmentation (two samples per group). B, In situ
analysis of DNA fragmentation (bar length, 25 µm).
Apoptotic primary spermatocytes are indicated by
arrowheads.
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DISCUSSION
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The present studies demonstrate that an anchored fusion receptor
approach can be used to generate soluble ligand-binding regions of
glycoprotein hormone receptors of the seven-transmembrane protein
family. The soluble receptor fragments are capable of binding specific
ligands and blocking signal transduction induced by FSH, LH/hCG, and
TSH, thus serving as potent functional antagonists. Reproductive
dysfunction due to aberrant gonadotropin secretion (such as polycystic
ovarian syndrome and central precocious puberty) has been treated with
potent GnRH analogs to suppress serum levels of both LH and FSH. The
availability of FBP and LBP capable of specific neutralization of
circulating gonadotropins could allow formulation of novel treatment
protocols for patients with these diseases and could be used for
treating patients with ovarian hyperstimulation syndrome by
neutralizing the endogenous gonadotropins (18). In addition, FBP and
LBP may be useful in the treatment of gonadal steroid-dependent
prostate and breast cancers. Similar to the gonadotropin-binding
proteins, the soluble TSH-binding protein can be used to block excess
stimulation of thyroid function by endogenous TSH in patients with
TSH-secreting tumors.
The present receptor antagonists bind specific hormones and are
unlikely to show agonistic activity in direct contrast to hormone
antagonists that interact with target organ receptors and usually
exhibit agonistic activity at high concentrations. Furthermore,
receptor antagonists do not have to be delivered to the target tissues
and, due to their similarity to wild type receptors, probably have low
antigenicity. Although immunization against hCG and other gonadotropins
has been proposed as a potential immunocontraceptive device, the immune
approach suffers from irreversibility and large individual variability.
If the delivery system is optimized, the present gonadotropin-binding
proteins could serve as reversible and reliable contraceptives in both
sexes. Of interest, chimeric molecules containing the Fc region of IgG
and the extracellular ligand-binding region of single-transmembrane
receptors for tumor necrosis factor, hepatic growth factor, and related
hormones, have been shown to be useful receptor antagonists (19, 20, 21).
Indeed, splicing variants of GH receptors containing only the
extracellular region are natural binding proteins found in circulation
(13).
Stimulatory and inhibitory antibodies against the ecto-domain of TSH
receptor cause thyroid dysfunction (22, 23), whereas premature ovarian
failure has also been associated with circulating antibodies against
FSH receptors (24, 25). The present anchored receptor approach might
allow future generation of receptor fragments for the treatment of
different autoimmune disorders. Patients with Graves disease could be
treated with mutant forms of TBP defective in TSH binding but still
capable of neutralizing the stimulatory effect of the stimulating IgG
because the binding domains for TSH and Graves IgG on the ecto-domain
of the TSH receptor are not identical (26).
Many other seven-transmembrane, G protein-coupled receptors also have
large ecto-domains, including receptors for the PTH family (6, 7, 8),
metabotropic glutamate receptor (9), and parathyroid calcium
ion-sensing receptor (10). The ecto-domains of PTH (11) and CRF
receptors are important for ligand binding, but the exact role of this
region for other receptors is unclear. It is likely that the
ecto-domains of many of these receptors can be generated using the
present approach to neutralize the action of endogenous ligands and
allow modulation of vascular (receptors for calcitonin gene-related
peptide and vasoactive intestinal peptide), metabolic (receptors for
glucagon and glucagon-like peptide 1), and other conditions. Recent
data suggested that the entry of human immunodeficiency virus into
target cells (27, 28) requires several seven-transmembrane chemokine
receptors that, similar to the related C5a receptor, also contain
ecto-domains believed to be involved in ligand recognition (29). Thus,
the present approach could allow characterization of the ecto-domains
for diverse G protein-coupled receptors and potential derivation of
soluble binding proteins to neutralize the actions of specific ligands,
antibodies, or viral proteins.
The present study demonstrates successful derivation of soluble,
ligand-binding domains of receptors in the large G protein-coupled
receptor family to serve as functional antagonists. Expression of the
ecto-domains of glycoprotein hormone receptors and related proteins
would also allow analysis of interactions between the ligand-binding
region and the transmembrane, signal transduction domain (30). In
addition, the generation of soluble receptor fragments is useful for
elucidating the three-dimensional structure of ligand-binding regions
for gonadotropin, TSH, and other seven-transmembrane receptors.
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MATERIALS AND METHODS
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Construction of Chimeric Receptors
Chimeric receptor cDNAs were generated using overlapping PCR and
confirmed by dideoxy sequencing. For chimeric receptor FtCD8, the
ecto-domain of human FSH receptor (amino acids 1 to 358; 31 was
fused to the transmembrane and cytoplasmic region of CD8 (amino acid
162 to C terminus; 32 through a stretch of the thrombin receptor
sequence using the plasmid SK-ATE-CD8 containing the thrombin cleavage
site (amino acids 36 to 66 of thrombin receptor) (33, 34). The
resulting junction between FSH receptor and thrombin receptor encoded
the sequence (. ...NPCED/ATLDP. ...), whereas the junction
between thrombin receptor and CD8 encoded the sequence
(. ...NESGL/IYIWA. ...). For LtCD8, the ecto-domain of human LH
receptor (amino acids 1 to 355; 35 was also fused to CD8 through
a stretch of the thrombin receptor sequence. The resulting junction
between LH receptor and thrombin receptor encoded
(. ...NPCED/ATLDP. ...), whereas the junction between thrombin
receptor and CD8 remained the same as that in FtCD8. For TtCD8, the
ecto-domain of human TSH receptor (amino acids 1 to 410; 4 was
fused to CD8 through a stretch of the thrombin receptor sequence and
the resulting junction encoded (. ...NPCED/ATLDP. ...). For
expression of wild type and chimeric receptors in human embryonic
kidney 293 cells, receptor cDNAs were subcloned into pcDNA3
(Invitrogen, San Diego, CA).
Cell Culture and Transient Expression of Wild Type and Chimeric
Receptors
Cell culture and transient expression were performed as
previously described (36). Briefly, 293 cells were cultured in the
DMEM/F12 medium supplemented with 10% serum and antibiotics. After
replacement of the medium by DMEM supplemented with 10% serum and
antibiotics, 293 cells were transfected using 10 µg expression vector
containing wild type and chimeric receptor cDNAs by the calcium
phosphate precipitation method (37).
RRA and Cross-Linking Analysis
Human CG (CR129) and human (h) FSH (NIDDK-I-1) were obtained
from the National Hormone and Pituitary Agency (Baltimore, MD) and
iodinated using the lactoperoxidase method as previously described
(36). The specific activities of the tracers were approximately 100,000
cpm/ng for [125I]-FSH and approximately 50,000 cpm/ng for
[125I]hCG. Cells transiently transfected with wild type
receptors were incubated with specific radioligand in 300 µl PBS
containing 0.1% BSA at 23 C for 18 h. Nonspecific binding was
determined by adding excess unlabeled hormones (100 IU hCG/tube or 4 IU
FSH/tube). Following estimation of optimal binding conditions, RRAs for
cells transfected with FtCD8 and LtCD8 were performed at 23 C for
5 h and 2 h, respectively. After incubation, cells were
washed twice followed by centrifugation and determination of
radioactivity using a
-counter. To determine equilibrium binding
constants (Kd), cells were incubated with labeled ligands
(FSH or hCG) with increasing amounts of unlabeled hCG or FSH. Data from
displacement analysis were analyzed using Scatchard plots (38).
To solubilize the ecto-domain of gonadotropin receptors, 293 cells
expressing anchored chimeric receptors were treated with
-thrombin
(10 IU/ml for 1 h) before incubation of the conditioned media
(10 x concentrated using Centricon 30, Amicon, Bedford, MA) with
saturating amounts of radiolabeled ligands. Media from
FtCD8-transfected cells were incubated with labeled FSH (150,000
cpm/tube) at 23 C for 5 h whereas media from LtCD8-transfected
cells were incubated with labeled hCG (100,000 cpm/tube) at 4 C for
1618 h. For studies on solubilized TSH receptor fragment, media from
TtCD8-transfected cells were incubated with 125I-labeled
bovine TSH (Kronus, San Clemente, CA) for 5 h at 23 C. Complexes
formed between labeled hormones and solubilized receptor fragments were
cross-linked using disuccinimidyl suberate (2 mM) for
1 h, and the reaction was terminated by the addition of 3.6
mM Tris-HCl, pH 7.4. After the addition of Laemmeli buffer
without reducing reagents, cross-linked complexes were visualized
following fractionation using PAGE (10%) and autoradiography. In some
experiments, increasing amounts of nonlabeled hCG or FSH were included,
and radioactivities corresponding to high molecular mass complexes were
determined to derive a competition curve for the estimation of
Kd values (38).
Cyclic AMP Production
To analyze the ability of solubilized gonadotropin receptor
fragments to interfere with signal transduction induced by FSH or hCG,
293 cells expressing wild type FSH or LH receptors were incubated in
DMEM/F12-0.1% BSA with increasing amounts of specific ligands in the
presence or absence of conditioned media from cells transfected with
FtCD8 or LtCD8 (107 cells equivalent per well). After
treatment for 3 h at 37 C, total cAMP production was determined by
RIA (39). The concentration of solubilized receptor fragments from each
preparation was estimated based on its ability to inhibit the binding
of respective labeled ligands to wild type receptors. Similar
approaches were used to evaluate the ability of solubilized TSH
receptor fragments to interfere with cAMP production induced by human
recombinant TSH (Genzyme, Cambridge, MA) in 293 cells transiently
expressing human TSH receptors.
In Vivo Studies
To generate large amounts of FSH receptor fragments, we
expressed FtCD8 in insect cells. FtCD8 cDNA was subcloned into pFast
Bac vector, and recombinant baculovirus was prepared using the
Bac-to-Bac baculovirus expression system (GIBCO-BRL, Gaithersburg, MD)
according to the protocol of the manufacturer. SF9 cells (3 x
106/ml) in the SF900II medium containing 5% serum were
infected with the recombinant viruses. After a 72-h culture at 27 C in
media containing 5% serum and thrombin (10 IU/ml), cell debris was
centrifuged and removed. The conditioned media were concentrated 30
times using DIAFLO ultrafiltration membrane XM50 (Amicon) and filtered
with 0.2 µm filters (UNIFLO, Schleicher & Schuell, Keene, NH).
Immature Sprague-Dawley male rats (21 days of age) were injected
subcutaneously (every 6 h) with conditioned media of SF9 cells
(108 cells equivalent per injection) containing solubilized
FSH receptor fragments. Based on the ability to displace binding of
labeled FSH to wild type FSH receptors, the amount of FBP in the
conditioned media was equivalent to 2 µg FSH/injection. Two days
later, testis weight was determined and testis cell apoptosis
quantitated using a 3'-end-labeling method (16). Some animals were
treated with a potent GnRH antagonist (Org30850, 50 µg/rat) to
suppress pituitary release of both LH and FSH (40) whereas others were
treated with conditioned media of SF9 cells infected with the wild type
baculovirus. SF9 cells infected with the wild type baculovirus were
treated with thrombin followed by ultrafiltration using identical
conditions as for the preparation of FBP. In situ analysis
of specific cell types undergoing apoptosis was performed using Apotag
kit (Oncor, Gaithersburg, MD) as previously described (17). Animal care
was in accordance with institutional guidelines.
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ACKNOWLEDGMENTS
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We thank Dr. Shaun Coughlin (UC San Francisco) for providing the
thrombin receptor-CD8 construct, Dr. G. Vassart (Belgium) for TSH
receptor cDNA, Dr. H. Kloosterboer for the GnRH antagonist, and
Emerald Perlas for technical assistance.
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FOOTNOTES
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Address requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5317.
This study was supported by NIH Grant HD-23273.
Received for publication May 19, 1997.
Revision received July 8, 1997.
Accepted for publication July 11, 1997.
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REFERENCES
|
---|
-
Lefkowitz RJ, Caron MG 1988 Adrenergic receptors. Models
for the study of receptors coupled to guanine nucleotide regulatory
proteins. J Biol Chem 263:49934996[Free Full Text]
-
McFarland K, Sprenge R, Phillips S, Rosemblit N,
Nikolics K, Segaloff DL, Seeburg P 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G
protein-coupled receptor family. Science 245:494499.[Medline]
-
Loosfelt H, Misrahi M, Atger M 1989 Cloning and sequencing of
porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.
Science 245:525528[Medline]
-
Nagayama Y, Rapoport B 1992 The thyrotropin receptor 25 years
after its discovery: new insight after its molecular cloning. Mol
Endocrinol 6:145156[Abstract]
-
Kobe B, Deisenhofer J 1993 Crystal structure of porcine
ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366:751756[CrossRef][Medline]
-
Juppner H 1994 Molecular cloning and characterization of a
parathyroid hormone/parathyroid hormone-related peptide receptor: a
member of an ancient family of G protein-coupled receptors. Curr Opin
Nephrol Hypertens 3:371378[Medline]
-
Chen R, Lewis K, Perrin MH, Vale WW 1993 Expression cloning
of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci
USA 90:89678971[Abstract]
-
Lin HY, Harris TI, Flannery MS, Kaji E, Gorn A, Lodish
HF, Goldberg SR 1991 Expression cloning of an adenylate cyclase-coupled
calcitonin receptor. Science 254:10221024[Medline]
-
Takahashi K, Tsuchida K, Tanabe Y, Masu M, Nakanishi S 1993 Role of the large extracellular domain of metabotropic glutamate
receptors in agonist selectivity determination. J Biol Chem 268:1934119345[Abstract/Free Full Text]
-
Brown EM, Pollak M, Seidman CE, Seidman JG, Chou YH, Riccardi
D, Hebert SC 1995 Calcium-ion-sensing cell-surface receptors. N
Engl J Med 333:234240[Free Full Text]
-
Juppner H, Schipani E, Bringhurst FR, McClure I, Keutmann HT,
Potts Jr JT, Kronenberg HM, Abou-Samra AB, Segre GV, Gardella TJ 1994 The extracellular amino-terminal region of the parathyroid hormone
(PTH)/PTH-related peptide receptor determines the binding affinity for
carboxyl-terminal fragments of PTH-(134). Endocrinology 134:879884[Abstract]
-
Heaney ML, Golde DW 1993 Soluble hormone receptors. Blood 82:19451948[Medline]
-
Leung EW, Spencer SA, Cachianes G, Hammonds G, Collins C,
Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor
and serum binding protein: purification, cloning and expression. Nature 330:537543[CrossRef][Medline]
-
Tsai-Morris CH, Buczko E, Wang W, Dufau ML 1990 Intronic
nature of the rat luteinizing hormone receptor gene defines a soluble
receptor subspecies with hormone binding activity. J Biol Chem 265:1938519388[Abstract/Free Full Text]
-
Xie YB, Wang H, Segaloff DL 1990 Extracellular domain of
lutropin/choriogonadotropin receptor expressed in transfected cells
binds choriogonadotropin with high affinity. J Biol Chem 265:2141121420[Abstract/Free Full Text]
-
Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJW 1993 Hormonal
control of apoptotic cell death in the testis: gonadotropins and
androgens as testicular cell survival factors. Mol Endocrinol 7:643650[Abstract]
-
Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M,
Hsueh AJW 1995 Apoptosis in testis germ cells: developmental
changes in gonadotropin dependence and localization to selective tubule
stages. Endocrinology 136:512[Abstract]
-
Fauser BC, Van Heusden AM 1997 Manipulation of human ovarian
function: physiological concepts and clinical consequences. Endocr Rev 18:71106[Abstract/Free Full Text]
-
Eason JD, Wee S, Kawai T, Hong HZ, Powelson JA, Widmer MB,
Cosimi AB 1995 Inhibition of the effects of TNF in renal allograft
recipients using recombinant human dimeric tumor necrosis factor
receptors. Transplantation 59:300305[Medline]
-
Mark MR, Lokker NA, Zioncheck TF, Luis EA, Godowski PJ 1992 Expression and characterization of hepatocyte growth factor
receptor-IgG fusion proteins. Effects of mutations in the potential
proteolytic cleavage site on processing and ligand binding. J Biol
Chem 267:2616626171[Abstract/Free Full Text]
-
Peppel K, Crawford D, Beutler B 1991 A tumor necrosis factor
(TNF) receptor-IgG heavy chain chimeric protein as a bivalent
antagonist of TNF activity. J Exp Med 174:14831489[Abstract]
-
Tomer T, Davies TF 1993 Infection, thyroid disease, and
autoimmunity. Endocr Rev 14:107120[Abstract]
-
Magner JA 1990 Thyroid-stimulating hormone: biosynthesis, cell
biology, and bioactivity. Endocr Rev 11:354385[Medline]
-
Chiauzzi V, Cigorraga S, Escobar ME, Rivarola A, Charreau EH 1982 Inhibition of follicle-stimulating hormone receptor binding by
circulating immunoglobulins. J Clin Endocrinol Metab 54:12211228[Abstract]
-
Dias JA, Gates SA, Reichert Jr LE 1982 Evidence for the
presence of follicle-stimulating hormone receptor antibody in human
serum. Fertil Steril 38:330338[Medline]
-
Kosugi S, Ban T, Akamizu T, Kohn LD 1992 Identification of
separate determinants on the thyrotropin receptor reactive with
Graves thyroid-stimulating antibodies and with thyroid-stimulating
blocking antibodies in idiopathic myxedema: these determinants have no
homologous sequence on gonadotropin receptors. Mol Endocrinol 6:168180[Abstract]
-
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima
KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton WA 1996 HIV-1 entry
into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667673[CrossRef][Medline]
-
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M,
DiMarzio P, Marmon S, Sutton RG, Hill CM 1996 Identification of a major
co-receptor for primary isolates of HIV-1. Nature 381:661666[CrossRef][Medline]
-
Wells TNC, Proudfoot AE, Power CA, Lusti-Narasimhan M, Alouani
S, Hoogewerf AJ, Peitsch MC 1996 The molecular basis of the
chemokine/chemokine receptor interactionScope for design of chemokine
antagonists. Methods: A Companion to Methods in Enzymology 10:126134[CrossRef]
-
Ji I, Ji TH 1991 Exons 110 of the rat LH receptor encode a
high affinity hormone binding site and exon 11 encodes G-protein
modulation and a potential second hormone binding site. Endocrinology 128:26482650[Abstract]
-
Tilly JL, Aihara T, Nishimori K, Jia XC, Kowalski KI, Perlas
EA, Hsueh AJW 1992 Expression of recombinant human follicle-stimulating
hormone receptor: species-specific ligand binding, signal transduction,
and identification of multiple ovarian messenger ribonucleic acid
transcripts. Endocrinology 131:799806[Abstract]
-
Littman DR, Thomas Y, Maddon PL, Chess L, Axel R 1985 The
isolation and sequence of the gene encoding T8: a molecule defining
functional classes of T lymphocytes. Cell 40:237246[Medline]
-
Vu TK, Hung DT, Wheaton VI, Coughlin SR 1991 Molecular cloning
of a functional thrombin receptor reveals a novel proteolytic
mechanism of receptor activation. Cell 64:10571068[Medline]
-
Chen J, Ishii M, Wang L, Ishii K, Coughlin S 1994 Thrombin
receptor activation. Confirmation of the intramolecular tethered
liganding hypothesis and discovery of an alternative intermolecular
liganding mode. J Biol Chem 269:1604116045[Abstract/Free Full Text]
-
Jia X-C, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJW 1991 Expression of human luteinizing hormone (LH) receptor: interaction
with LH and chorionic gonadotropin from human but not equine, rat, and
ovine species. Mol Endocrinol 5:759768[Abstract]
-
Kudo M, Osuga Y, Kobilka BK, Hsueh AJW 1996 Transmembrane
regions V and VI of the human luteinizing hormone receptor are required
for constitutive activation by a mutation in the third intracellular
loop. J Biol Chem 271:2247022478[Abstract/Free Full Text]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Scatchard G 1949 The attraction of proteins for small
molecules and ions. Ann NY Acad Sci 51:660666
-
Davoren JB, Hsueh AJW 1985 Vasoactive intestinal peptide: a
novel stimulator of steroidogenesis by cultured rat granulosa cells.
Biol Reprod 33:3752[Abstract]
-
Decker GH, de Graaf JH, Kloosterboer HJ, Loozen HJ 1992 Properties of a potent LHRH antagonist (Org 30850) in female and male
rats. J Steroid Biochem Mol Biol 42:705712[CrossRef][Medline]