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J Biol Chem, Vol. 273, Issue 22, 13835-13840, May 29, 1998
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.
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 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 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.
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.
The Amino-terminal Region of the Luteinizing
Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human
Choriogonadotropin
I. MUTATIONAL ANALYSIS*
ABSTRACT
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Introduction
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Discussion
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INTRODUCTION
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Abstract
Introduction
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- and 
-subunits of hCG. In addition,
another three sequences near the junctions of exons 4-5, 6-7, and
9-10 influence hormone binding.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-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.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (73K):
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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.
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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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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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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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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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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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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.
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.
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FOOTNOTES |
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* This work was supported by Grants HD-18702 and DK-51469 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 307-766-6272;
Fax: 307-766-5098; E-mail: ji{at}uwyo.edu.
1 The abbreviations used are: LH, luteinizing hormone; CG, choriogonadotropin; hCG, human CG; LH/CG-R, LH/CG receptor; PBS, phosphate-buffered saline.
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REFERENCES |
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S. Hong, I. Ji, and T. H. Ji The {alpha}-Subunit of Human Choriogonadotropin Interacts with the Exodomain of the Luteinizing Hormone/Choriogonadotropin Receptor Endocrinology, June 1, 1999; 140(6): 2486 - 2493. [Abstract] [Full Text]
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T. Phang, G. Kundu, S. Hong, I. Ji, and T. H. Ji The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin. II. PHOTOAFFINITY LABELING J. Biol. Chem., May 29, 1998; 273(22): 13841 - 13847. [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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Y. S. Song, I. Ji, J. Beauchamp, N. W. Isaacs, and T. H. Ji Hormone Interactions to Leu-rich Repeats in the Gonadotropin Receptors. II. ANALYSIS OF LEU-RICH REPEAT 4 OF HUMAN LUTEINIZING HORMONE/CHORIONIC GONADOTROPIN RECEPTOR J. Biol. Chem., January 26, 2001; 276(5): 3436 - 3442. [Abstract] [Full Text] [PDF]
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H. Zeng, T. Phang, |
