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(Received for publication, October 10, 1995) From the
The lutropin/choriogonadotropin receptor (LH/CG-R) contains a
relatively large extracellular domain, in addition to the seven
transmembrane helices (TMH), three extracellular loops (ECL), and three
intracellular loops typical of G protein-coupled receptors. While high
affinity ligand binding has been attributed to the N-terminal
extracellular domain, there is evidence that portions of the three ECLs
may function in ligand binding and transmembrane signaling. We have
investigated the role of several ionizable amino acid residues of rat
LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP
production. COS-7 cells were transfected with the pSVL expression
vector containing cDNAs of either wild-type or mutant rat LH/CG-R.
Several point mutants of Lys
The LH/CG-R ( The LH/CG-R, FSH-R, and
TSH-R are members of the glycoprotein hormone receptor family,
characterized by a relatively large extracellular N-terminal region and
a membrane-embedded C-terminal region containing seven TMHs. This
C-terminal region is homologous to the small ligand binding members of
the G protein-coupled receptor superfamily. However, unlike the small
ligand binding receptors, where binding occurs in a cleft formed by the
TMHs, the glycoprotein hormone receptors utilize their extracellular
domain as the high affinity binding site for the heterodimeric
glycoprotein hormones with molecular masses of 30-37 kDa (6, 7, 8, 9, 10) . This
structural difference classifies these receptors as a distinct
subfamily of the G protein-coupled receptor superfamily (11) . The N- and C-terminal domains of the rat LH/CG-R each contain over
300 amino acid residues, the latter distributed over three ECLs, seven
TMHs, three intracellular loops, and a cytoplasmic tail(1) . If
the LH/CG-R spans the membrane similar to the small ligand binding G
protein-coupled receptors, one would expect the seven putative TMHs to
form a pocket like that of the bacteriorhodopsin and rhodopsin
receptors(12, 13) ; the six hydrophilic connecting
loops are essential in maintaining this conformation. Additionally,
although the high affinity binding site of the LH/CG-R is located in
the ECD, there is evidence to support the presence of a lower affinity
binding site in the C-terminal domain of the
receptor(14, 15) . Therefore, the ECLs represent
potential hormone contact sites. In an attempt to define the role of
the LH/CG-R ECLs in hormone binding and signaling, several Arg and Lys
residues were replaced: Arg
Figure 1:
Schematic representation of the major
portion of the C-terminal region of LH/CG-R, including the seven
putative transmembrane helices, the three exoplasmic loops, and the
three cytoplasmic loops. The single mutations of the full-length
LH/CG-R prepared and characterized in this study are indicated:
Arg
The mean K
The Lys
Figure 2:
hCG
binding to COS-7 cells transfected with wild-type and mutant cDNAs to
LH/CG-R. Competition binding of
Figure 3:
cAMP
levels in COS-7 cells transfected with wild-type and mutant cDNAs to
LH/CG-R in the presence and absence of hCG. Panels A-D give data on six single mutant forms of LH/CG-R; in each case,
results are provided for cells expressing wild-type LH/CG-R and for
control cells.
To investigate
the side-chain specificity of Lys Competitive binding assays
were unable to detect significant cell surface binding of
Figure 4:
Western blots of lysates from COS-7 cells
transfected with cDNAs to wild-type LH/CG-R (WT) and
(Lys
Arg Our results on the substitution of Lys The finding that the [Arg There is no information on the type of
interaction between Lys Ji et al.(27) reported that
Asp Whatever the exact role of Asp Lys Two other positively charged residues of LH/CG-R, which are
invariant in the glycoprotein hormone receptors, Arg Our results on Western
analysis of wild-type LH/CG-R and (Lys In summary, these results enable
us to conclude that Lys
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 925-930
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
in the Third Extracellular Loop of the
Lutropin/Choriogonadotropin Receptor Is Critical for Signaling (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, located at the interface of
ECL III and TMH VII, bound hCG like wild-type receptor but exhibited
greatly diminished ligand-mediated signaling. Neither the point mutant,
Lys
Asp (ECL I), nor the double mutant,
Asp
Lys/Lys
Asp (ECLs I and
III, respectively), showed significant hCG binding to intact cells; in
detergent-solubilized cells, only the double mutant bound hCG. The
mutants Arg
Glu (interface of the extracellular
domain and TMH I) and Lys
Glu (ECL II) proved to
be similar to wild-type receptor in binding and signaling. Our results
establish that Lys
is important in signaling but not
ligand binding. Its location on the opposite side of the membrane from
G
precludes a direct interaction, thus emphasizing the
importance of a conformational change in the receptor and suggesting
that ligand binding to receptor and ligand-mediated receptor activation
are dissociable phenomena.
)(1) is expressed on several
types of gonadal cells (2) and has a crucial role in
reproductive processes. Upon binding to their common receptor, LH and
CG increase adenylate cyclase activity. Although cAMP appears to be the
principal mediator of the actions of gonadotropins on most gonadal
cells, there is evidence to support activation of the phospholipase C
pathway as well, which results in the formation of inositol
1,4,5-trisphosphate and increased [Ca]
levels (3, 4, 5) .
(at the boundary between the
ECD and TMH I), Lys
(ECL I), Lys
(ECL II)
and Lys
(ECL III). Additionally, a reciprocal mutation of
Asp
and Lys
was characterized. The relative
positions of these amino acid residues are shown in Fig. 1.
These particular residues are invariant at the homologous positions in
the LH/CG-R and the FSH-R of all known species; in the TSH-R, the
positions equivalent to residues 401 and 583 are His and Gly,
respectively, and the other residues are invariant. Our results
revealed that Lys
is not involved in hormone binding but
is essential for full receptor activation.
Glu (R341E), Lys
Asp (K401D), Lys
Glu (K488E),
Lys
Glu(K583E), Lys
Gln (K583Q), Lys
Arg (K583R), and
Lys
Pro (K583P) LH/CG-R. In addition, a
reciprocal mutation was investigated: Asp
Lys/Lys
Asp (D397K, K583D)
LH/CG-R.
Materials
I-hCG (100-150
µCi/µg) was purchased from ICN Biochemicals Inc. (Costa Mesa,
CA) and DuPont NEN. [
-S]dATP
(1000-1500 Ci/mmol) and the
I-cAMP radioimmunoassay
kit were products of DuPont NEN. hCG was a gift from Dr. Steven Birken
(Columbia University, New York, NY). The Transformer(TM) mutagenesis
system was obtained from Clontech (Palo Alto, CA), the Sequenase
version 2.0 kit was purchased from United States Biochemical Corp., and
the Wizard Minipreps DNA purification system was a product of Promega
(Madison, WI). The plasmid Maxiprep DNA purification kit was obtained
from Qiagen, Inc. (Chatsworth, CA). Lipofectamine, fetal bovine serum,
trypsin-EDTA, Waymouth's medium, gentamicin, and
penicillin-streptomycin were from Life Technologies, Inc. DEAE-dextran,
isobutylmethylxanthine, chloroquine, triacetylchitotriose, BSA, protein
molecular size markers, and Nonidet P-40 were purchased from Sigma. The
Enhanced Chemiluminescence Western blotting analysis system was from
Amersham Life Sciences, and the BCA protein assay system was purchased
from Pierce. Agarose-bound wheat germ agglutinin was a product of
Vector Laboratories (Burlingame, CA), and Centricon-30 columns were
obtained from Amicon (Beverly, MA). Immobilon P transfer membrane was
purchased from Millipore (Bedford, MA), and glass fiber filters were
from Whatman (Maidstone, UK). Polyclonal anti-LH/CG-R was kindly
provided by Dr. Deborah Segaloff (University of Iowa, Iowa City, IA).
Most other reagents were purchased from Sigma, Life Technologies, Inc.,
or Fisher.
Mutant cDNAs of the Rat LH/CG-R
The cDNA for
the rat LH/CG-R, inserted into the XbaI-BamHI site of
the expression vector pSVL, was the generous gift of Dr. William Moyle
(Robert Wood Johnson Medical School, Piscataway, NJ). The 21-base
deoxyoligonucleotides coding for the appropriate codon changes were
synthesized by Dr. Rudolf Werner (University of Miami, Miami, FL) and
by the Molecular Genetics Instrumentation Facility at the University of
Georgia. In vitro mutagenesis was performed (16) and
mutant clones identified by dideoxy sequencing(17) . Mutant
cDNAs were amplified and DNA was obtained using the Qiagen plasmid
Maxiprep kit.Expression of the Rat LH/CG-R
COS-7 cells were
kindly provided by Dr. Nevis Fregien (University of Miami, Miami, FL)
and also purchased from the American Type Culture Collection
(Rockville, MD). The cells, maintained at 37 °C in humidified air
containing 5% CO
in 90% DMEM and 10% fetal bovine serum,
with 100 units/ml each penicillin and streptomycin, were transiently
transfected with the eukaryotic expression plasmid pSVL-LH/CG-R,
wild-type and mutants, using the DEAE-dextran method (18) or
the Lipofectamine method as recommended by Life Technologies, Inc. For
DEAE-dextran transfection, 20 µg each of pSVL-LH/CG-R and
pSV-
-gal, to estimate transfection efficiencies, were added to a
10-ml solution of 90% DMEM, 10% NuSerum transfection medium containing
a 0.4 mg/ml DEAE-dextran and 0.1 mM chloroquine, and the
washed cells (4
10
) were incubated for
3.5-4 h at 37 °C. After removal of the transfection medium,
the cells were shocked for 2 min with 10% MeSO; then COS-7
growth medium was added to the washed cells. For transfections with
Lipofectamine, a 7.85-ml solution of serum-free DMEM, containing
Lipofectamine (57 µl/ml), 10 µg each of the pSVL-LH/CG-R
construct and the pSV--gal plasmid and 10% (v/v) Opti-MEM media,
was incubated with cells (2-3
10) for 5
h at 37 °C. The transfection medium was removed, the cells were
washed, and COS-7 growth medium was added. With both types of
transfection, the COS-7 cells were incubated overnight at 37 °C in
humidified air with 5% CO.
5-Bromo-4-chloro-3-indolyl--D-galactoside staining of
gluteraldehyde-fixed cells was used to estimate transfection
efficiencies, which were roughly 10% for DEAE-dextran and 40% for
Lipofectamine.
The COS-7 cells were maintained for 16 h after
transfection and then replated (5 I-hCG Cell-surface Binding to Transfected
Cells
10
cells/well,
six-well tissue culture plates). Some 48-51 h post-transfection,
the cells were about 70% confluent. Cells were then washed twice with
serum-free Waymouth's medium containing 1 mg of BSA/ml, and 1 ml
of this media was added to each well. Increasing concentrations of
unlabeled hCG were then added to each well, followed by addition of 25
pMI-hCG (approximately 10
cpm).
Total and nonspecific binding were determined by addition of I-hCG in the absence and presence of excess unlabeled hCG
(54 nM). The plates were incubated at 25 °C for
16-18 h with gentle shaking. The cells were washed twice with
cold phosphate-buffered saline, then trypsinized, collected, and
counted in a
counter. All determinations were performed in
duplicate. Binding affinities and maximal binding capacities were
calculated using the Ligand program (19) .
The transfected cells were maintained for 16 h after
transfection and then replated (2-2.5 I-hCG Binding to Detergent-soluble
Extracts
10
cells/dish, 10-cm tissue culture dishes). The protocol is adapted from
that described by others(6, 20, 21) . About
48 h post-transfection, the transfected cells were placed on ice for 15
min and then washed twice with 5 ml of ice-cold 0.15 M NaCl,
20 mM Hepes, pH 7.4 (buffer A). Cells were scraped into 2 ml
of cold buffer A containing 1 mM phenylmethylsulfonyl
fluoride, 2 mM EDTA, and 5 mMN-ethylmaleimide and pelleted by centrifugation at 2000
g for 20 min at 4 °C. The pellet was resuspended
in 0.25 ml of 1% Nonidet P-40, 20% glycerol in buffer A containing
protease inhibitors and incubated on ice for 15 min. This mixture was
centrifuged at 16,000 g for 15 min at 4 °C. The
supernatant was diluted with 2.25 ml of 20% glycerol in buffer A; 0.5
ml of the extract was incubated for 16-18 h at 4 °C with 50
pMI-hCG (10
cpm). Total and
nonspecific binding were determined in the presence and absence of
excess unlabeled hCG, respectively. Bound radioactivity was separated
from unbound by filtration through Whatman GF/B filters that were
previously soaked in 0.3% polyethylenimine in 10 mM Tris-HCl,
pH 9.1 (22) . The filters were washed five times with 0.1 M NaCl, 10 mM NaN
, 1 mg of BSA/ml in
phosphate-buffered saline and counted in a counter. All
determinations were performed in duplicate.
Intracellular cAMP Assay
Some 16-18 h after
transfection, the transfected cells were replated (1
10 cells/well, 12-well tissue culture plates). At
48-51 h post-transfection, the cells were washed twice with DMEM
containing 1 mg of BSA/ml and incubated in 0.5 ml of this medium with
0.8 mM isobutylmethylxanthine for 15 min at 37 °C.
Increasing concentrations of hCG were then added and the incubation was
continued for 30 min at 37 °C. The cells were washed twice with
fresh medium without isobutylmethylxanthine and then lysed in 100%
ethanol at -20 °C overnight. The extract was collected, dried
under nitrogen gas, and resuspended in the buffer of the I-cAMP assay kit, and cAMP concentrations were determined
by radioimmunoassay. All measurements were performed in duplicate;
means and standard errors were calculated using the Prism program.
Partial Purification of the
LH/CG-R
Transfected cells were maintained for 16-20 h
after transfection and then replated (2-2.5 10 cells/dish, 10-cm tissue culture dishes). Some 48-51 h
post-transfection, detergent-soluble extracts were prepared as
described above. The procedures for cell lysis and partial purification
of wild-type and mutant LH/CG-Rs were based on reports from other
laboratories(21, 23, 24) . Following cell
lysis with 1% Nonidet P-40 and centrifugation as described above, the
supernatant was diluted 2-fold with 10% glycerol in buffer A, then
loaded onto a small agarose-bound wheat germ agglutinin column
equilibrated with 0.1% Nonidet P-40 and 10% glycerol in buffer A, and
rotated at 4 °C overnight. Following extensive washing with this
buffer, the LH/CG-R was eluted with one column volume of 3 mM
triacetylchitotriose (24) containing 1 mM
phenylmethylsulfonyl fluoride, 2 mM EDTA, and 5 mMN-ethylmaleimide. The eluted material was concentrated to
approximately 0.1 ml in a Centricon-30 spin column, and the protein
concentration was determined using the BCA assay.SDS-PAGE and Western Blots
Equal amounts of
purified cell lysates in sample buffer with no reducing agent were
applied to a 7% SDS-polyacrylamide gel without boiling. After the
electrophoresis, the proteins were electrophoretically transferred to
polyvinylidene difluoride membranes, which were then washed twice in
Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl) and blocked for 2 h at room temperature in blocking solution
(10% glycerol, 5% instant nonfat dry milk, 0.2% Tween 20 in
phosphate-buffered saline). The filters were incubated overnight at
room temperature with the same blocking solution containing 3 µg/ml
rabbit anti-LH/CG-R IgGs, obtained by protein A-Sepharose purification
of anti-LH/CG-R antiserum(23) . The membranes were washed five
times for 5 min each with blocking solution and then incubated for 1 h
at room temperature with a 1:5000 dilution of a horseradish
peroxidase-labeled donkey anti-rabbit IgG whole antibody in blocking
solution. Next, the membranes were washed as follows: twice with TBS,
twice with 1% Nonidet P-40 in TBS, then once each with TBS, 1% Nonidet
P-40 in TBS, and TBS alone. The membranes were exposed to an Amersham
Enhanced Chemiluminescence developer solution for 1 min, wrapped in
Saran Wrap, and exposed to Kodak XAR-5 film for 1 min. The films were
densitometrically scanned (PDI System, Huntington, NY).
from multiple independent
transfections (n = 9) for hCG binding to wild-type
LH/CG-R on transfected intact COS-7 cells was 0.14 nM, with a
range of 0.07-0.25 nM (Table 1), there being no
difference between transfections with DEAE-dextran and Lipofectamine.
On the other hand, receptor numbers/cell (uncorrected for transfection
efficiencies) were greater with Lipofectamine transfection (mean of 2
10
) than with DEAE-dextran transfection (mean of
0.5 10). The difference in receptor number/cell,
however, had no significant impact on the maximal cAMP production
elicited by hCG at 100 ng/ml in COS-7 cells transfected with the cDNA
to wild-type LH/CG-R; the mean was 20.6 (range =
12.3-31.7) pmol of cAMP/10 cells under the conditions
used and was independent of receptor density over the range
investigated (Table 1). Likewise, the effective mean
concentration of hCG necessary to increase the intracellular cAMP
concentration from basal values to 50% of the maximal value in COS-7
cells expressing wild-type LH/CG-R was 0.19 nM (range =
0.05-0.4 nM) (Table 1); this too was independent
of receptor density. For ease in comparing the different experiments,
the maximal hCG-mediated cAMP production over basal is normalized to
100% for each wild-type LH/CG-R control, and the value obtained with
the various receptor mutants, also corrected for basal, is expressed as
a percentage of that of wild-type. There was no evidence for a
functional endogenous LH/CG-R in nontransfected COS-7 cells: the mean
(range) of the basal cAMP production was 3.9 (1.6-6.7) pmol
cAMP/10 cells and that of control cells in the presence of
100 ng/ml hCG was 4.6(3.8-5.2) pmol cAMP/10 cells.
Glu mutation resulted in a LH/CG-R that
specifically bound
I-hCG, and this binding was inhibited
by unlabeled hCG in a concentration-dependent manner (Fig. 2A). Although the estimated number of receptors
varied in the transfected cells, e.g. 2
10 for wild-type and 1 10 for mutant LH/CG-R
(uncorrected for transfection efficiency), the mutant LH/CG-R yielded a
binding affinity equivalent to that of wild-type LH/CG-R (Table 1). The Lys
Glu replacement resulted
in markedly decreased production of cAMP in response to added hCG, e.g. 15% that of wild-type LH/CG-R at 100 ng/ml hCG (Fig. 3A, Table 1). Since this result could be
attributed to the decreased number of cell surface receptors, this
mutant LH/CG-R was also transfected using Lipofectamine to obtain
higher efficiency and increased receptor expression. The mutant
receptor bound hCG with an affinity comparable to wild-type LH/CG-R (Table 1). There was an increase in cell surface expression, e.g. 2
10 receptors/cells (uncorrected for
transfection efficiency), for both wild-type and mutant LH/CG-R, but
the mutant receptor again stimulated cAMP production only 15% that of
wild-type LH/CG-R at 100 ng/ml hCG (Table 1).
I-hCG and hCG to
wild-type LH/CG-R and six single mutants of LH/CG-R are given in panels A-D. The binding of
I-hCG with no
added hCG was normalized to 100% in each
case.
, replacements were also
made with Arg, Gln, and Pro. In each case the mutations yielded
LH/CG-Rs that bound
I-hCG with affinities comparable to
that of wild-type LH/CG-R (Fig. 2B, Table 1), but
these mutant receptors also resulted in cAMP accumulation <30% that
of wild-type LH/CG-R upon addition of hCG to transfected cells (Fig. 3B, Table 1).
I-hCG to cells transfected with cDNAs to the Lys
Asp single mutation and the Asp
Lys/Lys
Asp reciprocal mutation (Table 2).
I-hCG binding to detergent-soluble extracts of
transfected cells was found for the reciprocal mutant but not the point
mutant (Table 2). However, Western blot analysis indicated that
the (Lys
Asp) LH/CG-R was expressed (Fig. 4). Wild-type and mutant LH/CG-Rs were about equally
distributed among three bands of apparent molecular mass 101, 93, and
82 kDa; the total protein in the mutant LH/CG-R was much less than that
of wild-type LH/CG-R.
Asp)LH/CG-R (K401D). Equivalent
amounts of cellular protein were analyzed in each case; control cells, i.e. nontransfected, exhibited no immunostaining bands (data
not shown). The apparent molecular mass values of the three
immunoreactive bands are indicated; these were based on comparisons of
the mobilities with those of prestained standards (rabbit muscle
myosin, 205 kDa; Escherichia coli
-galactosidase, 116
kDa; BSA, 66 kDa; chicken egg ovalbumin, 45 kDa; bovine erythrocyte
carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 20 kDa; bovine
milk -lactalbumin, 14.2 kDa; bovine milk aprotinin, 6.5
kDa).
and Lys
were
each replaced with Glu; both mutations yielded LH/CG-Rs that bound
I-hCG with affinities comparable to that of wild-type
LH/CG-R (Fig. 2, C and D, Table 1).
These mutant LH/CG-Rs were capable of stimulating cAMP production in a
dose-dependent manner, quite similar to wild-type LH/CG-R (Fig. 3, C and D, Table 1).
of the
rat LH/CG-R with 4 amino acid residues, positively charged Arg,
negatively charged Glu, polar but nonionizable Gln, and nonpolar Pro
(an imino acid), show that the mutant LH/CG-Rs bind hCG as well as
wild-type receptor, but coupling to adenylate cyclase is greatly
diminished. These findings are particularly intriguing since
Lys
is located extracellularly, at the boundary of ECL
III and TMH VII, and is unable to interact directly with G
.
Thus, one can conclude that ligand-mediated transmembrane signaling
involves a conformational change of the LH/CG-R in which
Lys participates in some manner. Furthermore, this
observation provides additional evidence that receptor
activation/signaling can be dissociated from high affinity hormone
binding in LH/CG-R. A similar conclusion was recently reached in
reference to another portion of the rat LH/CG-R, namely the
extracellular region just prior to TMH I(25) . In that study we
showed that individual replacements of Glu
and
Asp
with Lys yielded mutant receptors that also bound hCG
like wild-type LH/CG-R but exhibited greatly diminished signaling.
Interestingly, recent results with FSH indicate that different sites
are involved in receptor binding and signal transduction(26) .
]LH/CG-R mutant, in
which one positively charged side chain is replaced with another, does
not signal effectively indicates a stringent specificity for the lysine
side chain. Interestingly, a charged residue at the interface of ECL
III and TMH VII is not required for proper membrane localization since
the replacements of Lys
with Gln and Pro yielded mutants
that gave levels of cell surface expression comparable to that of
wild-type receptor.
of LH/CG-R and either the ligand
or another portion of the receptor. Since replacements of Lys
have no appreciable effect on hCG binding, one would conclude
that any such interaction between the hormone and Lys
makes a negligible contribution to the overall free energy of
binding. However, high affinity hormone binding may occur to the ECD,
followed by a conformational change that ``presents'' the
hormone to Lys
in ECL III where low affinity binding may
occur. Using overlapping synthetic peptides, Roche et al.(15) found that a peptide based on the sequence of rat ECL
III inhibited the binding of
I-hCG to rat luteal
membranes with an IC
of 0.2 mM. The most obvious
interaction of Lys
would be electrostatic, e.g. an ion-dipole or an ion-ion bond between the positively charged
amino group and a negatively charged carboxyl group on hCG or on the
receptor ECD or ECLs. Alternatively, a more hydrophobic environment
could result in an abnormal pK of Lys
, which
could lead to the formation of a strong hydrogen bond. Since our
results suggest some form of ligand-mediated conformational change of
the receptor, it is reasonable to expect that Lys
of the
LH/CG-R may flip from one type of interaction to another concomitant
with hormone binding and that this change accompanies, or perhaps even
triggers, a change in the conformation or relative interhelical
position of TMH VII.
of the LH/CG-R, located at the boundary of TMH II and
ECL I, formed an ion pair with Lys
of the
-subunit of
hCG. They postulated that ligand binding may alter the conformation of
the unoccupied receptor by reorienting TMH II (28) and
concluded that this residue was important in signaling but was not
essential for hormone binding(29) , i.e. as we found
for Lys.
in LH/CG-R function, we asked whether it and Lys
could participate in an ion-ion interaction. This was based in
part on the close proximity of TMHs II and VII suggested by projection
maps of rhodopsin (13) and by reciprocal ion-pair mutations in
the gonadotropin-releasing hormone receptor(30) . Moreover,
interhelical interactions of TMH I, in proximity to II and VII, have
been shown for the adrenergic receptors(31) . We found that the
reciprocal mutation failed to express in a functional form at the cell
surface, although hormone binding could be detected in
detergent-solubilized cells. Thus, Asp
and Lys
can be individually replaced with oppositely charged amino acid
residues and cell surface expression is obtained, but replacing both
residues results in intracellular trapping.
is
invariant in the gonadotropin receptors, i.e. LH/CG-R and
FSH-R, but in TSH-R the equivalent position is occupied by
Gly(2) . In a revealing study, ECL III of the TSH-R was
replaced with that from the
-adrenergic receptor; it
was found that the mutant TSH-R bound TSH with high affinity, but the
sensitivity of TSH-stimulated cAMP production and the maximal level of
TSH-mediated cAMP production were significantly diminished in the
mutant TSH-R(32) . Coupled with our studies on Lys in ECL III of the LH/CG-R, these results indicate an important
role of ECL III in glycoprotein hormone signaling and a possible region
of specificity delineating gonadotropin receptors from the TSH-R.
(located in the ECD at the interface with TMH I) and Lys
(ECL II) were each replaced with Glu with no observable effect on
hCG binding or receptor activation. Lys
(ECL I) was
replaced with Asp, and the mutant receptor failed to exhibit
significant hCG binding to intact or detergent-solubilized cells,
suggesting that this amino acid residue may be involved in hormone
binding. However, binding may occur but is difficult to detect if there
is a low level of mutant receptor expression, if the receptor is
rapidly degraded or if the K
is significantly
increased. Lys is present at this position in LH/CG-R and FSH-R, while
in TSH-R it is occupied by His; depending upon the pK of the His, the
potential exists for retention of a positive charge at this location in
ECL I of all glycoprotein hormone receptors.
Asp)
LH/CG-R revealed the presence of three bands of apparent molecular mass
101, 93, and 82 kDa. The original Western blot experiments on expressed
LH/CG-R were performed using human 293 cells stably transfected with
the wild-type LH/CG-R cDNA(24) . A major 85-kDa form,
considered the mature receptor, and a minor 68-kDa form, which appeared
to be the incompletely glycosylated form, were found. The apparent
93-kDa band observed in transiently transfected COS-7 cells corresponds
to the M
of purified rat ovarian
LH/CG-R(2) , although wide variations have been reported in the M of LH/CG-R from various sources. In another
study from our laboratory using a slightly different set of protein
standards, we found major and minor bands of apparent molecular mass 93
kDa (80%) and 78 kDa for expressed wild-type LH/CG-R(25) .
The greater amount of protein loaded in that study could easily prevent
resolution of the bands of 93 and 101 kDa reported herein.
Interestingly, the Lys
Asp LH/CG-R mutant also
gave the same apparent M
forms as wild-type
LH/CG-R, but at much lower levels. of the rat LH/CG-R is critical in
receptor activation following hormone binding. Since Lys
(ECL III) is located on the opposite side of the membrane from G
proteins and does not appear to be involved in hormone binding, these
findings offer considerable weight to the concept that receptor binding
and activation are dissociable phenomena. Of interest are other
observations we recently made that certain amino acid residues in TMH
VII are also critical for signal transduction (33) . Thus, this
region of the receptor appears to be important in transmembrane
signaling subsequent to hormone binding.
)
We thank Drs. Jianing Huang and Prema Narayan for
their interest in this work and for helpful discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 K. D. G. Pfleger, A. J. Pawson, and R. P. Millar Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations Endocrinology, June 1, 2008; 149(6): 3118 - 3129. [Abstract] [Full Text] [PDF]
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S. M. L. Smith, Y. Lei, J. Liu, M. E. Cahill, G. M. Hagen, B. G. Barisas, and D. A. Roess Luteinizing Hormone Receptors Translocate to Plasma Membrane Microdomains after Binding of Human Chorionic Gonadotropin Endocrinology, April 1, 2006; 147(4): 1789 - 1795. [Abstract] [Full Text] [PDF]
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H. Jaeschke, S. Neumann, G. Kleinau, S. Mueller, M. Claus, G. Krause, and R. Paschke An Aromatic Environment in the Vicinity of Serine 281 Is a Structural Requirement for Thyrotropin Receptor Function Endocrinology, April 1, 2006; 147(4): 1753 - 1760. [Abstract] [Full Text] [PDF]
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M. Claus, H. Jaeschke, G. Kleinau, S. Neumann, G. Krause, and R. Paschke A Hydrophobic Cluster in the Center of the Third Extracellular Loop Is Important for Thyrotropin Receptor Signaling Endocrinology, December 1, 2005; 146(12): 5197 - 5203. [Abstract] [Full Text] [PDF]
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 B. Karges, S. Gidenne, C. Aumas, F. Haddad, P. A. Kelly, E. Milgrom, and N. de Roux Zero-Length Cross-Linking Reveals that Tight Interactions between the Extracellular and Transmembrane Domains of the Luteinizing Hormone Receptor Persist during Receptor Activation Mol. Endocrinol., August 1, 2005; 19(8): 2086 - 2098. [Abstract] [Full Text] [PDF]
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 D. A. Roess and S. M.L. Smith Self-Association and Raft Localization of Functional Luteinizing Hormone Receptors Biol Reprod, December 1, 2003; 69(6): 1765 - 1770. [Abstract] [Full Text] [PDF]
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M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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H. Shinozaki, F. Fanelli, X. Liu, J. Jaquette, K. Nakamura, and D. L. Segaloff Pleiotropic Effects of Substitutions of a Highly Conserved Leucine in Transmembrane Helix III of the Human Lutropin/Choriogonadotropin Receptor with Respect to Constitutive Activation and Hormone Responsiveness Mol. Endocrinol., June 1, 2001; 15(6): 972 - 984. [Abstract] [Full Text] [PDF]
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D. A. Roess, R. D. Horvat, H. Munnelly, and B. G. Barisas Luteinizing Hormone Receptors Are Self-Associated in the Plasma Membrane Endocrinology, December 1, 2000; 141(12): 4518 - 4523. [Abstract] [Full Text] [PDF]
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K. Angelova, P. Narayan, J. P. Simon, and D. Puett Functional Role of Transmembrane Helix 7 in the Activation of the Heptahelical Lutropin Receptor Mol. Endocrinol., April 1, 2000; 14(4): 459 - 471. [Abstract] [Full Text]
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 H. Biebermann, T. Schöneberg, A. Schulz, G. Krause, A. Grüters, G. Schultz, and T. Gudermann A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling FASEB J, November 1, 1998; 12(14): 1461 - 1471. [Abstract] [Full Text]
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N. Bhowmick, P. Narayan, and D. Puett The Endothelin Subtype A Receptor Undergoes Agonist- and Antagonist-Mediated Internalization in the Absence of Signaling Endocrinology, July 1, 1998; 139(7): 3185 - 3192. [Abstract] [Full Text] [PDF]
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 T. Okamoto, N. Sekiyama, M. Otsu, Y. Shimada, A. Sato, S. Nakanishi, and H. Jingami Expression and Purification of the Extracellular Ligand Binding Region of Metabotropic Glutamate Receptor Subtype 1 J. Biol. Chem., May 22, 1998; 273(21): 13089 - 13096. [Abstract] [Full Text] [PDF]
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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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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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S. Nishi, K. Nakabayashi, B. Kobilka, and A. J. W. Hsueh The Ectodomain of the Luteinizing Hormone Receptor Interacts with Exoloop 2 to Constrain the Transmembrane Region. STUDIES USING CHIMERIC HUMAN AND FLY RECEPTORS J. Biol. Chem., February 1, 2002; 277(6): 3958 - 3964. [Abstract] [Full Text] [PDF]
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