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(Received for publication, March 15, 1995; and in revised form, July
31, 1995) From the
The lutropin/choriogonadotropin receptor (LH/CG-R) is a G
protein-coupled receptor with a relatively large extracellular domain.
The cDNAs of LH/CG-R wild type and 15 point and double mutants, which
encoded residues of opposite charge to that of wild type, were
transiently transfected into COS-7 cells. Human choriogonadotropin
(hCG) binding was determined, as was hCG-mediated cAMP production. Most
of the replacements resulted in no substantive effect on the binding
affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production,
although the mutants expressed at a lower level than LH/CG-R wild type.
The most interesting observation was noted with two point mutants of
LH/CG-R, Glu
The four glycoprotein hormones act through three homologous G
protein-coupled receptors, each characterized by a relatively large
glycosylated extracellular domain (Segaloff and Ascoli, 1993). Lutropin
and human choriogonadotropin bind to a common receptor
(LH/CG-R)( Considerable data have been obtained to delineate specific amino
acid residues and regions on the hCG The present
study addresses 11 of the ionizable groups on the extracellular portion
of rat LH/CG-R: Arg
An anti-LH/CG-R rabbit antiserum (Rosemblit et
al., 1988), kindly provided by Dr. Deborah Segaloff (University of
Iowa,), was diluted 4-fold with cold PBS (pH 8.0) and absorbed to a
slurry of protein A-Sepharose in a small column at 4 °C. The column
was then washed extensively with PBS, and the IgG fraction was eluted
by addition of a 50 mM glycine buffer, pH 3.0, containing 0.1 M NaCl, and collected in tubes of 1 ml each containing 75
µl of 0.2 M Tris-HCl and 23.3 µl of 0.85 M Hepes buffer. The most concentrated fractions were pooled and
stored at -70 °C. Western blots were based on the
technique of Hipkin et al. (1992) with slight modifications.
Prestained M The DNA sequences of the rat LH/CG-R wild type and mutants
were determined for the mutated and adjacent regions in all clones by
dideoxynucleotide sequencing. Following this confirmation of the
desired wild type and mutant clones, transfection efficiencies were
monitored by transient cotransfection of pSVL-LH/CG-R wild type and
mutants into COS-7 cells with pSVL- hCG binding to
transfected cells was determined via competition of
[
Fig. 1Fig. 2Fig. 3show the results of
competition and saturation binding of the LH/CG-R point mutants, and
the K
Figure 1:
hCG binding to COS-7 cells transfected
with pSVL vectors containing the wild type and mutant LH/CG-R cDNAs.
Competition binding of [
Figure 2:
hCG binding to COS-7 cells transfected
with pSVL vectors containing the wild type and mutant LH/CG-R cDNAs.
Competition binding of [
Figure 3:
hCG binding to two point mutants of
LH/CG-R. Saturation binding of [
Two double mutants of LH/CG-R, E65K,E68K and
E332K,D333K, were also characterized by direct and competitive binding (Fig. 4). The K
Figure 4:
hCG binding to COS-7 cells transfected
with pSVL vectors containing mutant LH/CG-R cDNAs. Competition binding
of [
Soluble binding assays were performed on several of the point
LH/CG-R mutants that exhibited a relatively low number of surface
receptors (Table 2). Compared to wild type LH/CG-R, the
Lys
To assess the relative amounts of mutant receptors in
cells with a low number of surface receptors, e.g. <25%
LH/CG-R wild type, Western blots were obtained. As reported by others
(Segaloff and Ascoli, 1993), purified rat LH/CG-R gives an apparent
molecular mass of about 93 kDa. Transiently transfected COS-7 cells
express a major band of about 93 kDa (Fig. 5), which is presumed
to correspond to the 85-kDa form obtained by Hipkin et al. (1992) using stably transfected human embryonic kidney 293 cells.
In addition, a minor band of apparent molecular mass of 78 kDa is also
detected from LH/CG-R wild type, probably corresponding to the 68-kDa
band reported by Hipkin et al.(1992), as are apparent higher
and lower M
Figure 5:
Western blots of COS-7 cells transfected
with pSVL vectors containing wild type and several mutant LH/CG-R
cDNAs. Results are shown for LH/CG-R mutants exhibiting <25% of the
receptors/cell relative to wild type LH/CG-R. These include the mutants
Lys
The functionality of each of the mutant receptors was
evaluated by cAMP production in response to added hCG. Fig. 6and 7 show typical results for those receptor point
mutants that exhibited ED
Figure 6:
Effects of hCG on cAMP production in COS-7
cells transfected with pSVL vectors containing mutant and seven wild
type LH/CG-R cDNAs. Results are given for replacements of
Arg
Figure 8:
Lack of an effect of hCG on cAMP
production in COS-7 cells transfected with pSVL vectors containing
mutant LH/CG-R cDNAs. Results are shown for two point mutants and one
double mutant with lysine replacements of Glu
Figure 7:
Effects of hCG on cAMP production in COS-7
cells transfected with pSVL vectors containing five point mutants and
one double mutant of LH/CG-R. Data are presented for lysine
replacements of Glu
The results presented herein enable us to conclude that
replacement of either Glu The two M Single
replacements of LH/CG-R at several conserved sites with oppositely
charged amino acid residues have no significant effect on the apparent K With the exception of the replacements of
Glu It is
interesting and somewhat surprising that cAMP responsiveness appears
independent of receptor density over a fairly wide concentration range.
Excluding replacements at positions 332 and 333, LH/CG-R wild type and
the various mutants are present at average receptor numbers/cell
ranging over 2 orders of magnitude; yet, the maximal amount of cAMP
produced at high concentrations of hCG is about the same. These results
suggest that the amount of G In
summary, Glu
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 30023-30028
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Lys and Asp
Lys,
which bound hCG but failed to give increased cAMP production. Several
of the mutant forms of LH/CG-R that expressed at low levels were
further analyzed by soluble binding assays and Western blots. There was
no evidence of any significant degree of intracellular trapping of
hCG-binding mutant receptors. The expected major (93 kDa) and minor (78
kDa) forms were found for LH/CG-R wild type and several of the mutants.
The Lys
Asp and Asp
Lys
mutants exhibited primarily the lower M
form,
indicating that receptor processing was impaired or that the mutant
higher M form was more rapidly degraded than
LH/CG-R wild type. These results demonstrate that Glu and
Asp
, which are located near the first transmembrane
helix, are important in receptor activation, while other conserved
ionizable residues of LH/CG-R appear important in cell surface
expression or stability but not in binding or signaling.
), of which cDNAs are available from four species:
rat (McFarland et al., 1989), pig (Loosfelt et al.,
1989), human (Minegishi et al., 1990), and mouse (Gudermann et al., 1992). Rat LH/CG-R is encoded by 11 exons (Koo et
al., 1991; Tsai-Morris et al., 1991), and the
extracellular domain contains a number of pseudo-leucine-rich repeats
(McFarland et al., 1989; Braun et al., 1991). 
and 
subunits that
participate in receptor binding and signal transduction. The recent
availability of a crystal structure of partially deglycosylated hCG
(Lapthorn et al., 1994; Wu et al., 1994) adds a new
dimension to the analysis of glycoprotein hormone structure-function
relationships. In contrast, relatively little is known about the
conformation of the receptor and specific amino acid residues that may
be involved in hormone binding and signaling, although mutagenesis has
been used to map certain of these regions of LH/CG-R (Xie et
al., 1990; Ji and Ji, 1991, 1993; Segaloff and Ascoli, 1993; Ji et al., 1993; Quintana et al., 1993; Wang et
al., 1993a, 1993b). A perusal of the available amino acid
sequences of LH/CG-R from several species shows the presence of a
number of conserved ionizable amino acid residues, many of which are
also conserved in the follitropin receptor and the thyrotropin
receptor. Such conserved residues may be important in signaling or as
hormone contact sites, e.g. to oppositely charged conserved
side chains on LH, CG, follitropin, and thyrotropin. and Arg
(encoded by exon
1), Lys
(encoded by exon 2), Glu
and
Glu
(encoded by exon 3), Arg
and
Lys
(encoded by exon 5), Glu
and
Lys
(encoded by exon 9), and Glu
and
Asp
(encoded by exon 11). Site-directed mutagenesis was
used to prepare single and double mutants where each ionizable side
chain was replaced by one of opposite charge. The data suggest that two
point mutants of LH/CG-R, Lys
and Lys
, bind
hCG with nearly the same affinity as LH/CG-R wild type but exhibit
diminished signaling.
Supplies
The Transformer
site-directed mutagenesis kit was obtained from Clontech Laboratories,
Inc, the Sequanase version 2.0 kit was a product of U. S. Biochemical
Corp., the plasmid kit was from Qiagen Inc. (Chatsworth, CA), and the
Magic
Minipreps DNA Purification System was from Promega.
Bovine serum albumin, isobutylmethylxanthine, DEAE-dextran,
chloroquine, dextran sulfate, dimethyl sulfoxide, and Nonidet P-40 were
purchased from Sigma. hCG (CR-121) was provided by National Institutes
of Health and by Dr. Steven Birken (Columbia University, NY);
[
I]hCG (100-150 µCi/µg) was from
ICN (Horsham, PA). The cAMP radioimmunoassay kit was from DuPont NEN.
Dulbecco's modified Eagle's medium and fetal bovine serum
were obtained from Life Technologies, Inc.; NuSerum was from
Collaborative Biomedical Products (Bedford, MA).
Mutagenesis of the LH/CG-R cDNA
The cDNA
for the rat LH/CG receptor was kindly provided by Dr. William Moyle
(Piscataway, NJ) in the expression vector pSVL. The desired 21-base
deoxyoligonucleotides were synthesized by Dr. Rudolf Werner (University
of Miami, FL) on an Applied Biosystems model 380B DNA synthesizer and
by the Molecular Genetics Core Facility of the University of Georgia. In vitro mutagenesis was performed using the
Transformer site-directed mutagenesis kit as described
(Deng and Nickoloff, 1992), and mutated plasmid clones were identified
by double-stranded sequencing.
Expression of LH/CG-R
COS-7 cells
(African green monkey kidney cells), obtained from ATCC CRL, were grown
at 37 °C in humidified air containing 5% CO
in the
following medium: Dulbecco's modified Eagle's medium (90%),
fetal bovine serum (10%), and 100 units/ml each of penicillin and
streptomycin. Cells were transiently transfected at 60-80%
confluency by the DEAE-dextran method. Transfection efficiencies were
estimated by cotransfecting a pSV--galactosidase plasmid with the
pSVL vectors containing the LH/CG-R wild type and mutant cDNAs. 48 h
after transfection, the cells were stained in situ for
-galactosidase. This was accomplished by first washing the cells
with PBS five times, then fixing them in a 0.05% (v/v) glutaraldehyde
solution in PBS for 10-15 min. After three additional washes with
PBS, the cells were incubated in a solution containing 1 mg/ml
5-bromo-4-chloro-3-indoyl -D-galactoside 1 mM MgCl, 20 mM potassium
ferricyanide/ferrocyanide in PBS for at least 1 h at 37 °C. The
cells transfected with the pSV--galactosidase control plasmid were
readily visualized under the microscope and counted. From the total
number of cells in the field, transfection efficiencies were obtained.[
At 48 h post-transfection, the cells were assayed for
[I]hCG Binding to COS-7
Cells
I]hCG binding, along with untransfected cells
as control. Cells (1
10
cells/well) were washed
twice with serum-free Waymouth's medium containing 1 mg of bovine
serum albumin/ml, then 1 ml of this medium was added to each well. For
competitive binding assays, [I]hCG (10
cpm) and different concentrations of unlabeled hCG were added to
each well, while for direct binding assays, cells were incubated with
increasing concentrations of [I]hCG.
Nonspecific binding was determined by inclusion of a 1000-fold excess
of unlabeled hormone. The cells were incubated overnight with gentle
shaking at room temperature. On the following day, cells were washed
twice with PBS, then trypsinized, collected, and counted in a
counter. Specific binding of [
I]hCG in the
absence of unlabeled hormone gave 4,000-5,000 cpm, and
nonspecific binding was about 5%. The results were analyzed by In-Plot
(Graphpad Software, San Diego, CA), by Ligand Program (Munson and
Rodbard, 1980), and by graphical analysis, the agreement between the
various methods being good. Unless indicated otherwise, the data from a
typical experiment are shown as mean ± S.E.
[
This procedure is
based on that described by others (Xie et al., 1990; Thomas
and Segaloff, 1994). Control cells and transfected cells (48 h) were
placed on ice and washed twice with cold 0.15 M NaCl, 20
mM Hepes, pH 7.4. They were then scraped into a small volume
of the same buffer containing 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, and 5 mMN-ethylmaleimide. The cells were centrifuged (1500
I]hCG Binding to
Detergent-solubilized COS-7 Cell Extracts
g, 15 min, 4 °C) and lysed by vortexing in a
solution containing the same buffer with 1% Nonidet P-40 and 20%
glycerol. After incubation on ice for 15 min, the cell lysate was
centrifuged (12,000 g, 15 min, 4 °C), and the
supernatant was diluted to 0.1% Nonidet P-40 and incubated overnight
with [I]hCG (2-3
10 cpm) in the presence and absence of a 1000-fold excess of hCG.
Then, the binding solution was added to Whatman GF/B filters that had
been soaked in 0.3% polyethylenimine (pH 9.1) for at least 1 h (Roche et al., 1985). After filtration and washing five times, the
filters were counted in a counter. The specific binding of
[
I]hCG to solubilized LH/CG-R was between 5 and
15
10
cpm, and nonspecific binding was about 25% of
the total binding.cAMP Assay
After transfection (48 h), the
COS-7 cells were washed twice with Dulbecco's modified
Eagle's medium containing 1 mg of bovine serum albumin/ml and
incubated in 0.5 ml of the medium containing isobutylmethylxanthine
(0.8 mM) at 37 °C for 15 min. Increasing concentrations of
hCG were then added, and the incubation was continued for 30 min at 37
°C. After removing the medium, the cells were lysed in 100% ethanol
at -20 °C overnight. The extract was collected, dried under
flowing nitrogen gas, and resuspended in the buffer of the cAMP assay
kit. cAMP concentrations were determined with the
[I]cAMP radioimmunoassay kit. Unless indicated
otherwise, the results of a typical experiment are given as mean
± S.E.
Western Blot Analysis of Partially Purified
LH/CG-R
Following transient transfection (48 h) of COS-7
cells with pSVL plasmids containing the LH/CG-R cDNAs (wild type and
mutants), cell lysates were prepared as described above, and LH/CG-R
was partially purified using wheat germ agglutinin-agarose
chromatography (Rosemblit et al., 1988; Thomas and Segaloff,
1994). LH/CG-R was eluted with 3 mM triacetylchitotriose
(Hipkin et al., 1992) in a 20 mM Hepes buffer, pH
7.4, containing 0.15 M NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide,
5 mM EDTA, 0.1% Nonidet P-40, and 20% glycerol. The partially
purified LH/CG-R fractions were concentrated by Centricon-30 spin
columns. standards (ovalbumin, bovine serum
albumin, -galactosidase, and myosin from Bio-Rad) and equal
amounts of cell lysates in sample buffer, with no boiling and in the
absence of reducing agent, were applied to 7% SDS-polyacrylamide gels.
An equivalent amount of lysate from untransfected COS-7 cells was used
as a negative control. The proteins were electrophoretically
transferred to polyvinylidine difluoride membranes, which were then
washed twice (5 min each) with 20 mM Tris-HCl buffer, pH 7.5,
containing 0.5 M NaCl, and blocked for 2 h at room temperature
with PBS containing 5% instant nonfat dry milk, 0.2% Tween 20, and 10%
glycerol. Next, the filters were incubated overnight at room
temperature with the same blocking solution containing 3 µg/ml
rabbit anti-LH/CG-R IgG. The blot was then washed five times (5 min
each) with blocking solution, followed by incubation for 1 h at room
temperature with a 1:5000-fold dilution of a horseradish
peroxidase-labeled donkey anti-rabbit IgG whole antibody fraction in
the blocking solution. The second antibody was replaced, and the
filters were washed twice (5 min each) with 20 mM Tris-HCl, pH
7.5, 0.5 M NaCl; this was followed by two washes (5 min first
wash, 10 min second wash) with the same buffer containing 1% Nonidet
P-40. The filters were then washed for 5 min with the 20 mM Tris-HCl, 0.5 M NaCl (pH 7.5) solution, for 10 min with
the same buffer containing 1% Nonidet P-40, and finally for 5 min with
the buffer solution only. The blots were treated for 1 min with
Amersham ECL developer solutions 1 and 2 (combined in equal amounts),
then wrapped in Saran wrap and exposed to autoradiography film for
about 30 s, followed by immediate development. The apparent M and density of the various bands were obtained
after scanning densitometry (PDI, Huntington Station, NY).
-galactosidase. The
cotransfected cells were stained in situ, and transfection
efficiencies of 5-10% were estimated.I]hCG with hCG and direct binding of
[
I]hCG. The results from multiple studies using
competition and saturation binding with LH/CG-R wild type yielded an
apparent K
of 0.17 ± 0.02 nM (n = 11) and an average of about 1 ± 0.1
10
receptors/cell, uncorrected for transfection
efficiencies (Table 1). We also found that untransfected COS-7
cells, as expected (Loosfelt et al., 1989), do not contain
LH/CG-R (data not shown).
values and receptor numbers are given in Table 1. Generally, the K values are within
severalfold of that obtained for wild type LH/CG-R, indicating that no
major alteration in hormone affinity accompanied the mutations. Several
of the receptor point mutants, however, had K values slightly lower than that of wild type, e.g. Lys
Arg, Lys
Asp, and
Lys
Asp. In contrast, the number of receptors/cell
is, with the exception of the Arg
replacements and the
Lys
Arg replacement, considerably less than that
obtained for the wild type receptor. Most of the other LH/CG-R mutants
yield on the order of 1-4
10 receptors/cell,
although receptors with the Lys
Asp and
Asp
Lys replacements contained <1
10 receptors/cell, again uncorrected for transfection
efficiencies.
I]hCG and hCG to seven
point mutant forms of LH/CG-R with replacements of Arg
,
Arg
, Lys
, Glu
, and Glu
is shown. In each case wild type (Wt) receptor was
included for comparison.
I]hCG and hCG to six
point mutant forms of LH/CG-R with replacements of Arg
,
Lys
, Glu
, Lys
,
Glu
, and Asp
is shown. Wild type (Wt) receptor was also included for
comparison.
]hCG to COS-7
cells transfected with pSVL vectors containing mutant LH/CG-R cDNAs
that produce lysine replacements of Glu
and of
Asp
is shown.
of the former double mutant
was somewhat lower than that of LH/CG-R wild type, and the average
number of receptors/cell was 625 ± 106 (n = 3) ( Fig. 4and Table 1). No specific binding was detected with
LH/CG-R(Lys, Lys
) under the conditions
used.
I]hCG and hCG to two double mutants of
LH/CG-R, E65K,E68K (
) and E332K,D333K () is shown; wild
type (Wt,
) LH/CG-R was included as control (left
panel). Saturation binding of [I]hCG to
the double mutant with lysine replacing Glu
and
Glu
is shown (right
panel).
Asp and Lys
Asp mutants
yielded only very low levels of specific binding. In contrast, the
Glu
Lys and the Asp
Lys
mutants gave specific binding intermediate to that of control and wild
type LH/CG-R.
minor bands, which may be nonspecific.
Interestingly, the LH/CG-R point mutants, Lys
Asp
and Glu
Lys, as well as the double mutant,
E65K,E68K, yield both the 93- and 78-kDa forms in about equal amounts,
while the point mutants, Lys
Asp, Lys
Asp, and Asp
Lys, exhibit
predominantly the lower M
form ( Fig. 5and Table 2). Equal amounts of cell lysate protein were added from
cells transfected with wild type and mutant LH/CG-R cDNAs to facilitate
comparison of the relative total amount of immunoreactive receptor in
each case. This amount of protein, which was necessary to visualize the
relatively low levels of mutant receptors, resulted in overloading the
gel with wild type LH/CG-R and obscuring the resolution of the major
and minor bands. These were resolved, however, by densitometric
scanning of the blots and by adding reduced amounts of cell lysate
protein (data not shown). Comparable studies on cell lysates that were
not purified by lectin chromatography gave similar results (data not
shown), indicating that no other non-glycosylated forms of LH/CG-R are
present.
Asp (K-55-D), Lys
Asp (K-121-D), Lys
Asp (K-235-D), Glu
Lys (E-332-K),
Asp
Lys (D-333-K), and E65K,E68K (E-65 & 68-K). Results are also shown for control cells (Control) and cells transfected with the wild type LH/CG-R
cDNA (Wt). The two arrows denote apparent molecular
mass values of 93 kDa (upper) and 78 kDa (lower).
values and maximal cAMP levels
similar to LH/CG-R wild type. Interestingly, the
LH/CG-R(Asp
) mutant, which gave a low level of
expression, exhibited an ED
and maximal cAMP response like
that of wild type receptor. In contrast, the LH/CG-R point mutants
Lys
and Lys
were non-responsive to added
hCG (Fig. 8). Likewise, the double mutant,
LH/CG-R(Lys
, Lys
), for which no receptors
could be detected, exhibited no hCG-responsive cAMP production. The
ED
values for cAMP production by hCG in the transiently
transfected COS-7 cells are given in Table 1.
, Arg
, Lys
,
Arg
, and Lys
of LH/CG-R. Data of wild type (Wt) LH/CG-R and untransfected (control) cells are shown for
comparison.
and
Asp
. The data are given as combined means ± S.E.
from several independent experiments. The cAMP values at each
concentration of hCG are slightly higher than control values (not
significant), i.e. in untransfected cells, and significantly
lower than values obtained with wild type LH/CG-R (cf. Fig. 6and Fig. 7for typical
responses).
, Glu
(single mutants of
each and a double mutant), and Glu
and for aspartic acid
replacement of Lys
. Results are also shown for wild type (Wt) LH/CG-R and for untransfected (control) cells. The cAMP
response to LH/CG-R(Asp
) was lower than that of wild type
receptor in this experiment, but this finding was not
reproducible.
or Asp
with Lys
in LH/CG-R yields mutant receptors that bind hCG as well as LH/CG-R
wild type but exhibit diminished signaling. Thus, this region of the
extracellular domain of the receptor, which is <10 amino acid
residues from the first transmembrane helix, appears critical in
gonadotropin-mediated receptor activation. While receptor numbers are
reduced with these particular mutants relative to LH/CG-R wild type,
other single and double receptor mutants with comparable receptor
densities, e.g. Asp
, Asp
,
Asp
, Lys
, and Lys
gave cAMP
responses like that of LH/CG-R wild type. Thus, the reduced
hormone-mediated signaling noted with Lys
and Lys
is believed to be an intrinsic property of the mutant receptors
and not a result of diminished receptor number.
forms of LH/CG-R expressed in COS-7 cells, i.e. 93 and 78 kDa as obtained herein, were first reported by
Ascoli and colleagues (Hipkin et al., 1992) who expressed the
receptor in stably transfected human embryonic kidney 293 cells,
although the molecular mass values they determined were somewhat
different than the values we obtained, e.g. 85 and 68 kDa,
respectively. They reported data showing that the lower M form was a precursor to the higher M species; in addition, they suggested that the
lower M form bound hormone with reduced affinity
relative to the major 85-kDa form. Since a variety of M values has been reported for LH/CG-R purified from various
tissues/species and expressed in cell lines (cf. Segaloff and
Ascoli, 1993), the glycosylation patterns may differ depending upon the
source of the receptor. In any case, we find that the lower M form of rat LH/CG-R expressed in COS-7 cells is
predominant for most of the mutants that lead to low receptor
densities. Since there is no evidence of significant intracellular
accumulation of these mutant forms, this observation suggests that the
mature mutant receptors have a short half-life relative to LH/CG-R wild
type, that processing proceeds less efficiently, or that the mRNA is
degraded more rapidly or translated less efficiently. relative to LH/CG-R wild type. That these
particular replacements in LH/CG-R result in little or no change in the
apparent K of hCG binding does not necessarily
mean that they are not contact sites. For example, Clackson and
Wells(1995) replaced each of the 33 side chains on the extracellular
domain of human growth hormone receptor, known to be involved in single
ligand binding (de Vos et al., 1992), and found that fewer
than half of the mutant receptors exhibited any significant loss in
binding affinity. The greatest contributions to hormone-receptor
binding were attributed to six hydrophobic amino acid residues on the
receptor and, to a lesser extent, to five ionizable side chains. These
particular 11 contact sites form a contiguous region in which the
hydrophobic groups are surrounded by the charged groups. Thus, about
two-thirds of the receptor amino acid residues that are rendered
solvent-inaccessible upon hormone binding contribute little to binding
affinity. Whether a similar relationship will emerge for LH/CG-R
remains to be determined. and Asp
with Lys, the charge reversals
we made on LH/CG-R did not alter receptor functionality as judged by
cAMP production in response to added hCG. There was, however, a
dramatic effect of most of these mutations on receptor density. A
reduction in receptor number could result from inefficient translation
of the mRNA, instability of the mutant receptor, and/or inefficient
assembly into the plasma membrane, as has been noted for several point
and deletion mutants of LH/CG-R (Segaloff and Ascoli, 1993).
proteins or adenylate cyclase
is limiting with regard to hCG-LH/CG-R-mediated cAMP production. and Asp
, which are located
<10 amino acid residues from the beginning of the first
transmembrane helix and are invariant in the known LH/CG, follitropin,
and thyrotropin receptors, appear to participate in transmembrane
signaling. Thus, these particular amino acid residues may have
fundamental roles in signaling for all of the glycoprotein hormone
receptors.
)
We thank Drs. Steven Birken, William Moyle, and
Deborah Segaloff for providing important materials. We also thank Drs.
Prema Narayan and Lizette Fernandez for helpful discussions and Dr.
K.-C. Peng for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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N. Bhowmick, P. Narayan, and D. Puett Identification of Ionizable Amino Acid Residues on the Extracellular Domain of the Lutropin Receptor Involved in Ligand Binding Endocrinology, October 1, 1999; 140(10): 4558 - 4563. [Abstract] [Full Text]
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C. A. Alvarez, P. Narayan, J. Huang, and D. Puett Characterization of a Region of the Lutropin Receptor Extracellular Domain Near Transmembrane Helix 1 That Is Important in Ligand-Mediated Signaling Endocrinology, April 1, 1999; 140(4): 1775 - 1782. [Abstract] [Full Text]
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 S. Y. Hsu, S.-G. Liang, and A. J. W. Hsueh Characterization of Two LGR Genes Homologous to Gonadotropin and Thyrotropin Receptors with Extracellular Leucine-Rich Repeats and a G Protein-Coupled, Seven-Transmembrane Region Mol. Endocrinol., December 1, 1998; 12(12): 1830 - 1845. [Abstract] [Full Text]
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S. S. Stavrou, Y.-S. Zhu, L.-Q. Cai, M. D. Katz, C. Herrera, M. DeFillo-Ricart, and J. Imperato-McGinley A Novel Mutation of the Human Luteinizing Hormone Receptor in 46XY and 46XX Sisters J. Clin. Endocrinol. Metab., June 1, 1998; 83(6): 2091 - 2098. [Abstract] [Full Text]
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 K. Ryu, H. Lee, S. Kim, J. Beauchamp, C.-S. Tung, N. W. Isaacs, I. Ji, and T. H. Ji Modulation of High Affinity Hormone Binding. HUMAN CHORIOGONADOTROPIN BINDING TO THE EXODOMAIN OF THE RECEPTOR IS INFLUENCED BY EXOLOOP 2 OF THE RECEPTOR J. Biol. Chem., March 13, 1998; 273(11): 6285 - 6291. [Abstract] [Full Text] [PDF]
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F.-P. Zhang, A. S. Rannikko, P. R. Manna, H. M. Fraser, and I. T. Huhtaniemi Cloning and Functional Expression of the Luteinizing Hormone Receptor Complementary Deoxyribonucleic Acid from the Marmoset Monkey Testis: Absence of Sequences Encoding Exon 10 in Other Species Endocrinology, June 1, 1997; 138(6): 2481 - 2490. [Abstract] [Full Text] [PDF]
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S. Hong, K.-S. Ryu, M.-S. Oh, I. Ji, and T. H. Ji Roles of Transmembrane Prolines and Proline-induced Kinks of the Lutropin/Choriogonadotropin Receptor J. Biol. Chem., February 14, 1997; 272(7): 4166 - 4171. [Abstract] [Full Text] [PDF]
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C. Wu, P. Narayan, and D. Puett Protein Engineering of a Novel Constitutively Active Hormone-Receptor Complex J. Biol. Chem., December 6, 1996; 271(49): 31638 - 31642. [Abstract] [Full Text] [PDF]
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R. L. Gilchrist, K.-S. Ryu, I. Ji, and T. H. Ji The Luteinizing Hormone/Chorionic Gonadotropin Receptor Has Distinct Transmembrane Conductors for cAMP and Inositol Phosphate Signals J. Biol. Chem., August 9, 1996; 271(32): 19283 - 19287. [Abstract] [Full Text] [PDF]
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L. M. Fernandez and D. Puett Lys[IMAGE] in the Third Extracellular Loop of the Lutropin/Choriogonadotropin Receptor Is Critical for Signaling J. Biol. Chem., January 12, 1996; 271(2): 925 - 930. [Abstract] [Full Text] [PDF]
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Y. S. Song, I. Ji, J. Beauchamp, N. W. Isaacs, and T. H. Ji Hormone Interactions to Leu-rich Repeats in the Gonadotropin Receptors. I. ANALYSIS OF LEU-RICH REPEATS OF HUMAN LUTEINIZING HORMONE/CHORIONIC GONADOTROPIN RECEPTOR AND FOLLICLE-STIMULATING HORMONE RECEPTOR J. Biol. Chem., January 26, 2001; 276(5): 3426 - 3435. [Abstract] [Full Text] [PDF]
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S. D. Mahale, J. Cavanagh, A. Schmidt, R. MacColl, and J. A. Dias Autologous Biological Response Modification of the Gonadotropin Receptor J. Biol. Chem., April 6, 2001; 276(15): 12410 - 12419. [Abstract] [Full Text] [PDF]
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