The Six N-linked Carbohydrates of the Lutropin/Choriogonadotropin Receptor Are Not Absolutely Required for Correct Folding, Cell Surface Expression, Hormone Binding, or Signal Transduction
David P. Davis,
Tim G. Rozell,
Xuebo Liu and
Deborah L. Segaloff
Department of Physiology and Biophysics The University of Iowa
College of Medicine Iowa City, IA 52242
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ABSTRACT
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Using two separate methods, we have determined
that all six potential sites for N-linked glyco-sylation on the rat
lutropin/choriogonadotropin receptor (rLHR) contain carbohydrates.
The functional roles of the carbohydrates were analyzed initially
through the use of two nonglycosylated receptor mutants
rLHR(N77,152,173,269,277,291Q) and
rLHR(N77,152,269,277,291Q;T175A).
Although Western blot analyses demonstrated both mutant recep-tors
to be stably expressed, little or no hCG binding activity could be
detected in detergent solubilized extracts of 293 cells expressing
either nonglycosylated LHR mutant. Although this loss of hCG binding
was concluded to be due to misfolding, it was unknown whether this
misfolding was due to the absence of carbohydrates or to the multiple
amino acid substitutions that had been introduced into the polypeptide.
To differentiate between these possibilities, hCG binding assays were
performed with nonglycosylated receptors obtained after tunicamycin
treatment of cells expressing the wild-type rLHR. Even though these
wild-type receptors were confirmed to be devoid of all N-linked
carbohydrates by Western blots, they were found to bind hCG with a
normal high affinity. In addition, tunicamycin-derived, nonglycosylated
LHRs were present at the cell surface and exhibited a phenotype
consistent with mature receptors due to their capability to mediate
hCG-stimulated cAMP production as well as bind oLH with high affinity.
These results indicate that the loss of high affinity hormone binding
by rLHR(N77,152,173,269,277,291Q) and
rLHR(N77,152,269,277,291Q;T175A)
is simply due to the collective amino acid substitutions rather than to
the absence of carbohydrates. Therefore, N-linked carbohydrates are
not absolutely required for the proper folding of the rLHR into a
mature receptor capable of binding hormone and signaling. These results
are in marked contrast to the follitropin receptor (FSHR), a very
similar receptor which has been shown to strictly require N-linked
carbohydrates for folding of the nascent protein.
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INTRODUCTION
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The three glycoprotein hormone receptors comprising the TSH
receptor (TSHR), the FSHR and the LHR share several structural
similarities. All three receptors are glycoproteins containing a large
extracellular N-terminal domain with several consensus sequences for
N-linked glycosylation (1). Numerous studies have shown these
extracellular domains to be sufficient for high affinity binding of
their respective hormones (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The N-terminal domains are followed
by a seven-transmembrane helical motif ending with an intracellular
C-terminus. All three glycoprotein hormone receptors have been shown to
transduce hormone binding via coupling to the heterotrimeric Gs and Gq
or other G proteins that activate phosholipase C (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Because the
FSHR and LHR have fundamental reproductive roles in both sexes, there
has been a steadfast interest on the mechanisms by which these
receptors bind hormone and transduce this binding to activate the
appropriate effectors.
The functional significance of N-linked glycosylation with respect to
the FSHR and LHR has been addressed in multiple studies. It has
recently been demonstrated that removal of N-linked carbohydrates from
the mature, wild-type rFSHR does not affect FSH binding (9, 23).
However, our studies have also shown that prevention of rFSHR
glycosylation by mutagenesis or tunicamycin treatment resulted a
nonglycosylated rFSHR devoid of FSH binding activity (9). It was
further shown that hormone binding could only be maintained if the
nascent rFSHR was glycosylated on at least one of its two glycosylated
sites. We concluded from these studies that instead of being directly
involved in hormone binding, N-linked rFSHR glycosylation is required
to ensure the correct folding of the nascent receptor. Once this
conformation has been attained, the N-linked rFSHR carbohydrates are no
longer required with respect to hormone binding.
Studies addressing the role of N-linked LHR carbohydrates have,
however, provided conflicting views of their function. For example,
some reports have suggested that N-linked LHR carbohydrates are not
required for hormone binding (2, 24, 25, 26, 27), while others have suggested a
requirement of these receptor carbohydrates with respect to hormone
binding and/or correct folding of the nascent LHR (28, 29). The present
report is, therefore, derived from an attempt to explain the
conflicting conclusions that have surrounded the analyses of N-linked
LHR carbohydrates, while at the same time providing a more complete
understanding of their functional significance. Our data demonstrate
that unlike the structurally similar rFSHR, the rLHR is heavily
glycosylated and yet does not seem to rely on its carbohydrates to the
same degree as the rFSHR.
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RESULTS
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Analysis of the rLHR cDNA previously identified six consensus
sequences for N-linked glycosylation within the extracellular,
N-terminal domain of the receptor located at amino acids 77, 152, 173,
269, 277, and 291 (30). To identify which sites are actually
glycosylated, we created two sets of rLHR glycosylation mutants (series
I and II) listed in Table 1
. The mutant receptors
comprising series I allowed the individual retention of each
glycosylation consensus sequence while the other five potential sites
were disrupted by mutagenesis. For example,
rLHR(N152,269,277,291Q;T175A) has the consensus
sequence at Asn-77 maintained, while the other five consensus sequences
at Asns 152, 173, 269, 277, and 291 have been disrupted by
substitution of the essential Asn or Ser/Thr residue at each site. To
determine which of the series I receptor mutants are glycosylated,
detergent-solubilized extracts prepared from 293 cells expressing these
receptor mutants were first incubated in the presence or absence of
PNGaseF (an enzyme that removes N-linked carbohydrates from
glycoproteins, see Ref.31), followed by resolution on SDS-PAGE gels
under reducing conditions. After transfer to PVDF membranes, the
receptors were detected by probing with a previously described
anti-rLHR antibody (designated anti-LHRO2; see Refs. 32 and 33). As
shown in Fig. 1
, the wild type rLHR (lanes 2 and 11)
migrates as two distinct broad bands of 89 and 68 kDa which have been
shown to correspond to the mature and immature forms of the receptor,
respectively (4, 34). Lanes 3 and 12 demonstrate that after PNGaseF
treatment, the wild type receptor migrates as a single band of 59 kDa.
This PGNaseF treatment (15 h at 37 C with 32 U/ml PNGaseF) or one
utilizing even higher concentrations of enzyme does not seem to remove
all N-linked carbohydrates from the wild type rLHR since the molecular
mass of the PNGaseF-treated wild type rLHR is larger than the
nonglycosylated mutant rLHR(N77,152,173,269,277,291Q)
(compare lanes 3 and 4). Nonetheless, although the PGNaseF treatment
was unable to remove all the N-linked carbohydrates from the wild type
rLHR, as shown below, it could efficiently remove N-linked
carbohydrates from the mutant rLHRs containing fewer potential
sites of N-linked glycosylation. Each of the series I rLHR mutants
(each containing only one potential site for N-linked glycosylation)
were tested for their sensitivity to PNGaseF. Looking first at
rLHR(N152,269,277,291Q;T175A), which can only
be glycosylated at the potential Asn-77 site, this mutant migrates as a
single band with a mass of 53 kDa before PNGaseF treatment (lane 6).
After PNGaseF treatment, its mass is reduced to 51 kDa (lane 7).
These data demonstrate that Asn-77 of the rLHR must contain N-linked
carbohydrate. A similar examination of the other mutant receptors in
this series demonstrated that they all exhibit a reduction in their
molecular mass upon PNGaseF treatment. As such, the results from the
series I glycosylation mutants suggest that all six of the rLHR
consensus sites normally have an N-linked carbohydrate attached (see
Fig. 1
, Gels A and B).

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Figure 1. Identification of rLHR N-linked Glycosylation Sites
Using rLHR Series I Mutants
Nonclonal, stably transfected 293 cells expressing the empty pcDNAI/neo
vector (lanes 1 and 10), the wild type rLHR (lanes 2, 3 and 11, 12),
rLHR(N77,152,173,269,277,291Q) (lanes 4, 5 and 13, 14),
rLHR(N152,269,277,291Q;T175A) (lanes 6 and 7),
rLHR(N77,269,277,291Q;T175A) (lanes 8 and 9),
rLHR(N77,152,269,277,291Q) (lanes 15 and 16),
rLHR(N77,152,277,291Q;T175A) (lanes 17 and 18),
rLHR(N77,152,269,291Q;T175A) (lanes 19 and 20),
or rLHR(N77,152,269,277Q;T175A) (lanes 21 and
22) were detergent solubilized. Equal amounts of protein were incubated
15 h at 37 C in the presence or absence of 32 U/ml PNGaseF (noted
as ± PNGaseF, respectively). After this incubation, the samples
were resolved under reducing conditions on two 8% SDS-PAGE gels,
transferred to PVDF membranes, and probed with anti-rLHRO2 antibody
(32, 33). The N-linked glycosylation consensus sequence that was
maintained for a particular mutant receptor is noted
above each receptor.
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To further substantiate the above conclusion, we also examined the rLHR
glycosylation pattern utilizing the series II glycosylation receptor
mutants listed in Table 1
. Unlike series I, series II receptor mutants
were constructed such that only one or two glycosylation consensus
sequences were disrupted within each mutant rLHR. For example, the
mutant rLHR(N77Q) would prevent glycosylation at Asn-77
while not affecting glycosylation at any of the other five sites. The
series II mutant receptors were expressed in 293 cells and extracted by
detergent solubilization as above. However, before resolution by
SDS-PAGE and subsequent Western blotting, these samples were digested
with N-chlorosuccinimide (NCS). This reagent cleaves
proteins after tryptophan residues (35), which theoretically should
cause the rLHR to be digested into six fragments. The largest of these
fragments, consisting of amino acids 1307, contains all six potential
sites for N-linked glycosylation and could be detected using the
anti-LHRO2 antibody. The NCS digestion increased the ratio of
carbohydrate relative to protein in the total mass of this rLHR
fragment, thereby providing a sharper resolution of the potential
differences in molecular masses between the mutants and the wild type
rLHR. After NCS digestion, the wild type receptor was found to migrate
as a broad band of 50 kDa (lanes 1 and 12 of Fig. 2
).
With the exception of rLHR(N173Q), and
rLHR(N291Q) (lanes 5 and 10), all the mutants were
expressed at levels comparable to cells expressing with wild type rLHR.
Although rLHR(N291Q) became more visible on longer
exposures of the film, rLHR(N173Q) could never be detected,
suggesting that it was not stably expressed. It is interesting to note
that the deglycosylated rLHR mutant, in which all glycosylation
consensus site asparagines were substituted with alanines, was stably
expressed (lanes 4, 5, 13, and 14). We can only speculate that one or
more of the other substitutions in the multiple mutant somehow
compensates for the conformational changes made by the N173Q
substitution, which now allow the protein to escape rapid degradation.
As shown below, the lack of expression of rLHR(N173Q),
however, did not preclude our ability to determine the potential
addition of carbohydrate to Asn-173. It has previously been
demonstrated that one can disrupt the consensus sequence for N-linked
glycosylation by either deleting or substituting the Asn residue to
which the carbohydrate is attached, or alternatively, by deleting or
substituting the Ser/Thr residue that is essential for hydrogen bonding
during the transfer of the precursor oligosaccharide (36, 37, 38, 39). By
creating rLHR(T175A), we could then examine whether the
lack of expression of rLHR(N173Q) was due to the potential
lack of carbohydrate at Asn-173 or due to mutation of Asn-173. As shown
in lane 6 of Fig. 2
, rLHR(T175A) was stably expressed,
confirming that the lack of expression of rLHR(N173Q) was
due to substitution of Asn-173 for Gln and not to the potential absence
of carbohydrate at that site. As shown in Fig. 2
, all of the series II
mutants containing only a single disrupted consensus site exhibited a
molecular mass less than the wild type receptor. Consistent with this
result, those mutants with two disrupted consensus sites
((rLHR(N77,152Q) and rLHR(N269,277Q)) exhibited
an even greater decrease in molecular mass than the rLHR mutants with a
single disrupted site. Therefore, as with the series I rLHR
glycosylation mutants, the results from the series II rLHR
glycosylation mutants clearly demonstrate the N-linked glycosylation of
all six consensus sites present on the rLHR.

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Figure 2. Identification of rLHR N-linked Glycosylation Sites
Using rLHR Series II Mutants
293 cells transiently expressing the empty pcDNAI/neo vector (lane 11),
the wild type rLHR (lanes 1 and 12), rLHR(N77Q) (lane 2),
rLHR(N152Q) (lane 3), rLHR(N77,152Q) (lane 4),
rLHR(N173Q) (lane 5), rLHR(T175A) (lane 6),
rLHR(N269Q) (lane 7), rLHR(N277Q) (lane 8),
rLHR(N269,277Q) (lane 9), or rLHR(N291Q) (lane
10) were detergent solubilized. Equal amounts of protein were incubated
2 h at 25 C in the presence of 50 mM
N-chlorosuccinimide. After this incubation, the samples were resolved
under reducing conditions on a 10% acrylamide SDS-PAGE gel,
transferred to PVDF membranes, and probed with anti-rLHRO2 antibody.
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The possible requirement of N-linked rLHR carbohydrates with respect to
correct folding of the nascent receptor and acquisition of hormone
binding has been investigated previously by ourselves and others
through the use of receptor mutants in which consensus sites for
N-linked glycosylation were either individually or collectively
disrupted (25, 28, 40, 41). We reported earlier that normal hCG binding
by a nonglycosylated receptor mutant could be maintained provided the
asparagine residue at 173 was not substituted with a glutamine (41).
These results suggested that N-linked rLHR carbohydrates are not
required for proper folding and subsequent hCG binding. However, we
have since discovered the mutant receptor thought to be
rLHR(N77,152,269,277,291Q;T175A) actually
contained two intact glycosylation con-sensus sequences at
Asn-77 and Asn-152. As such, this mutant was really rLHR(N269,
277,291Q;T175A). Therefore, a correct
rLHR(N77,152,269,277,291Q;T175A) was created
as well as a nonglycosylated
rLHR(N77,152,175,269,277,291Q), and both were determined to
be nonglycosylated by Western blotting (data not shown).
Hormone-binding assays were performed with detergent-solubilized
extracts of cells expressing either the two nonglycosylated receptor
mutants or rLHR(N269,277,291Q;T175A). The
results summarized in Table 2
show that
rLHR(N269,277,291Q;T175A) bound hCG with a
slightly reduced affinity compared with the wild type rLHR. In marked
contrast, however, there was no detectable hCG-binding activity
observed in detergent extracts of cells expressing either
nonglycosylated rLHR mutant. These results correct our previously
reported conclusions and demonstrate that the nonglycosylated mutant
receptor rLHR-(N77,152,269,277,291Q;T175A)
does not bind hCG.
The lack of hCG binding observed with the nonglycosylated mutants
rLHR(N77,152,269,277,291Q;T175A) and
rLHR(N77,152,173,269,277,291Q) markedly contrasts with the
hCG binding activity seen with
rLHR-(N269,277,291Q;T175A), in which only
the glycosylation consensus sequences at Asns 77 and 152 were
maintained. These data suggest that carbohydrates at Asns 77 and/or
152 might be essential for maintaining hCG binding activity. To test
this hypothesis, rLHR(N77,152Q) was created and stably
expressed in 293 cells. The N-linked carbohydrate content of this
mutant was again analyzed by Western blots. Lane 4 of Fig. 3
demonstrates that rLHR(N77,152Q) migrates
as a single band with a molecular mass between that of the immature
wild type rLHR and the PNGaseF-treated, deglycosylated wild type
receptor (lanes 2 and 3, respectively). The decreased mass of
rLHR-(N77,152Q) was expected due to the absence of
N-linked carbohydrates at Asns 77 and 152. Upon PNGaseF
treatment, a further reduction in the size of
rLHR(N77,152Q) occurred (lane 5) due to removal of
carbohydrates present at the remaining four glycosylation sites. To
determine the binding affinity of rLHR(N77,152Q) for hCG,
competition binding assays were performed with detergent-solubilized
extracts of cells expressing this mutant. As seen in Table 2
,
rLHR(N77,152Q) bound hCG with a slightly reduced
affinity relative to the wild type receptor. This small
decrease in affinity cannot explain the more drastic loss in
hCG binding affinity observed with either
rLHR(N77,152,173,269,277,291Q) or
rLHR(N77,152,269,277,291Q;T175A).
Therefore, the maintenance of the glycosylation consensus sites at
Asns 77 and 152 does not seem to be required for high-affinity hCG
hormone binding. These data also suggest that the inability to bind
hormone by either of the nonglycosylated rLHR mutants is more likely
due to the disruption of these rLHR mutants peptide backbone rather
than the prevention of glycosylation at Asns 77 and 152.

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Figure 3. Analysis of rLHR(N77,152Q) N-linked
Carbohydrate Content
Detergent-solubilized extracts of 293 cells stably expressing the empty
pcDNAI/neo vector (lane 1), wild type rLHR (lanes 2 and 3), or
rLHR(N77,152Q) (lanes 4 and 5) were incubated for 15 h
at 37 C in the presence or absence of 32 U/ml PNGaseF (denoted as
± PNGaseF treatment, respectively). The samples, adjusted to contain
equal amounts of protein, were then resolved under reducing conditions
on a 7% SDS-PAGE gel, transferred to a PVDF membrane, and probed with
an anti-rLHR antibody.
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There are two possible explanations that would account for the lack
of hCG-binding activity seen with the nonglycosylated receptor mutants
(rLHR-(N77,152,173,269,277,291Q) and
rLHR(N77,152,269,277,291Q;T175A). One
possibility is that, as with the structurally similar rFSHR (9),
N-linked rLHR carbohydrates may be necessary for the nascent receptor
to attain a conformation enabling it to bind hormone with high
affinity. Alternatively, the loss of hCG-binding activity by
rLHR(N77,152,173,269,277,291Q) and
rLHR-(N77,152,269,277,291Q;T175A) may be due
to the multiple amino acid substitutions introduced into the
polypeptide rather than to the lack of N-linked carbohydrates. To
differentiate between the above hypotheses, we created a
nonglycosylated receptor population by tunicamycin treatment of cells
expressing the wild type rLHR and analyzed their ability to bind hCG.
rLHR(wt-1) cells were cultured for 3 days in the absence or
presence of 10 µg/ml of tunicamycin and then detergent solubilized.
Results documenting the absence of carbohydrates on the rLHR isolated
from tunicamycin-treated cells are shown in Fig. 4
. In
this experiment we tested the susceptibility of rLHR solubilized from
tunicamycin-treated rLHR(wt-1) cells and untreated rLHR(wt-1) cells to
PNGaseF and to endoglycosidase H (endoH), a glycosidase that
specifically removes high mannose-containing carbohydrates from
proteins (36). We chose to utilize two distinct endoglycosidases in
this experiment to determine by two independent means whether or not
carbohydrates were present on the tunicamycin-derived rLHR. Looking at
the results from untreated cells first, as has been shown before (4, 34), endoH treatment causes a decrease in molecular mass of the
immature form of the rLHR only, and PGNaseF treatment results in a
decrease in the molecular masses of both the immature and mature forms
of the receptor to a single 58 kDa species. As shown in Fig. 4
, the
rLHR from tunicamycin-treated cells is resistant to both glycosidases,
suggesting that this receptor species is non-glycosylated. The
molecular mass of the rLHR from tunicamycin-treated cells (51 kDa),
however, is somewhat larger than that of the nonglycosylated mutant
rLHR(N77,152,173,269,277,291Q) (49 kDa) and is comparable
to that of rLHR(N77,152,173,277,291Q), which contains a
single N-linked carbohydrate at Asn-269. One possible explanation for
this discrepancy is that the rLHR from tunicamycin-treated cells is
indeed still partially glycosylated, but the PGNaseF and endoH
treatments are not removing the remaining carbohydrates. We consider
this possibility unlikely, however, due to the observation that the
same glycosidase treat-ments can clearly show a decrease in mass in
rLHR(N77,152,173,277,291Q), a mutant containing only a
single N-linked carbohydrate at Asn-269 and which remains trapped
intracellularly in an immature form (Figs. 1
and 5
).
Furthermore, concentrations of tunicamycin as high as 50 µg/ml did
not cause any further reduction in mass (data not shown). Therefore, we
conclude that the more likely reason why the rLHR from
tunicamycin-treated cells migrates with a slightly higher mass on SDS
gels, as compared with the nonglycosylated mutant
rLHR(N77,152,173,269,277,291Q), is that the polypeptide
backbone of the rLHR from tunicamycin-treated cells is unaltered
whereas rLHR-(N77,152,173,269,277,291Q) contain six
amino acid substitutions.

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Figure 4. Tunicamycin treatment prevents N-linked
glycosylation of the rLHR
Clonal rLHR(wt-1) cells stably expressing the wild type rLHR were
cultured for 3 days in the absence (wt) or presence (wt + tunic) of 10
µg/ml tunicamycin. Nonclonal 293 cells stably expressing the empty
cDNAI/neo vector (neo), the nonglycosylated mutant
rLHR(N77,152,175,269,277,291Q) (all CHO sites mutated), or
the mutant rLHR(N77,152,277,291Q;T175 A) (all
CHO sites mutated except N269) were used as controls.
Detergent-solubilized extracts of cells were then treated with or
without 300 mU/ml endoH or 32 U/ml PNGaseF for 15 h at 37C.). The
samples, adjusted to contain equal amounts of protein, were resolved
under reducing conditions on a 7% SDS-PAGE gel, transferred to a PVDF
membrane, and probed with an anti-rLHR antibody.
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Figure 5. Western Blots of Tunicamycin-Treated Cells
Expressing the Full-Length or Truncated rLHR when Comparing Equal
Amounts of Binding Activity
The cell lines rLHR(wt-1) and rLHR (t338) (gels A and B, respectively)
were cultured for 3 days in the absence (lanes 1 and 2) or presence
(lanes 3 and 4) of 10 µg/ml tunicamycin. The cells were then
detergent solubilized, and hCG competition binding assays were
performed to calculate the amount of hCG- binding activity per
milligram of total protein. Equal quantities of hCG-binding activity
for each sample were resolved on a 7% SDS-PAGE gel under reducing
conditions after incubation at 37 C for 15 h in the absence (lanes
1 and 3) or presence (lanes 2 and 4) of 32 U/ml PNGaseF. The samples
were transferred to a PVDF membrane and probed with an anti-rLHR
antibody.
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After determining the tunicamycin treatment conditions necessary
for preventing N-linked glycosylation of the wild type rLHR, we then
assayed the ability of these tunicamycin-derived, nonglycosylated
receptors to bind hCG. Due to the possible effect of tunicamycin
treatment on cell surface localization of the rLHR, hCG-binding assays
were performed with detergent-solubilized cell extracts. As shown in
Table 3
, the tunicamycin-derived, nonglycosylated rLHR
bound hCG with an affinity similar to that of the control rLHR
solubilized from nontreated cells. To ensure that the hCG-binding
activity present in the tunicamycin-treated rLHR(wt-1) cells was
not due to a small population of partially glycosylated rLHR
contaminating the nonglycosylated rLHR, the experiment shown in the
top panel of Fig. 5
was performed. As before, rLHRs were
solubilized from control and tunicamycin-treated rLHR(wt-1) cells. The
samples were incubated with or without PGNaseF, run on SDS gels, and
analyzed by Western blotting. However, in this experiment the samples
were adjusted to contain equal amounts of hCG-binding activity before
the PGNaseF incubations. The rationale for doing so was as follows.
Assume the 51 kDa rLHR from tunicamycin-treated cells was contaminated
with an imperceptibly small amount of larger rLHR containing
carbohydrate, and it was this small amount of higher molecular mass
receptor that contained binding activity. If an equal amount of binding
activity as compared with rLHR from untreated cells was then loaded
onto the gel, one would expect to see this larger sized band, and it
should be of comparable intensity as the PGNaseF-treated rLHR from
untreated cells. The 51-kDa nonglycosylated band would, in turn, be
expected to be of much greater intensity than the PGNaseF-treated rLHR
from untreated cells. However, as shown in Fig. 5
, equal band
intensities were observed for both the PGNaseF-treated rLHR from
untreated cells and the tunicamycin-derived nonglycosylated receptor
samples (compare lanes 3 and 4 with lane 2). These results argue
against the existence of a small amount of glycosylated rLHR in the
sample derived from tunicamycin-treated cells and further show that the
hCG-binding activity of the rLHR from tunicamycin-treated cells is
indeed due to the nonglycosylated 51 kDa receptor species. One can
conclude, therefore, that N-linked glycosylation of the rLHR does not
seem to be absolutely required for the nascent receptor to fold into a
conformation capable of binding hCG with high affinity. As such, these
data further suggest that the lack of hCG binding by the
nonglycosylated receptor mutants
(rLHR-(N77,152,173,269,277,291Q) and
rLHR(N77,152,269,277,291Q;T175A) is the result
of the multiple amino acid substi-tutions rather than the absence of
N-linked carbohy-drates on these mutants.
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Table 3. High-Affinity hCG Binding to
Detergent-Solubilized Extracts of Tunicamycin-Treated Cells Expressing
Either Nonglycosylated Wild Type rLHR or Nonglycosylated rLHR
Extracellular Domain
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While this study was in progress, Zhang et al. (28) reported
that tunicamycin treatment of insect cells expressing the rLHR
extracellular domain (referred to as form B rLHR and encompassing amino
acids 1294) prevented hormone binding as detected using ligand blots.
We, therefore, examined the ability of a similarly truncated rLHR to
bind hCG when glycosylation was prevented by tunicamycin treatment.
rLHR-t338(c1), a previously described, clonal 293 cell line stably
expressing the extracellular domain (encompassing amino acids 1338)
of the wild type rLHR (4), was cultured in the presence of 10 µg/ml
tunicamycin as described above for the full-length rLHR. As shown in
Fig. 5
, these tunicamycin conditions were sufficient to prevent
glycosylation of rLHR(t338) as evident from the lack of any change in
molecular mass upon PNGaseF treatment before SDS-PAGE resolution
(compare lanes 3 and 4). Hormone-binding assays were then performed
with detergent-solubilized extracts of these cells. As shown in Table 3
, the non-glycosylated rLHR(t338) from tunicamycin-treated cells
exhibited a 5-fold decrease in hCG-binding affinity compared with
the glycosylated control rLHR(t338), but the binding of hCG was
clearly detectable. Therefore, although the extracellular domain when
expressed alone does seem to require N-linked carbohydrates to achieve
the mature conformation required for normal high-affinity binding, in
the absence of carbohydrates it can fold sufficiently well to bind hCG
at a somewhat reduced affinity. As such, our results do not agree with
those previously reported by Zhang et al. (28) (see
Discussion).
The results presented thus far demonstrate that although all six sites
on the rLHR contain N-linked carbohydrates, they are not absolutely
required for the folding of the receptor into a conformation capable of
binding hCG with high affinity. It is possible, however, that cell
surface localization and/or hCG-mediated signal transduction of the
nonglycosylated rLHR could be altered by the absence of N-linked
carbohydrates. To examine these possibilities, the following
experiments were performed. First, the presence of tunicamycin-derived,
nonglycosylated rLHR at the cell surface was analyzed by hCG
competition-binding experiments using intact cells. Intact cells that
had been pretreated with tunicamycin bound hCG with the same high
affinity as control intact cells (data not shown). However, the
tunicamycin treatment decreased the number of receptors on the cell
surface of the rLHR(wt-1) cells from 450,000 (42) to 21,100 (Table 4
). This reduction in cell surface receptors was fully
accountable by the reduction in total rLHR protein after tunicamycin
treatment as determined by Western blotting and is presumably due to a
general decrease in protein synthesis caused by the tunicamycin
treatment (43). To analyze the ability of the tunicamycin-treated cells
to respond to hCG with increased cAMP production, we matched them with
a control, non-tunicamycin-treated wild type rLHR cell line (referred
to as rLHR(wt-16)), which expresses a comparable number of cell surface
receptors. The rLHR(wt-16) cells are a clonal line of 293 cells stably
transfected with the same wild type rLHR cDNA used to create the
rLHR(wt-1) cells. The only difference between the two cell lines is the
number of cell surface rLHRs expressed. Hence, the rLHR should not be
glycosylated any differently in the rLHR(wt-16) cells. As evident from
the hCG dose-response curves shown in Fig. 6
and the
EC50 and Rmax values listed in Table 4
, there
was little or no decrease in the ability of the tunicamycin-derived,
nonglycosylated rLHR to stimulate cAMP production in response to hCG.
These results indicate that N-linked carbohydrates are not absolutely
essential for the proper folding and membrane insertion of the rLHR,
for its ability to bind hCG with high affinity, or for its ability to
activate Gs upon the binding of hCG.

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|
Figure 6. hCG-Stimulated cAMP Dose-Response Curve
Tunicamycin-treated rLHR(wt-1) cells, expressing 21,100 cell surface
receptors per cell, and a control rLHR(wt-16) cell line, stably
expressing 13,600 cell surface wild type receptors per cell, were
incubated with increasing concentrations of hCG to determine
hCG-stimulated cAMP production as described in Materials and
Methods. The data were calculated using the Delta Graph
computer program. One representative experiment is shown.
|
|
Recent studies from our laboratory have shown that maturation of the
rLHR involved not only changes in the N-linked carbohydrates from an
endoH-sensitive form to an endoH-insensitive form, but also changes in
the conformation of the receptor that affect its ability to bind oLH
(44). Whereas both the immature and mature forms of the rLHR bind hCG
with high affinity, oLH binds the immature form of the receptor with a
reduced affinity (50 nM) as compared with the mature cell
surface form of the receptor (5 nM). These results
demonstrate that the conformation of the immature receptor is distinct
from that of the mature receptor and suggest further alterations in
folding must ensue between the time of the receptors exit from the
endoplasmic reticulum and its insertion into the plasma membrane. Since
carbohydrates are thought to be the recognition sites for a chaperone
protein involved in the folding of many glycoproteins, it was possible
that, in the absence of carbohydrates, the tunicamycin-derived rLHR
might "escape" the cells normal quality control system and be
inserted into the plasma membrane in the immature conformation. To
examine this possibility, the binding affinity for oLH was determined
on intact rLHR(wt-1) cells that had either been untreated or treated
with tunicamycin to generate cell surface nonglycosylated rLHRs. It was
predicted that tunicamycin treatment of rLHR(wt-1) cells would result
in the cell surface expression of nonglycosylated receptors with either
a single high binding affinity for oLH (indicative of only mature
receptors at the cell surface), a single low binding affinity for oLH
(indicative of only immature receptors at the cell surface), or a
composite of two binding affinities for oLH (indicative of the presence
of both mature and immature receptors at the cell surface). Results of
these experiments showed that the tunicamycin-derived, cell surface,
nonglycosylated rLHRs bound oLH with a single high affinity
(Kd = 3.31 ± 0.340), comparable to that of the
glycosylated, mature receptor (Kd = 1.54 ± 0.035).
These results demonstrate that the cell surface population of
nonglycosylated receptors consists only of mature, fully folded
receptors. We conclude from these results that N-linked rLHR
carbohydrates are not required for ensuring the release of only mature
receptors from the endoplasmic reticulum and their subsequent
localization at the cell surface.
 |
DISCUSSION
|
---|
Using two distinct approaches, we have shown herein that all six
potential N-linked rLHR glycosylation sites normally contain
carbohydrates. In spite of the large extent of glycosylation of the
rLHR, however, our studies show that N-linked glycosylation is not
absolutely required for the correct folding and cell surface expression
of the rLHR. Thus, tunicamycin treatment of wild type rLHR-expressing
cells produced nonglycosylated receptors that were demonstrated to be
present at the cell surface in a form capable of binding hCG with a
high affinity and able to transduce hCG-mediated cAMP stimulation.
Finally, using the ability of oLH to bind the immature and mature forms
of the rLHR with distinct affinities, we demonstrated that the
nonglycosylated rLHR at the cell surface of tunicamycin-treated cells
did not arise from mislocalization of the nonglycosylated immature
receptor but was instead a properly folded, mature form of the
receptor. Therefore, the N-linked carbohydrates of the rLHR are not an
indispensible requirement for the proper folding of the rLH. This
conclusion does not a priori exclude a role for N-linked
carbohydrates in the folding of the rLHR. They may, for example, aid in
the kinetics of folding. Because we are measuring the steady state
levels of receptor, any effects of the N-linked carbohydrates on the
kinetics of this process would go unnoticed. Further experiments will
be required to address this issue. Nonetheless, the results with the
rLHR stand in marked contrast to the rFSHR, where the N-linked
carbohydrates are essential for the steady state acquisition of a
receptor pool capable of binding hormone with high affinity (9).
As mentioned above, the results on the lack of a critical need for
N-linked carbohydrates for the proper folding of the rLHR stand in
marked contrast to the closely related rFSHR (1, 13). Previous studies
have shown that two of the three potential sites for N-linked
glycosylation on the rFSHR are normally glycosylated. To attain a
functional rFSHR with respect to hormone binding, at least one of the
two glycosylated consensus sites must be glycosylated. This reliance on
N-linked glycosylation was demonstrated to be at the level of folding
since PNGaseF deglycosylated mature rFSHR-bound FSH with a normal high
affinity while nonglycosylated rFSHR mutants as well as the
tunicamycin-derived nonglycosylated wild type rFSHR were devoid of
hormone-binding activity (9). This conclusion is in contrast to the
present findings concerning the rLHR, in which carbohydrates are not
essential for receptor folding. Therefore, despite the high degree of
deduced amino acid identity between the FSHR and the LHR, our present
results imply the existence of structural differences between these
receptors that result in the observed dissimilar reliance on N-linked
glycosylation. The dichotomous importance of N-linked carbohydrates in
protein folding, as seen between the FSHR and LHR, has been reported
with other glycoproteins. In many cases, only one to five amino acid
differences have been shown to determine the need of a particular
glycoprotein for its carbohydrates in proper folding (45, 46). Whether
a similar scenario is also true in the case of the FSHR and LHR will
require further study.
Recent evidence from our laboratory has provided further evidence
to support the existence of structural variances between the rFSHR and
the rLHR (47). An analogous series of rFSHR and rLHR mutants were
constructed and then analyzed for their ability to correctly fold. The
mutations were introduced at positions on the receptors (transmembrane
loops and intracellular C-terminal tail) that are not directly involved
in hormone binding so that hormone binding could be used as a tool to
determine whether a particular mutant had been correctly folded. Both
rFSHR and rLHR mutants were retained in the endoplasmic reticulum as
demonstrated by their sensitivity to endoH. However, while the
intracellularly retained rLHR mutants exhibited normal high-affinity
hormone binding, the analogous intracellularly retained rFSHR mutants
were unable to bind hormone. Therefore, as the rLHR exits the
endoplasmic reticulum, it has already acquired a conformation allowing
it to bind hCG (although it is still in an immature conformation as
assessed by its decreased affinity for oLH). In contrast, as the
rFSHR exits the endoplasmic reticulum, it has not yet folded into a
conformation that can bind FSH even with decreased affinity. These
results suggest a temporal difference in the folding of these two
closely related receptors. Taken together with the absolute reliance of
the rFSHR on its N-linked carbohydrates to fold properly (9) and the
lack of reliance of the rLHR on its N-linked carbohydrates to fold
properly, it appears as if the rLHR can fold more readily than the
structurally related rFSHR.
It should be pointed out that our results on the tunicamycin-derived
rLHR are in apparent disagreement with those reported by Dufau and
co-workers (28). This group examined the ability of the wild type rLHR
extracellular domain expressed in insect cells to fold correctly when
glycosylation was prevented by culturing the cells with tunicamycin.
The resulting nonglycosylated receptors were found to be incapable of
hormone binding when assayed by ligand blots. It was, therefore,
concluded that N-linked glycosylation is required to allow the nascent
receptor to attain the conformation necessary for hormone binding (28).
One possible explanation for the discrepancy between this groups
conclusions and ours was their use of a truncated form of the
rLHR for their experiments. We attempted to control for this by
examining the effects of tunicamycin treatment on the folding of the
rLHRs extracellular domain expressed in rLHR-t338(c1) cells. After
culturing rLHR-t338(c1) cells with tunicamycin, this truncated receptor
was demonstrated to be devoid of carbohydrates by Western blots. Yet,
as with the full-length receptor, detergent-solubilized extracts from
tunicamycin-treated rLHR-t338(c1) cells bound hCG with a high affinity.
It is possible that the additional C-terminal 44 amino acids of
rLHR(t338) relative to the rLHR B form used by Dufau and co-workers
allows the correct folding of the nonglycosylated rLHR(t338). A
more likely explanation, however, is the difference in assay
techniques. Dufau and co-workers examined hormone binding by ligand
blots, which requires the denatured receptor resolved by SDS-PAGE to
correctly renature before it can bind hormone. This renaturation step
may require the presence of carbohydrates for the rLHR to correctly
refold and, therefore, prevent hormone binding by nonglycosylated
receptors. Support for this hypothesis can be found in the same report
by Dufau and co-workers (28). By comparing the effects of endoH and
PNGaseF treatment of the rLHR B form, they concluded the proximal
N-acetylglucosamine to be essential for renaturing the
receptor after resolution by SDS-PAGE. This conclusion would,
therefore, explain the inability of the tunicamycin-derived,
nonglycosylated rLHR B form to bind hCG when assayed by ligand blots.
Another earlier report by Ji et al. (24) used tunicamycin to
test the functional role of the N-linked carbohydrates on the LHR.
Their results showed that tunicamycin treatment of wild type
rLHR-expressing cells resulted in the absence of cell surface hormone
binding. These results could have been due to a loss of binding
activity of nonglycosylated cell surface receptors or to the absence of
cell surface nonglycosylated receptors. As shown herein, culturing
cells with tunicamycin not only prevents glycosylation but also results
in a reduction in receptor expression (coinciding with a decrease in
total protein expression). Because the studies by Ji and co-workers
were performed utilizing cells that express much lower levels of rLHR
than the rLHR(wt-1) cells, it is likely that the tunicamycin
treatment decreased the cell surface expression of the rLHR to
undetectable levels.
Our results demonstrating that all six sites of the rLHR are
glycosylated are also in apparent disagreement with the report by Dufau
and co-workers (28). Specifically, they reported the lack of any
N-linked glycosylation at the Asn-77 consensus site. As noted above,
Dufau and co-workers used insect cells to express the rLHR. It is
possible that the insect cells do not utilize the same sites for
N-linked glycosylation that mammalian cells do. It is also possible
that the conclusion of an absence of carbohydrate at Asn-99 by Dufau
and co-workers may have been due to a lack of detection rather than to
a true absence of carbohydrate. As demonstrated in Fig. 1
, the
individual glycosylation of Asn-77 or any of the other five sites
increases the rLHR molecular mass by at most 2 kDa. Therefore,
preventing glycosylation at one site may not produce a readily visible
difference in molecular mass. This effect may have been further
exacerbated due to the use of insect cells to express the mutant
receptors. Dufau and co-workers demonstrated in the same report that
insect cells do not glycosylate the rLHR to the same extent as
mammalian cells, which would decrease the contribution of the
carbohydrates toward the overall molecular mass of the receptor. By
utilizing mammalian cells to express rLHR glycosylation mutants in the
context of the full-length receptor, we have conclusively demonstrated
the glycosylation of Asn-77. This is most visible by analyzing the
additive contribution in molecular mass of the carbohydrates at Asn-77
and Asn-152. When glycosylation at both sites is prevented, there is a
greater decrease in molecular mass relative to the individual
disruption of each of these sites (compare lane 4 with lanes 2 and 3
in Fig. 2
).
Given that all six sites of the rLHR are normally glycosylated, it
might be possible to create a nonglycosylated mutant of the rLHR by
disrupting all six consensus sites for N-linked glycosylation. If the
mutations did not perturb the peptide backbone, one would predict
that this nonglycosylated mutant should fold normally and bind hormone
with high affinity because tunicamycin-derived nonglycosylated rLHR
can do so. However, two separate nonglycosy-lated mutants,
rLHR(N77,152,269,277,291Q;T175A) and
rLHR-(N77,152,173,269,277,291Q), were unable to bind
any detectable hCG when assayed in detergent-solubilized cell
extracts. The present study corrects a previous report from this
laboratory (41) and further suggests that the lack of binding activity
of these two mutants is the result of multiple amino acid
substitutions. Given that one could disrupt a consensus site for
N-linked glycosylation by substituting either the Ser/Thr or Asn within
the consensus sequence with any one of the remaining 19 amino acids,
there are a huge number of possible nonglycosylated rLHR mutants one
could construct. It may be possible, though formidable, to determine
whether appropriate combinations of amino acid substitutions exist that
would prevent N-linked glycosylation while at the same time not
affecting the correct folding and membrane insertion of a
non-glycosylated, mutant rLHR.
In summary, we have shown that, unlike the closely related rFSHR,
the rLHR does not absolutely require N-linked glycosylation for folding
into a mature conformation. Experiments are underway to examine the
folding of these glycoproteins in more detail and the role of chaperone
proteins in these processes.
 |
MATERIALS AND METHODS
|
---|
Construction of rLHR Glycosylation Mutants
Creation of the B series of rLHR mutants, as well as the
creation of rLHR(N77,152,173,269,277,291Q) and
rLHR-(N77,152,269,277,291Q;T175A), has been
described previously (41).
rLHR(N152,269,277,291Q;T175A) and
rLHR(N77,269, 277,291Q;T175A) were constructed
by splicing the corresponding fragments from the wild type pDLHR9 (41)
into the EcoRV/XmnI and the
XmnI/NsiI sites, respectively, of
rLHR-(N77,152,269,277,291Q;T175A).
rLHR(N77,152,269,277,291Q),
rLHR-(N77,152,277,291Q;T175A),
rLHR(N77,152,269,291Q;T175A), and
rLHR(N77,152, 269,277Q;T175A) were all
created by the PCR (48, 49) using
rLHR(N77,152,269,277,291Q;T175A) as a template.
rLHR(77,152Q) was constructed by oligonucleotide-directed
mutagenesis of the pDLHR9, which had been subcloned into pALTER-1
(Promega, Madison, WI). All of the resulting rLHR mutants were cloned
into the pcDNAI/neo expression vector (Invitrogen, San Diego, CA), and
their identities were confirmed by dideoxy sequencing the entire region
amplified by the PCR (50).
Cell Lines
Human embryonic kidney 293 (HEK293) cells (ATCC CRL 1573) were
maintained at 5% CO2 in a culture medium consisting of
DMEM containing 50 µg/ml gentamicin, 10 mM HEPES, and
10% newborn calf serum. Transfections were performed using the
CaPO4 method as described previously (41). For assays
utilizing transient rLHR expression, cells were harvested 48 h
posttransfection. Nonclonal cell lines stably expressing the wild type
or mutant rLHR, or the empty pcDNA/neo expression vector, were prepared
as previously described (9) by selection with 700 µg/ml G418 (GIBCO,
Grand Island, NY). These nonclonal cell lines, as well as the clonal
cell lines rLHR(wt-1) (42), rLHR(wt-16) (44), and rLHR-t338(c1) (4)
were maintained in culture medium containing 700 µg/ml G418.
Tunicamycin Treatment of rLHR(wt-1) and rLHR-t338(c1) Cell
Lines
The clonal cell lines rLHR(wt-1) and rLHR-t338(c1) were plated
with culture medium on dishes precoated with 0.1% gelatin [0.1%
gelatin (DIFCO Laboratories, Detroit MI] in CMF PBS (GIBCO) and
allowed at least 48 h to equilibrate and attach to dishes. The
culture medium was then replaced with fresh culture medium containing
10 µg/ml of tunicamycin (homologs A, B, C, and D). Fresh culture
medium with 10 µg/ml tunicamycin was readded 24 and 48 h later.
The cells were then detergent solubilized, as described herein, 72
h after the initial tunicamycin addition.
Western Blotting of Detergent-Solubilized Cell Extracts
Detergent-solubilized cell extracts were prepared using 0.5%
Nonidet P-40 in buffer A (150 mM NaCl, 20 mM
HEPES, pH 7.4) as described previously (9). After solubilization, the
cell extracts were subjected to incubations performed in the presence
or absence of PNGaseF or NCS as noted. PNGaseF treatment before gel
loading was attained by incubation of the detergent-solubilized cell
extracts for 15 h at 37 C with 32 U/ml PNGaseF.
Detergent-solubilized cell extracts digested with NCS before gel
loading were incubated for 2 h at 25 C with 50 mM NCS
in 4 M urea. With the exception of the experiments shown in
Fig. 5
, all samples were adjusted to contain equal amounts of protein
before loading on the SDS gel. In Fig. 5
, the samples were adjusted to
contain equal amounts of hCG-binding activity. A reducing, 6x SDS
Lammeli sample buffer (12% wt/vol SDS, 40% vol/vol glycerol, 109
mM EDTA, 1.5 M Tris/HCl, 98 mg/ml
dithiothreitol, and 6% vol/vol ß-mercaptoethanol) was added to the
extracts to achieve a final 1x concentration, and the samples were
incubated at room temperature for 1 h. The reduced and denatured
cell extracts were resolved by SDS-PAGE gel, transferred to a PVDF
membrane, and probed with the anti-rLHR antibody
anti-LHRO2, which was raised against a peptide
corresponding to amino acids 194207 of the rLHR (32, 33).
[125I]hCG Binding to
Detergent-Solubilized Cell Extracts
Saturation[125I]hCG bindings were performed by
incubating detergent-solubilized cell extracts with 100 ng/ml
[125I]hCG overnight at 4 C in the absence or presence of
50 IU/ml crude hCG. Equilibrium binding constants were derived by
competition [125I]hCG binding assays in which cell
extracts were incubated with a subsaturating concentration of
[125I]hCG (2 ng/ml) overnight at 4 C in the presence of
increasing concentrations of unlabeled hCG (04.3 µg/ml). For both
assays, the receptor-hormone complexes were separated from the unbound
hormone by vacuum filtration through polyethylenimine-treated filters
(51). Equilibrium binding parameters were calculated from the
competition binding assays using the LIGAND computer program (52).
Determination of Cell Surface Receptor Numbers and hCG-Mediated
cAMP Production
The number of cell surface receptors was determined by
performing competition hCG-binding assays with intact cells. The cells
were first cooled on ice for 15 min and then washed two times with cold
buffer B (Waymouths media without sodium bicarbonate and containing
0.1% BSA). A subsaturating concentration of [125I]hCG (2
ng/ml) and increasing concentrations of unlabeled hCG (04.3 µg/ml)
were added to the cells and incubated overnight at 4 C. The assay was
completed by scraping and washing the cells to remove unbound hormone,
and the resulting data were calculated using the LIGAND computer
program to determine the equilibrium-binding parameters (52). Human
CG-mediated stimulation of cAMP production was also performed as
described (53). Briefly, cells were washed with warm buffer B
containing sodium bicarbonate, preincubated with
3-isobutyl-1-methylxanthine, and then incubated for 30 min at 37 C in
the presence of increasing concentrations of hCG. The cells were
collected and the total cAMP determined by RIA.
Ovine LH Binding Assays with Tunicamycin-Derived, Nonglycosylated
rLHR
rLHR(wt-1) cells cultured in the presence or absence of
tunicamycin as described above were analyzed for oLH binding. After
cooling on ice for 15 min, the cells were washed two times with cold
buffer B, and a subsaturating concentration (2 ng/ml) of
[125I]hCG was added. Increasing concentrations of
unlabeled hCG (04.3 µg/ml) or unlabeled oLH (0105 µg/ml) were
added to the cells and incubated overnight at 4 C. The assay was
completed and the LIGAND computer program used to analyze whether the
resulting data best fit a one- or two-site ligand-binding model as
described previously (44).
Hormones and Supplies
Highly purified hCG (CR-127) was kindly provided by the National
Hormone and Pituitary Agency of the NIDDK (NIH) and was iodinated as
described previously (54). NP-40, crude hCG, and tunicamycin were
obtained from Sigma (St. Louis, MO) PNGaseF was purchased from
Boehringer Mannheim (Indianapolis, IN). PVDF membranes were obtained
from Bio-Rad (Richmond, CA) and the enhanced chemiluminescence (ECL)
detection kit was obtained from Amersham (Arlington Heights, IL).
Tissue culture plasticware and reagents were from Corning (Corning, NY)
and GIBCO (Grand Island, NY), respectively.
 |
ACKNOWLEDGMENTS
|
---|
We thank Julie Jacquette for expert technical assistance and Dr.
Mario Ascoli for critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology, The University of Iowa College of Medicine, Iowa City, Iowa 52242.
These studies were supported by NIH Grant HD-28970 (to D.L.S.). The
services and facilities provided by the Diabetes and Endocrinology
Research Center of the University of Iowa (supported by Grant DK-25295)
are also gratefully acknowledged. D.L.S. is a recipient of NIH Research
Career Award HD-00968.
Received for publication January 3, 1997.
Revision received February 13, 1997.
Accepted for publication February 18, 1997.
 |
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