From the Department of Molecular Biology, University of Wyoming,
Laramie, Wyoming 82071-3944
The luteinizing hormone/choriogonadotropin
receptor is a seven-transmembrane receptor. Unlike most
seven-transmembrane receptors, it is composed of two halves of equal
size, the N-terminal extracellular exodomain and the C-terminal
membrane-associated endodomain. The exodomain is exclusively
responsible for high affinity hormone binding, whereas receptor
activation occurs only in the endodomain. This mutually exclusive
physical separation of the two functional domains sets the lutropin
receptor and its subfamily of receptors apart from all other
seven-transmembrane receptors. The mechanisms of hormone binding and
receptor activation also appear to be different from those of other
receptors in that binding occurs in at least two steps. However, the
precise hormone contact sites in the exodomain are unknown. To
determine the hormone/receptor contact sites, we have examined the
receptor using progressive truncation from the C terminus, Ala
scanning, immunofluorescence microscopy, and antibody binding.
Progressive truncation from the C terminus of the receptor indicates
several discrete regions that impact hormone binding. These regions are
around the boundaries of exons 1-2, 4-5, 6-7, and 9-10. Ala
scanning of the Asp17-Arg26 region near
the exon 1-2 junction uncovered three alternating residues
(Leu20, Cys22, and Gly24) crucial
for hormone binding. Ala substitution for any one of these residues
abolished hormone binding, although the resulting mutant receptors were
successfully expressed on the cell surface. In contrast, Ala
substitution for their flanking and intervening residues did not impair
hormone binding. These results and the data in the accompanying article
(Phang, T., Kundu, G., Hong, S., Ji, I., and Ji, T. (1998)
J. Biol. Chem. 273, 13841-13847) indicate that this
region directly contacts the hormone and suggest a novel mode of
embracing the hormone.
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INTRODUCTION |
The LH1/CG receptor
belongs to a subfamily of glycoprotein hormone receptors within the
seven-transmembrane receptor family. Unlike most seven-transmembrane
receptors, it is composed of two equal halves: the 341-amino acid-long
extracellular N-terminal exodomain and the 334-amino acid-long
membrane-associated C-terminal endodomain, which includes seven
transmembrane helices (1, 2). The exodomain binds the hormone with high
affinity (3-7) without hormone action (5, 8). The exodomain-hCG
complex is thought to make a secondary contact with the endodomain,
thus generating a signal (9). Therefore, the high affinity interaction of the exodomain and hCG is the crucial first step leading to signal
generation and hormone action. Despite the importance, only limited
information is available concerning the precise hormone contact
residues and sites in the exodomain. Roche et al. (10) found
that three peptide mimics of the exodomain, peptide-(21-38), peptide-(102-115), and peptide-(253-266), attenuated
125I-hCG binding to membranes expressing the LH/CG
receptor. More recently, Segaloff and co-workers (11) observed that
receptors lacking any of several discrete exodomain sequences, the 11 N-terminal residues and leucine-rich motifs 1-6, were trapped in cells
and failed to bind hCG.
In this work and the accompanying article (12), the exodomain was
examined using several independent methods, including serial truncation
from the C terminus, Ala scanning, peptide mimics of the receptor,
photoaffinity labeling, affinity cross-linking, and immunofluorescence
microscopy. Our results show that the
Leu20-Pro38 sequence near the exon 1-2
junction contacts both the
- and
-subunits of hCG. In addition,
another three sequences near the junctions of exons 4-5, 6-7, and
9-10 influence hormone binding.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Functional Expression of LH/CG
Receptors--
Mutant LH/CG receptor cDNAs were prepared in the
pSELECT vector using the Altered Sites Mutagenesis system (Promega),
sequenced, subcloned into pcDNA3 (Invitrogen) as described (13),
and sequenced again to verify mutation sequences. This procedure does
not involve polymerase chain reaction. To produce truncated receptors,
a stop codon was introduced at the 3'-end of the exons. Mutant and
truncated LH/CG receptor constructs were transfected into human
embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in minimal essential medium containing 10%
horse serum and 500 µg/ml G418. These cells were used for hormone
binding, cAMP production, antibody binding, and fluorescence microscopy. All assays were carried out in duplicate and repeated four
to five times. Means ± S.D. were calculated.
125I-hCG Binding and Intracellular cAMP
Assay--
Stable cells were assayed for 125I-hCG binding
in the presence of 150,000 cpm of 125I-hCG (14) and
increasing concentrations of unlabeled hCG. The Kd
values were determined by Scatchard plots. hCG (batch CR 127) was
supplied by the National Hormone and Pituitary Program. For
intracellular cAMP assay, cells were washed twice with Dulbecco's modified Eagle's medium and incubated in the medium containing isobutylmethylxanthine (0.1 µg/ml) for 15 min. Increasing
concentrations of hCG were then added, and incubation was continued for
45 min at 37 °C. After removing the medium, the cells were rinsed
once with fresh medium without isobutylmethylxanthine, lysed in 70% ethanol, freeze-thawed in liquid nitrogen, and scraped. After pelleting
cell debris at 16,000 × g for 10 min at 4 °C, the
supernatant was collected, dried under vacuum, and resuspended in 10 µl of cAMP assay buffer (Amersham Pharmacia Biotech). cAMP
concentrations were determined with a 125I-cAMP assay kit
(Amersham Pharmacia Biotech) following the manufacturer's instructions
and validated for use in our laboratory.
125I-hCG Binding to Solubilized LH/CG
Receptor--
Transfected cells were washed twice with ice-cold 150 mM NaCl and 20 mM HEPES, pH 7.4 (buffer A).
Cells were scraped on ice, collected in buffer A containing protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 10 mM
EDTA), and pelleted by centrifugation at 1300 × g for
10 min. Cells from a 10-cm plate were resuspended in 0.6 ml of buffer A
containing 1% Nonidet P-40, 20% glycerol, and the above protease
inhibitors; incubated on ice for 15 min; and diluted with 5.4 ml of
buffer A containing 20% glycerol plus the protease inhibitors. The
mixture was centrifuged at 100,000 × g for 60 min. The
supernatant (500 µl) was mixed with 150,000 cpm of
125I-hCG and 6.5 µl of 0.9% NaCl and 10 mM
Na2HPO4, pH 7.4, containing increasing
concentrations of unlabeled hCG. After incubation at 4 °C for
12 h, the solution was thoroughly mixed with 250 µl of buffer A
containing bovine
-globulin (5 µg/ml) and 750 µl of buffer A
containing 20% polyethylene glycol 8000. After incubation at 4 °C
for 10 min, samples were pelleted at 1300 × g for 30 min, and supernatants were removed. Pellets were resuspended in 1.5 ml
of buffer A containing 20% polyethylene glycol 8000, centrifuged, and
counted for radioactivity.
Immunofluorescence Microscopy--
For fluorescence labeling of
LH/CG receptors, the Flag epitope (15), Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(5'-GAC TAC AAG GAC GAT GAC GAT AAG-3'), was inserted between
the C terminus (Ser26) of the signal sequence and the N
terminus (Arg27) of mature receptors. The Flag epitope (16)
has successfully been used as a marker to identify, trace, and purify
recombinant proteins carrying the tag without significantly impairing
their biological activities (17, 18). For labeling intact cells, cells
were cultured on coverslips in 6-well plates for 2 days and fixed with
4% formaldehyde in PBS for 10 min at 25 °C. For labeling
permeabilized cells, cells were fixed with 4% formaldehyde in PBS for
5 min at 4 °C and treated with 0.1% Triton X-100 in PBS for 5 min
at 4 °C. Fixed intact or permeabilized cells were washed five times
with PBS for 2.5 min each at 25 °C. They were sequentially treated
with 0.4% type IV gelatin isolated from calf skin (Sigma) in modified
Eagle's medium free of phenol red for 10 min at 25 °C and then with
5% goat serum and 1% fetal calf serum in the same medium for 20 min
at 25 °C. The treated cells were incubated with 500 µl/well
primary antibody solution (25 µg of mouse anti-Flag antibody in 1 ml
of modified Eagle's medium containing 5% goat serum and 1% fetal
calf serum) for 2 h at 37 °C. The cells were washed three times
with PBS for 2.5 min each at 25 °C and treated with a 400-fold
dilution of Texas Red-conjugated goat anti-mouse IgG (Molecular Probes,
Inc.). Finally, the cells were washed with PBS six times for 2.5 min
each at 25 °C. The coverslip containing processed cells was mounted
on slide glass using 50% glycerol in PBS and sealed using nail polish.
Specimens were examined under a Leica TCS-4D laser scanning confocal
microscope equipped with Scanware analysis software. Entire experiments
were completed in 1 day to prevent increasing background
fluorescence.
Radioimmunoassay for Flag-LH/CG Receptors--
Mouse anti-Flag
monoclonal antibody M2 (Eastman Kodak Co.) was iodinated with
125I according to the published procedure for
radioiodination of hCG (14), and 125I-anti-Flag antibodies
were purified on a Sephadex G-150 column. Binding of
125I-anti-Flag antibodies to 293 cells expressing Flag-LH
receptors was carried out according to the 125I-hCG binding
assay described above.
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RESULTS |
Progressive Truncation of the C Terminus--
The LH/CG receptor
is encoded by 11 exons (19, 20). Exons 1-10 comprise most of the
exodomain, whereas the endodomain is encoded in exon 11. As an initial
step to define important regions for hCG binding, individual exons from
11 to 2 were progressively truncated from the C terminus (Fig.
1D). These truncated receptor fragments represent exons 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and 1. None of the stably transfected cells bound hCG, and
therefore, they were solubilized in Nonidet P-40 and assayed for hCG
binding (Fig. 1, A and B). All of the expressed
receptor fragments, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5 1-4, 1-3 and
1-2, except for the exon 1 fragment, were capable of binding hCG, but
were trapped in cells.

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Fig. 1.
hCG binding to exodomain fragments. The
11 exons of the LH/CG receptor were progressively truncated from the C
terminus to produce receptor fragments consisting of exons 1-10, 1-9,
1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and 1 (D). These
exon fragments were stably expressed in 293 cells, solubilized in
Nonidet P-40, and assayed for 125I-hCG binding in the
presence of increasing concentrations of unlabeled hCG. The results are
presented as displacement of 125I-hCG binding
(A) and in a Scatchard plot (B). Experiments were
repeated four to five times in duplicate, and means ± S.D. were
calculated (C). Nontransfected cells did not show specific
binding of hCG. WT, wild type; NS, not
significant. Arrows indicate regions that influence hormone
binding.
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Interestingly, Kd values for the fragments increased
stepwise in groups rather than continuously as the C terminus was
progressively truncated (Fig. 1C). Truncation of exon 11 to produce the exon 1-10 fragment slightly reduced the
Kd value compared with that of the wild type. In
contrast, the exon 1-9 fragment, resulting from truncation of exons 10 and 11, showed a ~20-fold higher Kd value. As the
C terminus was successively truncated, the Kd values
increased in several discrete steps. This result suggests that the
ultimate effects of the truncated exons on hCG binding are not equal.
There are four potentially important regions around the junctions
between exons 1 and 2, 4 and 5, 6 and 7, and 9 and 10 (Fig.
1D). Among the four, the region near the exon 1-2 junction
appears to be most crucial for hCG binding. Therefore, the region was
further investigated by Ala scanning.
Ala Scanning of the Asp17-Arg26
Sequence--
As a first step to identify important residues near the
exon 1-2 junction,
Asp17-Gly18-Ala19-Leu20-Arg21-Cys22-Pro23-Gly24-Pro25-Arg26
was Ala-scanned (Fig. 2). Ala
substitution for Arg21, Pro23,
Pro25, or Arg26 did not impair hCG binding to
intact cells but increased the Kd values
approximately 5-6-fold (Fig. 2, A and B). All of
the mutant receptors that bound hCG on intact cells were capable of
inducing cAMP production (Fig. 2C). Their EC50
values for cAMP induction were similar to the wild type value except,
the R26A mutant, which has a 2.4-fold higher EC50 value
(Fig. 2, table). In contrast, Ala substitution for
Asp17, Gly18, Leu20,
Cys22, or Gly24 impaired hCG binding to intact
cells. EC50 values for cAMP production are generally lower
than Kd values of the corresponding receptors. This
is thought to be due to the fact that receptors are activated before
they are fully occupied by hormone. Sometimes, it happens when <5% of
receptors are occupied. Furthermore, the maximum receptor activation is
reached long before receptors are fully occupied.

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Fig. 2.
Ala substitution for
Asp17-Arg26. Residues from
Asp17 to Arg26, except Ala19, of
the LH/CG receptor were individually substituted with Ala, and the
resulting mutant receptors were stably expressed in 293 cells. Intact
cells were used for 125I-hCG binding in the presence of
increasing concentrations of unlabeled hCG (A and
B) and for cAMP production (C). Experiments were
repeated four to five times in duplicate as described in the legend to
Fig. 1. As a control to verify intended mutations and to identify
unintended changes, if any, in the cDNA sequence, mutant receptor
cDNAs were reverted to the wild type, and their sequences were
confirmed. The revertant cDNAs were used to transfect cells and
were assayed for hormone binding and cAMP induction. All revertants
behaved the same as the wild type (data not shown). NS, not
significant.
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Hormone Binding of Solubilized Receptors--
To determine whether
the non-binding mutants are defective in hCG binding or trapped in the
cytoplasm, cells expressing the mutants were solubilized in Nonidet
P-40 and assayed for hCG binding (Fig.
3). hCG bound to the D17A and G18A
mutants. Their Kd values are comparable to those of
the R21A, P23A, P25A, and R26A mutants, but ~5-8-fold higher than
the wild type value. This result suggests that the D17A and G18A
mutants are trapped in cells. In contrast to these binding-competent
mutants, the L20A, C22A, and G24A mutants failed to bind hCG.
Therefore, the L20A, C22A, and G24A mutants either were not synthesized
or were synthesized but incapable of binding hormone. Even if they were
expressed, our data shown in Figs. 2 and 3 could not pinpoint whether
the mutants were located either on the cell surface or within the cells. To distinguish these possibilities, we utilized two independent immunological methods, immunofluorescence microscopy and
125I-antibody that should bind to receptors expressed on
the cell surface and in cells.

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Fig. 3.
125I-hCG binding to non-binding
mutant receptors solubilized in Nonidet P-40. Cells transfected
with plasmids encoding the non-binding receptors shown in Fig. 2 were
solubilized in Nonidet P-40 and assayed for 125I-hCG
binding in the presence of increasing concentrations of unlabeled hCG.
NS, not significant.
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Immunofluorescence Microscopy--
For immunological studies, the
Flag epitope (16) was inserted between the C terminus of the signal
sequence and the N terminus of mature receptors. The resulting
receptors are the Flag-wild type LH/CG receptor,
Flag-LH/CG-RD17A, Flag-LH/CG-RG18A,
Flag-LH/CG-RL20A, Flag-LH/CG-RC22A, and
Flag-LH/CG-RG24A. Cells were transfected with the plasmids
encoding receptors carrying the Flag epitope. They were either examined
intact or after treatment with Triton X-100 to permeabilize the plasma
membrane and to allow the antibody to enter the cytosol. The cells were
treated sequentially with mouse anti-Flag and Texas Red-conjugated goat
anti-mouse IgG monoclonal antibodies. Confocal laser fluorescence
microscopy showed bright fluorescence of the Flag-wild type receptor on
intact cells and in permeabilized cells (Fig.
4). Cells expressing the wild type receptor lacking the Flag tag did not show fluorescence, regardless of
permeabilization. In addition, the cells expressing the Flag-wild type
receptor did not show fluorescence when treated for fluorescence labeling without anti-Flag antibody. These controls demonstrate that
the fluorescence staining is specific for the Flag epitope and that the
Flag-wild type receptor is expressed both on the cell surface and
within cells. Flag-LH/CG-RL20A,
Flag-LH/CG-RC22A, and Flag-LH/CG-RG24A were
also observed on intact and permeabilized cells, indicating they were
expressed on the cell surface and within cells. On the other hand,
Flag-LH/CG-RD17A and Flag-LH/CG-RG18A were
observed in permeabilized cells, but not intact cells, indicating that
they were not transported to the cell surface.

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Fig. 4.
Localization of Flag-LH/CG receptors using
anti-Flag antibody. Cells stably transfected with plasmids
encoding the wild type LH/CG receptor, the Flag-wild type LH/CG
receptor, Flag-LH/CG-RD17A, Flag-LH/CG-RG18A,
Flag-LH/CG-RL20A, Flag-LH/CG-RC22A, and
Flag-LH/CG-RG24A were fixed with 4% formaldehyde and
sequentially labeled with mouse anti-Flag antibody and Texas
Red-conjugated goat anti-mouse IgG. In addition to this labeling of
intact cells, cells were permeabilized with 0.1% Triton X-100 for
labeling receptors present inside of cells. Specimens were scanned
through multiple sections of cells using confocal laser fluorescence
microscopy.
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125I-Anti-Flag Antibody Binding to Intact
Cells--
125I-Anti-Flag antibody was used to verify the
surface expression of receptors. Fig. 5
shows that 125I-anti-Flag antibody bound to cells
expressing the Flag-wild type receptor, Flag-LH/CG-RL20A,
Flag-LH/CG-RC22A, or Flag-LH/CG-RG24A, but not
to those expressing Flag-LH/CG-RD17A or
Flag-LH/CG-RG18A. The Kd values were 29, 96, 106, and 135 nM for the Flag-wild type receptor,
Flag-LH/CG-RL20A, Flag-LH/CG-RC22A, and
Flag-LH/CG-RG24A, respectively. The numbers of binding
sites on the cell surface (27,000 and 187,000 per cell) are
significant, and therefore, these bindings are likely to be specific.
Taken together, these and the fluorescence microscopy results
demonstrate that the Flag-wild type receptor,
Flag-LH/CG-RL20A, Flag-LH/CG-RC22A, and
Flag-LH/CG-RG24A were expressed on the cell surface,
whereas Flag-LH/CG-RD17A and Flag-LH/CG-RG18A
were trapped in the cells.

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Fig. 5.
Binding of 125I-anti-Flag
antibody. Intact cells stably transfected with plasmids encoding
the Flag-wild type LH/CG receptor, Flag-LH/CG-RD17A,
Flag-LH/CG-RG18A, Flag-LH/CG-RL20A,
Flag-LH/CG-RC22A, and Flag-LH/CG-RG24A were
incubated with 125I-anti-Flag antibody in the presence of
increasing concentrations of unlabeled anti-Flag antibody. Results were
analyzed by Scatchard plots. NS, not significant.
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Activities of Flag-tagged Receptors--
To test whether the Flag
epitope might have interfered with normal processing so that the mutant
receptors carrying the Flag epitope were fortuitously expressed on the
cell surface, the activities of the Flag-LH/CG receptors (hCG binding,
and cAMP induction) were determined. The Flag-wild type receptor and
the wild type receptor on intact cells bound hCG with the same affinity
(Fig. 6, A and B).
In addition, the Flag-wild type receptor was capable of
hCG-dependent cAMP induction, although the EC50
value for cAMP induction was ~3-fold higher than the value of the
wild type receptor (Fig. 6C). These data show that the
Flag-LH/CG receptors are active, although their potency is somewhat
different compared with LH/CG receptors lacking the Flag epitope. With
this in mind, we examined Flag-LH/CG-RL20A,
Flag-LH/CG-RC22A, and Flag-LH/CG-RG24A. They did not bind hCG or induce cAMP production (data not shown). These results, along with the results obtained from the non-Flag mutants, demonstrate that LH/CG-RL20A, LH/CG-RC22A, and
LH/CG-RG24A are expressed on the surface of intact cells,
but are defective in hCG binding. In contrast, LH/CG-RD17A,
LH/CG-RG18A, LH/CG-RR21A, LH/CG-RG24A, LH/CG-RP25A, and
LH/CG-RR26A are capable of binding hCG.

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Fig. 6.
Activities of Flag-tagged receptors.
Hormone binding and cAMP induction by Flag-tagged receptors were
determined as described in the legend to Fig. 2 and under
"Experimental Procedures." NS, not significant.
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DISCUSSION |
Truncation at several discrete regions of the exodomain, near the
boundaries of exons 1-2, 4-5, 6-7, and 9-10, noticeably influences
hormone binding. It is unclear whether these regions represent hormone
contact sites or whether their truncation has allosteric effects on the
global structure of the exodomain and thus indirectly impacts hormone
binding. In the sequence near the exon 1-2 junction, three alternate
residues (Leu20, Cys22, and Gly24)
are important for hormone binding. It has been speculated that a
primary hCG-binding site is the putative crescent structure (21-23)
that is composed of Leu-rich motifs (24). However, the three residues
are upstream (but not part) of the Leu-rich motifs and crescent
structure of the exodomain. Therefore, this upstream region near the N
terminus of the receptor appears to be at least equally important for
hormone binding as the crescent structure. It will be interesting to
see whether this upstream sequence is a hormone contact site and, if
so, how it interacts with the hormone.
Role of Leu20, Cys22, and
Gly24--
The data in this work suggest two general
possible roles for the region covering the three residues. It may
contact hCG or be important for the exodomain to assume a structure
necessary for hormone binding without directly interacting with the
hormone. The latter possibility could be a result of misfolding of the L20A, C22A, or G24A mutant. However, the results of our specific photoaffinity labeling of both hCG
- and
-subunits by a peptide mimic of the receptor region indicate the direct interaction of the
receptor region with hCG (12). Besides, several observations described
in this study are consistent with the interaction of the receptor
region with hCG. The effect of Ala substitutions for the three
alternate residues on hormone binding are remarkably consistent with,
yet strikingly different from, the effects of Ala substitutions for the
intervening and flanking residues. For example, Ala substitutions for
the flanking and intervening residues of Leu20,
Cys22, and Gly24 reduced the hormone binding
affinity approximately 5-8-fold, but never abrogated hCG binding.
Therefore, the flanking and intervening residues appear to be important
for hormone binding, but are not as crucial as Leu20,
Cys22, and Gly24. The three residues are 20 amino acids from the N terminus, and the Flag epitope was recognized in
the Flag-wild type receptor as well as in Flag-LH/CG-RL20A,
Flag-LH/CG-RC22A, and Flag-LH/CG-RG24A. These
results suggest that the structure of the N-terminal region including
Leu20, Cys22, and Gly24 is similar
regardless of Ala substitutions for the three residues. The alternate
sequence of Leu20, Cys22, and Gly24
suggests a
-like structure, orienting the three residues on one side where hCG might contact. Our results are consistent with other
reports that the presence of the peptide mimic of receptor Arg21-Pro38 (10), substitution for
Cys22 (25), or deletion of exon 1 (11) interferes with
hormone binding.
In addition to the three residues, our data and the peptide mimic (10)
and deletion (11) studies predict the importance of the boundaries of
exons 1-2 and 4-5 in hormone binding. On the other hand, our data and
the peptide mimic study (10) suggest the potential role of the exon
9-10 boundary in hormone binding. In contrast, the deletion of
residues 212-341 covering the exon 9-10 boundary from the receptor
increased the Kd value merely by ~2-fold (11). Our
study and the deletion study (11), but not the peptide mimic study
(10), suggest a role of the exon 6-7 boundary in hormone binding.
These results suggest the usefulness and limitations of individual
methods by themselves.
Some of the mutant receptors such as R21A, P23A, P25A, Flag-L20A,
Flag-C22A, and Flag-G24A were expressed at significantly higher levels
compared with expression of the wild type receptor. It is unclear
whether the higher expression levels are caused by better plasmid
preparation, better transfection, more efficient translation and/or
processing, or a reduced degradation rate. We have experienced that the
expression level is dependent on the transfection efficiency, which, in
turn, is affected by the quality and amount of plasmid preparation as
well as the cell condition. If the transfection efficiency is equal, it
is possible that mutations could impact on the expression level.
Similarly, substitutions for a crucial amino acid can diversely
influence the machinery for the surface expression depending on the
side chain of the amino acid (26).
Importance of Asp17 and Gly18 in
Targeting--
When either Asp17 and Gly18 was
substituted with Ala, the corresponding mutant receptors were trapped
within cells and could not be detected on the cell surface. This total
lack of their surface expression implies the importance of these
residues in targeting the receptor to the plasma membrane. Furthermore,
the targeting machinery is extremely sensitive to a change in the
structure of this sequence. At least in the case of the D17A and G18A
substitutions, the targeting mechanism appears to be more sensitive
than hormone binding is. Therefore, targeting to the cell surface could
be used as an indicator of structural changes of the receptor including mutant receptors.