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(Received for publication, September 1, 1995; and in revised form, November 7, 1995) From the
The rat lutropin/choriogonadotropin receptor (rLHR) is a G
protein-coupled receptor which binds either human choriogonadotropin
(hCG) or lutropin (luteinizing hormone, LH) and, therefore, plays a
central role in reproductive physiology. In addition to the seven
transmembrane helices, three extracellular loops, three intracellular
loops, and a cytoplasmic tail characteristic of all G protein-coupled
receptors, the rLHR also contains a relatively large N-terminal
extracellular domain. Since high affinity hormone binding occurs to
this N-terminal extracellular domain and since G proteins are activated
by intracellular regions of the receptor, it has been hypothesized that
upon hormone binding a portion of the hormone or the receptor's
extracellular domain might interact with the receptor's
extracellular loops and/or transmembrane helices, thus evoking an
intracellular conformational change. To explore this possibility, we
prepared and characterized several mutants of the rLHR in which
portions of the extracellular loops were deleted. Ultimately, it was
not possible to examine the signal transduction properties of the
mutants because all but one mutant were retained intracellularly.
Although the intracellularly retained mutants must be somewhat
misfolded, all were found to bind hCG with high affinity if the cells
were first solubilized in detergent. However, the binding of oLH to the
detergent solubilized mutants was altered. Thus, whereas the wild-type
rLHR bound oLH with two apparent affinities, the solubilized deletion
mutants bound oLH with only one apparent affinity. Although these data
could be interpreted to suggest that an ovine LH (oLH) binding site on
the extracellular loops of the rLHR was deleted, data shown argue
against this hypothesis. Rather, the results presented suggest that the
two apparent affinities of the wild-type rLHR for oLH represent the
binding affinities of two populations of rLHR where the mature, cell
surface form binds oLH with a higher affinity than the immature,
intracellular form. Furthermore, we show that mutations of the rLHR
which cause intracellular retention of the receptor result in a
decrease from two to one apparent binding sites for oLH due to the
absence of the high affinity oLH binding component contributed by the
mature cell surface receptor. Therefore, whereas hCG cannot
discriminate between the mature cell surface wild-type receptor and an
intracellularly retained rLHR mutant, oLH can make this discrimination,
thus suggesting a conformational difference between the two forms of
the receptor.
The rat lutropin/choriogonadotropin receptor (rLHR) ( In addition to the
high affinity binding of hormone to the large N-terminal domain of the
rLHR, there is some evidence that the extracellular loops of the rLHR
may play a role in low affinity interactions with the hormone. Using
short synthetic peptides that represent extracellular portions of the
rLHR, it was shown that micromolar concentrations of a peptide
corresponding the third extracellular loop were able to inhibit the
binding of hCG to the rLHR, suggesting that hCG may bind with low
affinity to the third extracellular loop(11) . Also, rLHR
truncation mutants expressing the C-terminal portion of the receptor
and either a 10- or 49-amino acid N-terminal extension were able to
bind hCG with low affinity and stimulate cAMP production at high
concentrations of hCG(12) . Both of these studies suggest a
potential low affinity interaction of hCG with the extracellular loops
of the rLHR. To further examine whether the extracellular loops of
the rLHR play a role in the binding of glycoprotein hormones, we
created a series of rLHR extracellular loop deletion mutants, the
hypothesis being that if hormone interacted with a loop or a portion
thereof, deletion of a portion of that loop would disturb hormone
binding. The data presented demonstrate that, while mutations within
all three extracellular loops of the rLHR do not affect hCG binding to
the rLHR, they reduce the affinity of the rLHR for oLH from two
apparent affinities to one apparent affinity. However, we further show
that the reduction in oLH binding to one apparent affinity is not due
to the deletion of a binding site, but instead due to the absence of
the mature population of the rLHR.
For the creation of stable cell lines, cells were transfected as
described above. Sixteen to twenty hours following transfection, cells
were washed as described above and then plated with growth medium
containing 700 µg/ml geneticin. For nonclonal stables such as
rLHR-wt and rLHR-E3M, surviving cells were pooled, maintained in growth
medium containing 700 µg/ml geneticin, and utilized for
experiments. Clonal stable cell lines rLHR-wt12 and rLHR-wt16 were
kindly donated by Mario Ascoli (University of Iowa).
For
Western blots performed on temperature shifted cells, solubilized
extracts (0.5% Nonidet P-40, final concentration) were prepared from
293 cells stably transfected with rLHR-wt16 and rLHR-E3M preincubated
at 37 or 26 °C and from 293 cells stably expressing the empty pcDNA
I/neo vector as described for soluble binding assays. High molecular
weight standards and extracts were loaded onto a 7% polyacrylamide gel,
where each lane received an equal amount of hCG binding activity (2.6
ng of hCG bound per lane for extracts containing rLHR-wt and 5.1 ng of
hCG bound per lane for extracts containing rLHR-E3M).
To measure binding to intact cells, dishes were
placed on ice for 10 min and subsequently washed twice with cold
Waymouth's MB752/1 medium lacking bicarbonate, but supplemented
with 1 mg/ml BSA and 50 µg/ml gentamicin (assay medium). Dishes
were incubated overnight at 4 °C with a saturating concentration
(100 ng/ml, final concentration) of For
the soluble binding assays, cells were placed on ice for 15 min and
washed with cold buffer A (150 mM NaCl and 20 mM HEPES, pH 7.4). The cells were then scraped into cold buffer A
containing protease inhibitors(19) . Cells were collected by
centrifugation at 4 °C (1,500
For experiments with GTP For the
determination of hCG and oLH binding affinities to intact cells
expressing rLHR-wt, competition experiments were performed. Cells were
washed twice with cold assay medium, and then incubated overnight at 4
°C with a trace concentration of
To examine whether the extracellular loops of the rLHR are
involved in the binding of gonadotropin hormones, a series of deletion
mutants were created in which small portions, three to six amino acids
at a time, were removed from regions of the extracellular loops. The
deduced amino acid sequence of the rLHR and the specific amino acids
deleted in each mutant are shown in Fig. 1. The E in
the designations of the mutants is to indicate that these are deletions
in the extracellular loops. The number defines whether it is the first,
second, or third loop relative to the N terminus, and N, M, and C refer to the N-terminal, middle, and
C-terminal portions of each extracellular loop, respectively. For
example, rLHR-E1M corresponds to the full-length rLHR in which the
middle portion of the first extracellular loop of the rLHR was deleted.
Figure 1:
rLHR extracellular loop deletion
mutants. The orientation (4) and proposed topology of the
deduced amino acid sequence for the rLHR (1) is pictured with
the shaded area representing the transmembrane regions. Above
the transmembrane regions lies the extracellular portions of the rLHR,
and below lies the intracellular portion of the receptor. rLHR
extracellular loop deletion mutants were prepared as described under
``Materials and Methods'' and are outlined by heavy ovals where each oval represents a separate mutation. The specific amino
acids deleted for each mutant are the following: Gln
Initially, it was important to determine if the rLHR extracellular
loop deletion mutants were expressed. As shown in Fig. 2,
Western blotting of solubilized extracts from transiently transfected
293 cells revealed that all nine mutants were stably expressed. The
wild-type rLHR appears as a mature 85-kDa protein and an immature
68-kDa precursor protein(18, 22) . In addition to the
specific rLHR bands at 85 and 68 kDa, a prominent band appears between
the 85- and 68-kDa bands in the wild-type rLHR and in all the
extracellular loop deletion mutants (Fig. 2). However, this band
represents a nonspecific interaction of the antibody with an
unidentified protein since it was also detected in the untransfected
293 cells (Fig. 2). Further examination of the Western blot of
the rLHR extracellular loop deletion mutants reveal that E2N migrates
as two proteins with the same molecular masses as the wild-type rLHR.
All the other mutants, however, appear only as the smaller 68-kDa
protein (Fig. 2).
Figure 2:
Expression of rLHR mutant proteins by
Western blotting. Detergent solubilized extracts from 293 cells
transiently expressing the wild-type rLHR or one of the rLHR mutants
were run under non-reducing conditions on 7.5% SDS-gels and transferred
to poly(vinylidene fluoride) membranes. The membranes were probed with
a polyclonal antibody to the full length rLHR as described under
``Materials and Methods''(18) . Only the relevant
portion of the gel is shown.
The following experiments were performed to
determine if the receptor mutants could bind hCG and to determine if
they were properly localized to the cell surface or not. To address
these questions, intact 293 cells transiently expressing the
extracellular loop deletion mutants and solubilized extracts thereof
were tested for their ability to bind a saturating concentration of
The next set of experiments tested the crucial question concerning
the potential effects of the deletion of small portions of the rLHR
extracellular loops on the affinity of the receptor for hormone.
Previously, it was shown that hCG binds to the rLHR with picomolar
affinity(1) . It was also demonstrated that the extracellular
domain alone binds hCG with an affinity comparable to that of the
full-length rLHR(8) . Because of these prior results, the
possibility exists that even if hCG interacted with the extracellular
loops with low affinity, a decrease in the affinity for hCG in the
solubilized extracts of cells expressing the extracellular loop
deletion mutants might not be detected because of the high affinity
interaction of hCG with the N-terminal 340 amino acids of the rLHR.
Consequently, we chose to measure the affinity of the receptor mutants
for oLH, as well as for hCG, because oLH has a much lower affinity for
the rLHR(25) . To measure the binding affinities of the
wild-type rLHR and deletion mutants for hCG and oLH, competition assays
were performed using solubilized extracts from transiently transfected
293 cells using unlabeled hCG or oLH to displace
Figure 3:
Binding of hCG to the wild-type rLHR.
Detergent-solubilized extracts from 293 cells transiently expressing
the wild-type rLHR were incubated with a trace concentration of
Figure 4:
Binding of oLH to rLHR-wt and rLHR-E1M.
Detergent solubilized extracts from 293 cells transiently transfected
with the cDNA for rLHR-wt or rLHR-E1M were incubated with a trace
concentration of
Thus far, the shift from two apparent affinities to one apparent
affinity observed for all the extracellular loop deletion mutants, with
the exception of rLHR-E2N, could be explained by the deletion of a
binding site for oLH on the extracellular loops. However, reevaluation
of the Western blot in Fig. 2, cell surface expression data in Table 1, and the oLH binding affinity data in Table 3shows
that rLHR-E2N is the only extracellular deletion mutant that is
expressed as both the mature 85-kDa and the immature 68-kDa forms of
the receptor, that is expressed at the cell surface at levels
comparable to rLHR-wt, and that binds oLH with two apparent affinities.
These observations suggest an alternative hypothesis to explain the
shift from two apparent affinities to one affinity observed for all the
extracellular loop deletion mutants other than rLHR-E2N. Thus, the
introduction of mutations into the extracellular loops of the rLHR may
result in the improper folding and retention of the mutant receptors
intracellularly. This retention may result in a lack of processing of
the mutants to the mature 85-kDa cell surface receptor which may be
reflected by the absence of high affinity oLH binding. To examine this
further, the following experiments were performed. If the loss of
high affinity oLH binding to the intracellularly retained rLHR mutants
is due to the absence of the fully processed cell surface rLHR, one
would predict that expression of the extracellular loop deletion
mutants at the cell surface should restore high affinity oLH binding.
Incubation of some mislocalized proteins such as the
Figure 5:
Western blot of extracts from rLHR-wt and
rLHR-E3M-expressing cells incubated at 37 or 26 °C.
Detergent-solubilized extracts prepared from rLHR-wt or rLHR-E3M
expressing cells incubated at 37 or 26 °C were run under
nonreducing conditions on 7% SDS-gels and transferred to
poly(vinylidene fluoride) membranes. The membranes were probed as
described under ``Materials and Methods'' with the polyclonal
antibody Bugs prepared to the purified rLHR. Only the relevant portion
of the gel is shown.
Figure 6:
Binding of oLH to extracts from
rLHR-E3M-expressing cells incubated at 37 °C or 26 °C.
Nonclonal stably transfected 293 cells expressing rLHR-E3M were
incubated for 48 h at either 37 or 26 °C as described under
``Materials and Methods.'' Detergent-solubilized extracts
were prepared from these cells and incubated with a trace concentration
of
To further
examine the hypothesis that the absence of high affinity oLH binding
for the intracellularly retained mutants is due to the absence of the
mature, cell surface rLHR, a search was undertaken to identify other
rLHR mutants that were mislocalized intracellularly similar to the
extracellular loop deletion mutants, but that did not involve the
alteration of a putative hormone binding site. One such mutant,
rLHR-t616, is a truncation mutant of the cytoplasmic tail of the rLHR
after amino acid 616. One would predict that a truncation of the
cytoplasmic tail should not alter a hormone binding site.
Figure 7:
Binding of oLH to the truncation mutant
rLHR-t616. Detergent-solubilized extracts from 293 cells transiently
expressing rLHR-t616 were incubated with a trace concentration of
Thus far, the data suggest that a mature, cell surface rLHR is
required for high affinity oLH binding. The question remains, though,
whether the two apparent affinities for oLH represent two binding sites
for oLH present on the mature, cell surface rLHR, or whether the two
apparent affinities represent the binding affinities for two
populations of rLHR, where the 85-kDa cell surface population of rLHR
binds oLH with a high affinity, and the 68-kDa intracellular population
of rLHR binds oLH with a lower affinity. To attempt to discern if the
mature, cell surface rLHR binds oLH with two apparent binding
affinities or with single high binding affinity, equilibrium binding
assays were performed on intact rLHR-wt-expressing cells with the
rationale that only the mature, fully processed rLHR should be at the
cell surface and be able to bind hormone. As shown in Table 6,
intact cells expressing rLHR-wt bound oLH with two apparent affinities.
However, a comparison of the distribution of high and low affinity
sites observed with intact cells expressing rLHR-wt as compared to
solubilized extracts thereof revealed distinct differences in the
percentage of total receptor with a high or low affinity (Table 6). As measured in intact cells, approximately 90% of oLH
binding to rLHR-wt was of high affinity while <10% was of low
affinity. In contrast, when assayed in detergent solubilized extracts,
only 60% binding to rLHR-wt was of high affinity, while 40% was of low
affinity (Table 6). These data suggest that the cell surface rLHR
is predominantly in the high affinity form and that the cell surface
low affinity binding component is most likely contributed to by the
presence of a small percentage of incompletely folded rLHR on the cell
surface.
Taken altogether, these data clearly indicate that the
shift from two apparent oLH binding affinities for rLHR-wt to one
apparent affinity with most of the extracellular loop deletion mutants
is not due to the deletion of an oLH binding site on the rLHR. Instead,
deletion of portions of the extracellular loops and even part of the
cytoplasmic tail most likely perturb the normal folding of the rLHR
resulting in the retention of the mutant receptors intracellularly and
the absence of the mature cell surface receptor. These data also
suggest that the observation of two binding affinities for the binding
of oLH to rLHR-wt is not due to the presence of two binding sites on
the mature rLHR for oLH, but is instead most likely due to the presence
of two populations of rLHR with different binding affinities for oLH
where the mature, 85-kDa cell surface form of the receptor binds oLH
with a higher affinity than the immature, 68-kDa intracellular form of
the receptor. The present study was undertaken to identify potential low
affinity binding sites for hormone on the extracellular loops of the
rLHR. There is much evidence for the involvement of the extracellular
loops of other G protein-coupled receptors in ligand binding. For
example, experiments in which portions of the neurokinin-1 receptor
were substituted with the corresponding sequences of the neurokinin-2
and -3 receptors revealed that the first and second extracellular loops
of the neurokinin-1 receptor, in addition to a portion of the
N-terminal tail and transmembrane 3, bind substance P (30, 31) In addition, Glu-301 of the third
extracellular loop of the mammalian gonadotropin-releasing hormone
receptor has recently been shown to be involved in an electrostatic
interaction with Arg-8 of gonadotropin-releasing hormone(32) .
The second and third extracellular loops of the interleukin-8 type A
receptor have also been demonstrated to be part of a ligand binding
site(33, 34) . Although it has been shown that
hormone binds with high affinity to the N-terminal extracellular domain
of the
rLHR(8, 35, 36, 37, 38) ,
low affinity interactions of hormone with the extracellular loops
and/or transmembrane helices of the rLHR have also been
suggested(11, 12, 18, 25) . For
example, a low affinity interaction of hCG with the third extracellular
loop of the rLHR was suggested by the finding that micromolar
concentrations of a synthetic peptide corresponding to the third
extracellular loop of the rLHR was able to inhibit hCG binding to the
full-length rLHR(11) . In addition, it was reported that a rLHR
truncation mutant expressing the C-terminal portion of the receptor and
either a 10- or 49-amino acid N-terminal extension was able to bind hCG
with micromolar affinity and to stimulate low levels of cAMP production (12) . However, without data to demonstrate that this
truncation mutant was properly located to the plasma membrane, it is
difficult to distinguish such a low binding affinity for hCG and low
cAMP stimulation from nonspecific effects. Studies by Ascoli and
co-workers found that a substitution of Asp-383 for Asn in the second
transmembrane helix of the rLHR produced a receptor that had a
decreased affinity for oLH in the absence of sodium(25) ,
suggesting a binding site for oLH on the C-terminal half of the rLHR.
Lastly, our laboratory has shown that, unlike the full-length rLHR
which binds oLH with two apparent affinities, the rLHR extracellular
domain alone binds oLH with a single affinity(18) . In the
present studies, we examined the effects of deletions of portions of
the extracellular loops of the rLHR on hCG and oLH binding affinities.
Western blotting revealed that all of the mutants were expressed as
stable proteins. However, unlike rLHR-wt, which is expressed as both
the mature 85-kDa and immature 68-kDa forms, all the mutants, with the
exception of rLHR-E2N, were expressed as only the 68-kDa precursor form
of the rLHR. In addition, all the extracellular loop deletion mutants,
with the exception of rLHR-E2N, were severely or completely retained
intracellularly. Binding assays to detergent solubilized extracts of
cells showed that although the affinities for hCG were unaffected by
deletions of any portion of any of the extracellular loops, deletions
of portions of all the extracellular loops with the exception or
rLHR-E2N resulted in altered oLH binding. A shift from two apparent
binding affinities of oLH for rLHR-wt to a single binding affinity for
oLH was observed for all the mutants with the exception of rLHR-E2N.
The results of experiments presented herein, therefore, at first
suggested that the extracellular loops of the rLHR were contributing to
the binding of oLH. However, further experiments confirmed the
correlation between the presence of two binding affinities for oLH and
the expression of the cell surface 85-kDa form of the receptor. Taken
altogether, these data suggest that the shift from two apparent oLH
binding affinities for rLHR-wt to a single binding affinity for the
most of the mutants is not due to the loss of an oLH binding site on
the rLHR, but is due to the absence of the fully folded, cell surface
form of the receptor. We hypothesize that the mutations are causing a
misfolding of the receptor that causes them to be retained
intracellularly, thus preventing the further folding and processing of
the mutant receptor into the mature cell surface form. Our data also
show that the observation of two oLH binding affinities measured for
rLHR-wt does not represent two binding sites on an individual receptor.
Rather, the two oLH binding affinities for rLHR-wt represent the
binding affinities of oLH for two populations of rLHR. The 85-kDa
mature cell surface form of the rLHR binds oLH with a higher affinity,
while the 68-kDa intracellular precursor form binds oLH with a lower
affinity. For certain other G protein-coupled receptors, in
particular the adrenergic receptors, the affinity of agonist for the
receptor can be affected by the receptor's association with a G
protein(39) . This is manifest by the ability of exogenously
added GTP to decrease the binding affinity of agonist for the
receptor(39) . The possibility that perhaps the two apparent
binding affinities of the rLHR for oLH were arising from solubilization
of one pool of rLHR associated with G proteins and one pool not
associated with G proteins was, therefore, also considered. It is
generally not possible to observe a change in hCG binding affinity to
membranes containing the rLHR upon addition of
GTP(40, 41, 42, 43, 44) .
This is thought to be due to the high affinity interactions of hCG with
the extracellular domain of the rLHR(8) , a situation quite
unlike the binding of catecholamines to the transmembrane helices of
adrenergic receptors(45) . Thus, whereas it can be envisioned
how an intracellular conformational change in an adrenergic receptor
accompanying G protein activation could lead to an increased
dissociation of catecholamine from their binding site, a similar
conformational change in the rLHR would not be as likely to result in a
dissociation of hCG from the extracellular binding domain. The
possibility remained, however, that the binding affinity of oLH, which
is much lower than that of hCG for the rLHR, might be affected by G
protein activation, especially if there were additional contact sites
of oLH on the carboxyl half of the receptor. If so, then this might
also account for the two populations of oLH binding affinities observed
in detergent extracts of cells expressing the wild-type rLHR. To test
this possibility, we examined the effects of 200-300 µM GTP Previously, our laboratory reported that
rLHR-t338, a rLHR truncation mutant at amino acid 338 that expresses
only the N-terminal extracellular domain of the rLHR, was retained
intracellularly and bound oLH with a single apparent
affinity(18) . Based upon these observations, we proposed that
the loss of high affinity oLH binding was due to the loss of an oLH
binding site on the C-terminal half of the rLHR(18) . However,
the evidence presented herein suggests that the absence of high
affinity oLH binding to rLHR-t338 is most likely due to the misfolding
of this mutant resulting in its retention intracellularly and lack of
folding and processing to a mature form. The data presented herein
clearly illustrate important differences in the binding of hCG and oLH
to the rLHR. Unlike oLH, hCG is not capable of distinguishing between
the immature and mature forms of the rLHR. Consistent with reports on
other intracellularly retained rLHR mutants (5, 23, 24, 46) , hCG binds to the
intracellularly trapped extracellular loop deletion mutants with the
same high affinity as it binds to the cell surface form of the rLHR as
long as a hormone binding site has not been disturbed. Therefore,
although each of these mutations is causing a degree of misfolding
resulting in intracellular retention of the mutant protein, it cannot
be a gross perturbation of structure since hCG can still bind with
normal high affinity. Because hCG binds with equally high affinities to
the mature and immature forms of the rLHR, one might conclude that the
immature form of the rLHR was folded in the same conformation as the
mature cell surface form. However, our observations that oLH binds with
lower affinity to the immature receptor suggest otherwise and argue
that although the immature receptor is folded completely enough to bind
hCG, it must not be in exactly the same conformation as the mature cell
surface receptor. An interesting comparison can be made between
mutants of the rLHR and those of the rFSHR, a receptor which shares a
high degree of amino acid identity with the rLHR(47) . In this
respect, our laboratory has recently demonstrated that mutations of the
rFSHR which cause intracellular retention of the mutant rFSHR in the
endoplasmic reticulum result in a lack of detectable FSH binding even
if a hormone binding site has not been altered(48) . For
example, rFSHR-t637 is a truncation mutation of the cytoplasmic tail of
the rFSHR at amino acid 637 that, similar to rLHR-t616, should
theoretically not perturb a hormone binding site. Similar to rLHR-t616,
rFSHR-t637 is retained intracellularly, as evidenced by its sensitivity
to endoglycosidase H. Detergent-solubilized extracts of cells
expressing rFSH-t637, however, have no detectable FSH binding activity.
Thus, whereas intracellularly retained mutants of the rLHR show
comparable high affinity hCG binding and reduced oLH binding affinity
as compared to the cell surface mature rLHR, intracellularly retained
mutants of the rFSHR lack any detectable binding activity. Assuming
that mutants of the rLHR and rFSHR are retained in the same
intracellular compartment (i.e. the endoplasmic reticulum),
these observations suggest that the newly synthesized rLHR in the
endoplasmic reticulum is already folded into a conformation that can
bind hCG with high affinity. It must still be in a conformation
distinct from the cell surface mature receptor, though, since it does
not yet have the same high affinity oLH binding as the mature rLHR. In
contrast, newly synthesized rFSHR in the endoplasmic reticululum does
not appear to be folded appropriately enough to allow for any hormone
binding. Taken altogether, the data on intracellularly retained mutants
of the rLHR and rFSHR suggest that the rLHR folds more readily than the
highly related rFSHR. Clearly more studies will be required in order
to better understand the complex folding and maturation of the
gonadotropin receptors, as well as other G protein-coupled receptors.
Toward this end, the differential binding of oLH to mature versus immature rLHR should prove useful in elucidating the folding of
the rLHR.
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4518-4527
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is a 674-amino acid single polypeptide chain belonging to
the superfamily of G protein-coupled receptors(1) . Similar to
other members of this superfamily such as the adrenergic (2) and tachykinin receptors(3) , the rLHR is thought
to consist of seven transmembrane regions connected by alternating
intracellular and extracellular loops with an extracellular N-terminal
domain and an intracellular C-terminal tail(1, 4) .
The rLHR is also a member of the glycoprotein hormone receptor family,
a subfamily of the G protein-coupled receptors that binds large
28-38-kDa ligands(5) . This subfamily consists of the
LHR, follitropin receptor (FSHR), and the thyrotropin receptor. Unlike
the adrenergic receptors, which have a short N-terminal extension and
have been shown to bind their ligands within their transmembrane
helices(6, 7) , the glycoprotein receptors have the
unusual feature of a large, approximately 340-amino acid, N-terminal
domain which has been demonstrated to bind hormones with high
affinity(8, 9, 10) .
Supplies
Highly purified hCG (CR-125 and CR-127)
and oLH (NIADDK-oLH-26) were kindly provided by the National Hormone
and Pituitary Agency of the NIDDK (Baltimore, MD). hCG was iodinated as
described previously(13) . Crude hCG, Nonidet P-40,
fibronectin, and most of the other reagents were obtained from Sigma.
Tissue culture plastic ware and reagents were obtained from Corning
(Corning, NY) and Life Technologies, Inc., respectively. Modular
incubator chambers were obtained from Billups-Rothenberg, Inc. (Del
Mar, CA). Poly(vinylidene fluoride) membranes and electrophoresis
reagents were purchased from Bio-Rad. Enhanced chemiluminescence
blotting reagents were obtained from Amersham Corp.Preparation of rLHR and Mutants
For preparation of
the wild-type rLHR, the full coding sequence of the rLHR cDNA (1) was subcloned into pcDNA I/neo (InVitrogen, San Diego, CA).
The rLHR extracellular loop deletion mutants were created by polymerase
chain reaction using the overlap extension method (14, 15) and were subcloned into pcDNA I/neo. The
entire region that was synthesized by a polymerase chain reaction was
sequenced to verify the removal of the correct base pairs and the
integrity of the remaining sequence. rLHR-t616 was constructed and
initially characterized as described previously(16) .Cells and Transfection
Human embryonic kidney 293
cells (ATCC CRL 1573; American Type Culture Collection, Rockville, MD)
were maintained in a high glucose Dulbecco's modified
Eagle's medium supplemented with 50 µg/ml gentamicin, 10
mM HEPES, and 10% newborn calf serum (growth medium) and kept
in a sterile, humidified, 5% CO
environment. Using the
calcium phosphate precipitation method(17) , cells were
transiently transfected with pcDNA I/neo containing either the
wild-type rLHR cDNA or one of the extracellular loop deletion mutant
cDNAs, or with pCIS containing rLHR-t616 cDNA. Sixteen to twenty hours
after transfection, cells were washed twice with warm Waymouth's
MB752/1 medium containing 1 mg/ml BSA and 50 µg/ml gentamicin and
then replaced with growth medium. Twenty-four hours later, cells were
used for experiments as described below. Unless otherwise specified,
all experiments were performed on transiently transfected 293 cells. Western Blotting
High molecular weight standards,
soluble extracts from 293 cells expressing the wild-type rLHR or one of
the rLHR mutants and solubilized extracts from untransfected 293 cells
were applied to a 7.5% polyacrylamide gel. Solubilized extracts (0.25%
Nonidet P-40, final concentration) were prepared as described for
soluble binding assays. Extracts were loaded at a concentration of 256
µg of protein/lane (57.2 µg of protein/mm of gel). The Western
blots were performed as described previously(18) .Determination of the Percentage of Wild-type and
Extracellular Loop Mutant rLHRs Expressed on the Cell
Surface
Because both intracellular and surface receptors are
solubilized by detergent, I-hCG binding to
detergent-solubilized extracts is an indication of the total amount of
rLHR present. The percentage of receptor expressed at the cell surface
was calculated by dividing the amount of
I-hCG bound to
intact cells by the amount bound to detergent solubilized extracts of
these same cells.
I-hCG in the presence
or absence of an excess of unlabeled hCG (50 IU/ml). After 24 h, the
reaction was terminated by scraping the cells into Hanks'
balanced salt solution supplemented with 1 mg/ml BSA and 50 µg/ml
gentamicin. Cells were pelleted by centrifugation (1500
g, 10 min), washed with the same buffer, and counted in a
gamma counter. All determinations were performed in duplicate. g, 5 min), and the
pellet was resuspended in cold buffer A containing 1% Nonidet P-40 and
20% glycerol. The suspension was incubated for 15 min on ice and
subsequently centrifuged (12,000 g, 15 min, 4 °C).
The supernatant, which contains the solubilized receptor, was diluted
10-fold with cold buffer A containing 20% glycerol and protease
inhibitors to bring the detergent concentration to 0.1% Nonidet P-40. A
saturating concentration of I-hCG (100 ng/ml, final
concentration) was added to aliquots of the detergent extracts in the
presence or absence of excess unlabeled hCG (50 IU/ml). The binding
assay was terminated by filtration through polyethyleneimine-treated
filters(20) , and the filters were subsequently counted in a
gamma counter. All determinations were performed in duplicate.
Determination of Equilibrium Binding Constants
To
determine the equilibrium binding constants for hCG and oLH,
competition experiments were performed using detergent solubilized cell
extracts in buffer A containing 20% glycerol, 0.1% Nonidet P-40, and
protease inhibitors prepared as described above. Aliquots of extracts
were incubated overnight at 4 °C with a subsaturating concentration
(2 ng/ml, final concentration) of I-hCG and increasing
concentrations of unlabeled hCG (0-4.3 µg/ml) or unlabeled
oLH (0.03-105 µg/ml). Assays were terminated by filtration as
described above. All determinations were performed in duplicate. The
data from each experiment were analyzed for both one site and two site
models of ligand binding using the computer program
LIGAND(21) , and the best statistical fit for the data was
determined.
S, detergent-solubilized
extracts (0.1% Nonidet P-40, final concentration) of a nonclonal
rLHR-wt expressing cell line were prepared as described above and
incubated with a trace concentration of
I-hCG and
increasing concentrations of unlabeled hCG or oLH as described above in
the absence or presence of 100-200 µM GTP
S.
Reactions were incubated at 4 °C and terminated 24 h later by
filtration as described above. Filters were counted on a gamma counter
and determinations were performed in duplicate.
I-hCG and increasing
concentrations of unlabeled hCG or oLH. After 24 h, cells were scraped
and collected in Hanks' balanced salt solution supplemented with
1 mg/ml BSA and 50 µg/ml gentamicin (wash medium). Cells were
centrifuged (1500
g, 10 min, 4 °C), washed with
wash medium, vortexed, centrifuged again, and counted in a gamma
counter. All determinations were performed in duplicate.Temperature Shift Experiments
For temperature
shift experiments, cells stably expressing either rLHR-wt12 or rLHR-E3M
were propagated in a sterile, humidified, 5% CO environment
at 37 °C. Cells were plated on 60-mm dishes coated with fibronectin
and grown for 24 h under the same conditions as for propagation. After
24 h, dishes were transferred to modular incubator chambers filled with
a humidified 95% O, 5% CO mixture and then
placed in either a 26 or 37 °C incubator. Forty-eight hours later,
cells were harvested. Total I-hCG binding to intact cells
and detergent solubilized extracts thereof were performed to determine
cell surface expression as described above. Competition binding assays
utilizing detergent solubilized extracts were performed to determine
equilibrium binding constants as described above.
to
Gln
(E1N), Tyr
to Ile
(E1M), Asp
to Gly
(E1C), Lys
to Ile
(E2N), Leu
to Asp
(E2M), Val
to Leu
(E2C), Lys
to Ile
(E3N), Leu
to Thr
(E3M), and Thr
to Ser
(E3C). Although not shown, all mutants contain the
extracellular domain and the cytoplasmic tail of the
rLHR.
I-hCG. The results of these experiments are shown
summarized in Table 1. Intact cells expressing wild-type, E2N,
and E3M receptors bound hCG, but the intact cells expressing the other
mutants failed to bind hormone. However, solubilized extracts from
cells expressing the wild-type or any of the mutant rLHRs were able to
bind hCG. The absolute amounts of hormone bound depended on the
efficiency of transfection and varied from experiment to experiment.
However, by comparing the amount of
I-hCG bound to intact
cells with the total binding detected in solubilized extracts within a
given experiment, the percentage of rLHR expressed at the cell surface
can be calculated. As seen in Table 1, approximately 75% of the
rLHR-wt and rLHR-E2N are expressed on the cell surface. In contrast,
only 20% of rLHR-E3M and 1.5% or less of all the other extracellular
loop deletion mutants are expressed on the cell surface. The lack of
cell surface expression of most of these extracellular loop mutants
does not exclude the possibility that these receptors may bind hormone
with high affinity as it has been previously demonstrated that many
rLHR mutants that fail to reach the cell surface can still bind hCG
with high affinity(5, 23, 24, 25) .
I-hCG.
Consistent with previous reports, computer analyses of the data
utilizing the LIGAND program (21) indicated that a single class
of high affinity (360 pM) hCG binding sites was present in
extracts from cells expressing the wild-type rLHR ( Table 2and Fig. 3)(23, 25) . The results for the binding
of hCG to all the mutants are compiled in Table 2. Significantly,
the deletion of any portion of any of the three extracellular loops had
no effect on the affinity of the receptor mutants for hCG. When
competition assays were performed with unlabeled oLH as the displacing
ligand, analyses of data from extracts of cells expressing rLHR-wt
revealed two apparent classes of oLH binding sites, one of 7.1 nM and a lower affinity site of 64 nM (Table 3). Fig. 4, Panels A and B, shows the binding of
oLH to the wild-type rLHR, where the curvilinear nature of the
Scatchard plot is readily apparent. oLH binding affinities for the
wild-type rLHR and all rLHR extracellular loop deletion mutants are
summarized in Table 3. Solubilized extracts of cells expressing
rLHR-E2N bound oLH with two apparent affinities similar to the
wild-type rLHR. In contrast to the rLHR-wt and rLHR-E2N, however,
deletion of any of the other portions of any of the extracellular loops
resulted in oLH binding with only a single apparent binding affinity of
33-46 nM (see Table 3). As an example, Fig. 4, Panels C and D, illustrates the
binding of oLH to rLHR-E1M. A comparison of Panels B and D of Fig. 4clearly shows a shift in oLH binding from two
apparent affinities for rLHR-wt to a single affinity for rLHR-E1M.
I-hCG and increasing concentrations of unlabeled hCG as
described under ``Materials and Methods.'' The results are
representative of 11 independent experiments. Each point represents the
mean of duplicate determinations. The computer program LIGAND (21) was used to analyze the data. The program determined that
a single class of binding sites was the best statistical fit through
the data points (p < 0.05).
I-hCG and increasing concentrations of
unlabeled oLH as described under ``Materials and Methods.''
The computer program LIGAND (21) was used to analyze the data
to determine the best fit through the data points. A and B are a representative displacement curve and Scatchard plot,
respectively, for rLHR-wt from 11 independent experiments. For the
binding of oLH to rLHR-wt, a model of two classes of binding sites was
found to be the best statistical fit for the data points (p < 0.05). C and D are a representative
displacement curve and Scatchard plot, respectively, for rLHR-E1M from
four independent experiments. For the binding of oLH to rLHR-E1M, a
model of a single class of binding sites was determined to be the best
statistical fit (p < 0.05)
F508 CFTR
mutant(26, 27) , some vesicular stomatitis virus G
proteins with mutations in their glycosylation
sites(28, 29) , and some rLHR mutants ()at
reduced temperatures restored proper localization of these mutant
proteins to the cell surface. Therefore, I-hCG binding
assays were performed on nonclonal 293 cells stably transfected with
the different intracellularly trapped rLHR extracellular loop deletion
mutants after a 48-h preincubation at 26 versus 37 °C.
With the exception of rLHR-E3M, little or no cell surface binding was
detected for all of the intracellularly trapped extracellular loop
deletion mutants incubated at either temperature (data not shown),
indicating that these mutants continued to be mislocalized
intracellularly even when incubated at reduced temperature. However,
rLHR-E3M, which was expressed at <20% at the cell surface at 37
°C, was expressed at the cell surface with levels similar to the
wild-type receptor when preincubated at 26 °C (Table 4). As
shown in Fig. 5, Western blots of detergent solubilized extracts
from rLHR-E3M expressing cells preincubated at 37 °C exhibited only
the immature 68-kDa form of the receptor. However, extracts from cells
preincubated at 26 °C clearly showed both the 85- and 68-kDa foms
of the rLHR, similar to wild-type rLHR. Equilibrium binding assays were
subsequently performed with detergent-solubilized extracts prepared
from a clonal cell line stably expressing the wild-type rLHR or from a
nonclonal 293 cell line stably transfected with rLHR-E3M preincubated
at 37 versus 26 °C. Similar to experiments with extracts
from transiently transfected cells, one class of high affinity binding
sites was determined for the binding of hCG to extracts from rLHR-wt
and rLHR-E3M expressing cells preincubated at either 37 or 26 °C
(data not shown). A model of two apparent affinities was determined for
the binding of oLH to rLHR-wt regardless of whether the cells were
preincubated at 37 or 26 °C (Table 4). As shown in Table 4and Fig. 6, Panels A and B, a
model of one apparent affinity for oLH binding to rLHR-E3M was
determined for cells preincubated at 37 °C, similar to that
observed for equilibrium binding assays performed with extracts from
cells transiently expressing rLHR-E3M. Importantly, as shown in Table 4and Fig. 6, Panels C and D,
detergent solubilized extracts from rLHR-E3M-expressing cells
preincubated at 26 °C now bound oLH with two apparent affinities
similar to rLHR-wt. A comparison of the Scatchard plots of oLH binding
to rLHR-E3M from cells preincubated at 37 versus 26 °C (Fig. 6, Panel B versus Panel D) clearly demonstrates
the shift from one apparent affinity for rLHR-E3M expressing cells
preincubated at 37 °C to two apparent affinities for those
incubated at 26 °C. This correlation between the appearance of cell
surface hCG binding and the mature 85-kDa receptor when rLHR-E3M
expressing cells are preincubated at 26 °C with the shift from one
apparent oLH binding affinity at 37 °C to two apparent affinities
at 26 °C suggests that the detection of only one apparent oLH
binding affinity for most of the extracellular loop deletion mutants is
due to the absence of the mature population of rLHR.
I-hCG and increasing concentrations of unlabeled oLH.
A representative experiment from two such experiments is shown and all
points represent the mean of duplicate determinations. The computer
program LIGAND (21) was utilized to determine the best fit
through the data points. A and B are a representative
displacement curve and Scatchard plot, respectively, from rLHR-E3M
expressing cells grown at 37 °C. A single class of binding sites
was determined to be the best statistical fit for rLHR-E3M incubated at
37 °C (p < 0.05). C and D are from
the same cell line incubated at 26 °C. A model of two classes of
binding sites was determined to be the best statistical fit for
rLHR-E3M incubated at 26 °C (p <
0.05).
I-hCG binding assays to intact cells transiently
expressing rLHR-t616 and detergent-solubilized extracts thereof
revealed that <2% of rLHR-t616 was expressed at the cell surface,
suggesting the retention of this mutant intracellularly (Table 5). Similar to rLHR-wt and the extracellular loop deletion
mutants, a model of a single class of high affinity (337 pM)
binding sites was determined for the binding of hCG to extracts from
rLHR-t616-expressing cells. However, extracts of rLHR-t616-expressing
cells bound oLH with only one apparent affinity similar to that
observed for most of the intracellularly retained extracellular loop
deletion mutants ( Table 5and Fig. 7). Therefore, these
experiments with rLHR-t616 also suggest that the absence of high
affinity oLH binding is due to the absence of the mature, cell surface
rLHR.
I-hCG and increasing concentrations of unlabeled oLH as
described under ``Materials and Methods.'' A representative
experiment from three independent experiments is shown. Each point
represents the mean of duplicate determinations. The computer program
LIGAND (21) was utilized to determine the best statistical fit
for the data points. A model of a single class of binding sites was
determined to be the best statistical fit for the data (p <
0.05).
S on oLH binding to detergent soluble extracts of cells
expressing the wild-type rLHR. In three separate experiments, however,
the same results were observed regardless of the presence or absence of
GTP
S (data not shown). Therefore, it is highly unlikely that the
association or lack of association of G proteins with the rLHR
contribute to alterations in oLH binding to this receptor. As such, the
higher affinity binding of oLH to the mature cell surface receptor as
compared to the intracellular precursor form of the receptor does not
appear to be due to differences in the associations of these receptor
pools with G proteins.
)S,
guanosine 5`-O-(thiotriphosphate).
)
We thank Dr. Mario Ascoli for helpful discussions and
for critically reading the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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