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Molecular Endocrinology 20 (10): 2493-2503
Copyright © 2006 by The Endocrine Society

Biological Effect of a Novel Mutation in the Third Leucine-Rich Repeat of Human Luteinizing Hormone Receptor

Michael Yiu-Kwong Leung, Peter J. Steinbach, Deborah Bear, Vanessa Baxendale, Patricia Y. Fechner, Owen M. Rennert and Wai-Yee Chan

Laboratory of Clinical Genomics (M.Y.-K.L., D.B., V.B., O.M.R., W.-Y.C.), National Institute of Child Health and Human Development, and Center for Molecular Modeling (P.J.S.), Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892; Department of Pediatrics (P.Y.F.), Stanford University, Stanford, California 94305; and Department of Pediatrics (W.-Y.C.), Georgetown University, Washington, D.C. 20007

Address all correspondence and requests for reprints to: Dr. Wai-Yee Chan, Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 2A08, 49 Convent Drive, MSC 4429, Bethesda, Maryland 20892-4429. E-mail: chanwy{at}mail.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A novel heterozygous mutation A340T leading to the substitution of Phe for the conserved amino acid Ile114 was identified by nucleotide sequencing of the human LH/chorionic gonadotropin receptor (hLHR) of a patient with Leydig cell hypoplasia. This mutation is located in the third leucine-rich repeat in the ectodomain of the hLHR. In vitro expression studies demonstrated that this mutation results in reduced ligand binding and signal transduction of the receptor. Studies of hLHR constructs in which various amino acids were substituted for the conserved Ile114 showed that receptor activity is sensitive to changes in size, shape, and charge of the side chain. A homology model of the wild-type hLHR ectodomain was made, illustrating the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile114 by Phe might disrupt intermolecular contacts between hormone and receptor. This mutation might also affect an LHR-dimer interaction. Thus, the I114F mutation reduces ligand binding and signal transduction by the hLHR, and it is partially responsible for Leydig cell hypoplasia in the patient.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE LH/CHORIONIC GONADOTROPIN receptor (LHR) is a G protein-coupled receptor with a large ectodomain containing nine leucine-rich repeat (LRR) motifs and an endodomain containing seven transmembrane helices. LRR proteins possess a characteristic ß-region (1), 11 residues that form a ß-strand and the adjacent loops in known structures. Hydrophobic residues that pack in the protein core are well conserved among human LHR (hLHR), human FSH receptor (hFSHR), and human TSH receptor. The recently determined crystal structure of the hFSHR complexed with hormone (2) permits modeling of the hLHR ectodomain. Nonconserved amino acids of the ß-strands serve as determinants for hormone binding and hormone specificity (3, 4, 5). Hormone binding is mediated by hydrophobic interactions, ionic interactions, hydrogen bonding, and water-mediated hydrogen bonding (2, 6). Upon binding of hLH/human chorionic gonadotropin (hCG) to the LRRs of hLHR, the seven-transmembrane helices of the receptor reposition to generate the binding crevice on the extracellular side for reception of the ectodomain-bound hLH/hCG molecule (7), and a pocket on the cytoplasmic side for the Gs protein (8). The interaction between the ligand and the transmembrane helices elicits the guanine nucleotide exchange by the Gs protein and leads to cellular cAMP production and subsequent cellular events such as gene transcription. The activated cellular events are important for proper reproductive function and development, such as testosterone production and masculinization in males.

Inactivation of LH signaling due to mutation of the hLHR causes underdevelopment of Leydig cells and decreased production of testosterone, giving rise to male hypogonadism or pseudohermaphroditism called Leydig cell hypoplasia (LCH) or Leydig cell agenesis (9). Among the 17 inactivating mutations of the hLHR identified in LCH patients (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), only two affect the conserved hydrophobic amino acids (F194V and V144F) of the LRRs (10, 21). The structural and functional effects of mutations of conserved hydrophobic amino acids in the LRRs are not well characterized. The study of naturally occurring mutations in the LRRs of hLHR provides information about this receptor and, presumably, related LRR proteins.

In this report, we identify a novel heterozygous loss-of-function mutation (I114F) that affects a conserved hydrophobic amino acid of the third LRR of hLHR in a patient with LCH. Results obtained in vitro suggest that this mutation reduces binding of the hormone, thereby reducing receptor activity. The findings are interpreted with the aid of a homology model of the wild-type (WT) hLHR ectodomain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of I114F Mutation
Analysis of the genomic DNA from a patient with LCH identified a heterozygous A-to-T transition mutation at nucleotide 340 of the hLHR gene (Fig. 1Go). The mutation results in the substitution of Phe for the conserved Ile114 of the LRR3 in the ectodomain of the hLHR. The mutation was only detected in the patient. No corresponding mutation could be identified in the hLHR of the parents, suggesting that it is a new mutation in the family. The disease-causing mutation of the other allele of the hLHR had not been identified after scanning the entire coding sequence of the hLHR gene of the patient.


Figure 1
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  		Fig. 1. Heterozygous A340T Mutation of the hLHR Identified in the Patient with LCH

A, Father. B, Mother. C, Patient. This mutation was only found in the patient and not in her parents. It is apparently a new mutation. The position of the mutated base (340) is indicated by the arrow. The mutant nucleotide base (T) and the mutant amino acid (Phe) are shown in green. The amino acids encoded by the nucleotide sequence are shown with their position indicated by the numbers at the ends of the sequence.

 
Functional Study of I114F Mutant
The functional defect of the mutant receptor was confirmed by in vitro expression and cAMP assay. hLHR-WT and hLHR-I114F cDNAs were transiently expressed in human embryonic kidney (HEK) 293 cells. The transfection efficiency of the expression cassettes was normalized by the measurement of cotransfected ß-galactosidase activity. Both hLHR-WT- and hLHR-I114F-expressing cells responded to hCG in a dose-dependent manner. However, the maximum cAMP level of the hLHR-I114F transfectants was significantly lower than that of hLHR-WT transfectants (Fig. 2AGo).


Figure 2
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  		Fig. 2. Receptor Function and Ligand Binding

A, hCG-stimulated cAMP accumulation in transfected HEK293 cells. HEK293 cells transfected with either pcDNA3-hLHR-WT or pcDNA3-hLHR-I114F responded to hCG and produced cAMP in a dose-dependent manner. However, the maximum response of hLHR-I114F transfectants was significantly lower than that of hLHR-WT. Maximum cAMP response: hLHR-WT (15,995 ± 191 fmol/ß-gal unit) and hLHR-I114F (3,326 ± 481 fmol/ß-gal unit). EC50: hLHR-WT (4.6 ± 0.3 ng/ml) and hLHR-I114F (7.11 ± 0.2 ng/ml). B, Displacement of cell cytoplasmic surface membrane receptor bound 125I-labeled hCG with various amounts of unlabeled hCG. IC50: hLHR-WT (105 ± 9 ng/ml) and hLHR-I114F (22 ± 2 ng/ml). C, Displacement of glycerol/detergent-solubilized membrane receptor-bound 125I-labeled hCG with various amounts of unlabeled hCG. All determinations were done in triplicate. The saturated bound/free 125I-labeled hCG of I114F mutant receptor was less than 5% that of WT receptor in both cell surface binding and total membrane binding. IC50: hLHR-WT (116 ± 22 ng/ml) and hLHR-I114F (12 ± 4 ng/ml).

 
Cell surface receptor binding was examined by incubating transfected HEK293 cells with a fixed amount of 125I-labeled hCG, which was displaced by various amounts of unlabeled hCG. The saturated cell surface-bound/free 125I-labeled hCG of pcDNA3-hLHR-I114F transfectants was about 5% that of cells expressing the WT receptor (Fig. 2BGo). Glycerol/detergent solubilized whole-cell binding (total binding) was similar to that of cell surface binding. The total membrane-bound/free 125I-labeled hCG of pcDNA3-hLHR-I114F transfectants was about 4% that of cells expressing WT receptor (Fig. 2CGo). The binding of the mutant was slightly higher than that of the vector and was too low for accurate estimation of the Kd. The comparable bound/free 125I-labeled hCG ratios for cell surface (5%) and total-membrane (4%) hormone binding indicate that the impairment of ligand binding was the cause of the reduced hormone response of the mutated receptor.

Flow cytometry was used to further compare cell surface expression of WT and I114F hLHR. Enhanced green fluorescent protein (EGFP) fused to the N termini of hLHR-WT and -I114F was used as an epitope tag for expression analysis by flow cytometry. Dot plots of antibody-stained cells are shown in Fig. 3Go. pCDNA3-, pcDNA3-WT-, and pcDNA3-I114F-transfected cells were negative control and displayed less than 1% positively stained cells. Similar to negative control, isotype-matched control also displayed less than 1% positively stained cells. pSEGFP-WT- or pSEGFP-I114F-transfected cells when stained by both first and second antibodies displayed 6.0 ± 0.4 and 5.8 ± 0.5% of positively stained cells, respectively. The cell shifting patterns of pSEGFP-WT- and pSEGFP-I114F-transfected cells were similar. Although pSEGFP-WT-transfected cells displayed a slightly higher expression than that of pSEGFP-I114F-transfected cells, the difference was unable to account for the huge discrepancies observed in the binding and cAMP assays.


Figure 3
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  		Fig. 3. Dot Plots of Anti-GFP Antibody-Stained Cells

PM2 represents red fluorescence intensity, which is proportional to the level of receptors expressed on the cell surface, and FCS represents forward scattering. A, pCDNA3-, pcDNA3-WT-, and pcDNA3-I114F-transfected cells, equilibrated first with anti-GFP serum and followed by rhodamine-conjugated goat anti-rabbit IgG, represent negative control and displayed less than 1% positively stained cells. Cells appearing in the upper right quadrant are positive. pCDNA3-, pcDNA3-WT-, and pcDNA3-I114F-transfected cells have 0.6, 0.3, and 0.5% positive cells, respectively. B, Isotype-matched control, which provides a measurement of nonspecific binding of second antibody, displayed less than 1% positively stained cells. Both pSEGFP-WT and pSEGFP-I114F have 0.7% positive cells. C, pSEGFP-WT- and pSEGFP-I114F-transfected cells, when stained with both first and second antibodies, displayed 6.0 and 5.8% of positive cells. The shifting patterns of pSEGFP-WT- and pSEGFP-I114F-transfected cells are similar.

 
Receptor Stability and Membrane Insertion
To compare the stability of the mutant and the WT, hLHR cDNA was fused in-frame to the C terminus of EGFP (Fig. 4AGo). The chimeric DNA constructs (pSEGFP-hLHR-WT and pSEGFP-hLHR-I114F) were transiently expressed in HEK293 cells and were examined by fluorescence microscopy. Cells expressing SEGFP-hLHR-WT and SEGFP-hLHR-I114F exhibited comparable fluorescence intensity or stability when treated with or without proteasome inhibitor [N-acetyl-Leu-Leu-Nle-CHO (ALLN)] (Fig. 4Go, Ba–Bf). ALLN is a cell-permeable peptide aldehyde that inhibits the activity of proteasome. If the loss of ligand binding of I114F was a result of rapid clearance of the mutant receptor by proteasome degradation, the difference in fluorescence intensity between WT and I114F, both before and after ALLN treatment, should be more apparent. The results indicate the mutant and the WT receptor have similar susceptibility to proteasome degradation.


Figure 4
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  		Fig. 4. Receptor Stability

A, Schematic representation of pSEGFP-hLHR-WT/I114F expression construct. CMV is the cytomegalovirus early promoter. The first 42 codons of hLHR were used as the signal sequence to direct the fusion protein to ER. The entire amino acid encoding sequence of EGFP was used. {Delta}hLHR was the mature hLHR cDNA sequence from codon 26 to termination codon of WT/I114F. SV40 polyA was the simian virus early mRNA polyadenylation signal. B, Receptor stability studied by expressing the EGFP fusion constructs in HEK293 cells. Transfected cells were treated with 10 µM ALLN for 18 h at 24 h after transfection (right panels; b, d, and f). Proteasome-sensitive vector, which encoded a proteasome sensitive fluorescence protein, was used as a control (left panels; a, c, and e). The fluorescence of proteasome-sensitive vector-transfected cells increased after ALLN treatment (a and b). The fluorescence of WT (c and d) and mutant (e and f) was similar in both ALLN-untreated and -treated conditions. Thus, mutant and WT had similar susceptibility to proteasome degradation. C, Membrane insertion of receptor. a, pEGFP-N2 transfectant with EGFP expressed in cell cytoplasm had very intense fluorescence that filled up the entire cell. b, pSEGFP transfectant with SEGFP expressed in cisternae of ER had intensely fluorescing ER as SEGFP was trapped in the cisternae of ER. EGFP fused WT (c) and mutant (d) hLHR, which were inserted into the membrane of ER after translation, had similar fluorescence patterns and intensities, and the fluorescing intensity of the ER was much lower than that of SEGFP transfectant. The fluorescence of transfectants was observed 72 h after transfection with a fluorescence inverted microscope equipped with a x100 oil immersion lens. Scale bar, 20 µm.

 
The proper membrane insertion of the mutant receptors after protein translation was demonstrated by transfecting HEK293 cells with pEGFP-N2, pSEGFP, pSEGFP-hLHR-WT, and pSEGFP-I114F. Cells transfected with pEGFP-N2 had the expressed EGFP localized in the cell cytoplasm (Fig. 4CaGo), and the fluorescence of the cell cytoplasm was so strong that no cellular structures could be discerned. Cells transfected with pSEGFP had fluorescence localized in the lumen of the endoplasmic reticulum (ER) (Fig. 4CbGo). In cells expressing pSEGFP-hLHR-WT or pSEGFP-hLHR-I114F, fluorescence localized on the ER and cell surface membranes (Fig. 4Go, Cc and Cd). The fluorescence of pSEGFP-transfected cells was much stronger than that of pSEGFP-hLHR-WT or pSEGFP-hLHR-I114F transfectants, and the fluorescence pattern of pSEGFP-hLHR-WT and pSEGFP-hLHR-I114F transfectants were comparable.

Effect of the Amino Acid Side Chain at Position 114
To assess the effect of residue 114 on hormone binding, the Ile at this position was replaced by Gly, Val, Leu, Trp, and Asp. In vitro expression and cAMP assays of the mutants indicated that hLHR-I114G and hLHR-I114D had no activity. The Trp substitution retained receptor activity equivalent to 3.1% that of WT. Leucine and Val substitutions had activity between that of WT (Ile) and I114F (Fig. 5AGo).


Figure 5
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  		Fig. 5. Activity and Ligand Binding of Mutant Receptors

A, Effect of substituting Ile114 with different amino acids. HEK293 cells transfected with hLHR-WT, hLHR-I114F, hLHR-I114G, hLHR-I114V, hLHR-I114L, hLHR-I114W, hLHR-I114D, and empty vector. The substitution mutants hLHR-I114G, hLHR-I114W, and hLHR-I114D did not respond to hCG stimulation. The response curves of these mutants overlapped with that of the vector control and were not discernable in the figure. hLHR-I114V and hLHR-I114L had activities between WT and I114F. Maximum cAMP response: hLHR-WT (11,050 ± 778 fmol/ß-gal unit), hLHR-I114F (2,490 ± 279 fmol/ß-gal unit), hLHR-I114V (5,086 ± 531 fmol/ß-gal unit), hLHR-I114L (4,840 ± 255 fmol/ß-gal unit), hLHR-I114W (416 ± 19 fmol/ß-gal unit), hLHR-I114G [not determined (N/A)], and hLHR-I114D (N/A). EC50: hLHR-WT (5.9 ± 0.5 ng/ml), hLHR-I114F (8.2 ± 0.2 ng/ml), hLHR-I114V (1.7 ± 0.2 ng/ml), hLHR-I114L (1.7 ± 0.3 ng/ml), hLHR-I114W (9.7 ± 0.8 ng/ml), hLHR-I114G (N/A), and hLHR-I114D (N/A). B, Displacement curves of receptor-ligand binding of WT, I114F, I114V, I114L, I114W, I114G, and I114D mutants. Each determination was done in triplicate. The mean and SD values are shown. IC50: hLHR-WT (61 ± 4 ng/ml), hLHR-I114F (7 ± 1 ng/ml), hLHR-I114V (34 ± 4 ng/ml), hLHR-I114L (36 ± 3 ng/ml), hLHR-I114W (N/A), I114 G (N/A), and hLHR-I114D (N/A).

 
Receptor binding assays of glycerol/detergent-solubilized cell lysates from transfected cell preparations were used to investigate whether or not the change in the signaling activity of the mutants was a consequence of altered ligand-receptor binding. The binding results of the mutants were consistent with the results of cAMP assays (Fig. 5BGo). Results of EGFP fusion protein experiments indicated that the stability and membrane insertion properties of all the artificial mutants were similar to that of WT hLHR (Leung, M., and W. Chan, unpublished data). These results strongly suggest that the reduced activities of the mutants are not due to low receptor stability or poor membrane insertion of the receptor.

Molecular Modeling of hLHR LRRs
Figure 6AGo shows a stereo view of a homology model of LHR, residues 50–263. The conserved columns of Leu, Ile, and Phe residues point inward, characteristic of LRRs. Note that no charged side chains are in the model interior. Figure 6BGo shows residues 50–159 of the LHR model, viewed from the hormone-binding surface (essentially a 180° rotation relative to Fig. 6AGo). Conserved surface residues for which the corresponding residues in FSHR make direct contacts with the hormone {alpha}-chain in the recently solved crystal structure (2, 6) are shown as space filling, along with I114, which extends into the LHR core from the opposite side. The bulkier Phe side chain in the I114F mutant might decrease LH binding to LHR by disrupting the side-chain packing in the protein core so as to weaken one or more of these interactions with the LH {alpha}-chain.


Figure 6
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  		Fig. 6. Molecular Modeling of hLHR LRRs

A, Stereo view of homology model of LHR, residues 50–263. The main-chain ribbon is colored red for residues identical to those of the template (hFSHR receptor) and blue for all others. Phe (orange), Leu (green), and Ile (gray) side chains in the modeled interior are shown as ball-and-stick, as are all Asp/Glu (red), Lys/Arg (blue), and His (pink) side chains. Note the conserved column (spine) of Phe residues as well as the columns of Ile and Leu residues pointing inward from the ß-sheet, characteristic of LRRs. No charged side chains were modeled inside the protein. Three conserved Asn residues are marked with yellow spheres. Ile114 is shown in dark gray. B, Stereo view of LHR model, residues 50–159, viewed from the hormone-binding surface (rotated ~180° about the vertical axis relative to A). Again, the main-chain ribbon is colored red where identical in sequence to FSHR. Conserved surface residues for which the corresponding residues in FSHR make direct contacts with the hormone {alpha}-chain (2 6 ) are shown as space filling, along with I114, which extended into the LHR core from the opposite side. The bulkier Phe side chain in the I114F mutant might decrease LH binding to LHR by disrupting the side-chain packing in the protein core so as to weaken one or more of these interactions with the LH {alpha}-chain.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Mutation of the conserved amino acid Ile114 in the ectodomain of the hLHR caused a subnormal response of the receptor to hormone stimulation. Because LCH is an autosomal recessive disorder, this heterozygous mutation is only partly responsible for the patient’s LCH. Even though the entire coding sequence of the hLHR gene of the patient had been sequenced, the mutation of the other hLHR allele still eludes detection. The inactivating mutation of the other hLHR allele can occur at any of the noncoding regions of the hLHR gene.

Reduced hormone response of the mutated receptor is a consequence of reduction in the binding of the hormone, as shown by the studies with intact cells and detergent-solubilized cell lysates. Reduced hormone binding could be a consequence of rapid clearance of the misfolded mutant receptor or failure of the translated receptor to insert into the membrane of the ER. Cell surface expression studies demonstrated similar expression levels of the WT and mutant receptors. Results of the receptor stability studies indicated the mutant and the WT receptor have similar susceptibility to proteasome degradation. Membrane insertion studies showed that the trapping of SEGFP in the cisternae of the ER is consistent with the report of a previous study with rLHR-t338 (26). Both rLHR-t338 and SEGFP had no transmembrane domain but the signal peptide sequence. Without a transmembrane domain, rLHR-t338 and SEGFP did not insert into the membrane of the ER after translation and the signal peptide of LHR alone was not sufficient to direct the molecules to the extracellular environment. The higher fluorescence intensity of the ER of SEGFP-expressing cells could be explained by the fact that the capacity of cisternae of the ER for SEGFP is much higher than the capacity of the membrane of the ER for SEGFP-hLHR-WT and SEGFP-hLHR-I114F. This observation demonstrated the mutant receptor was inserted into the ER membrane after protein translation, which is not unexpected because, compared with mutation in the transmembrane domain, mutation in the LRR is less likely to affect proper membrane insertion of the receptor. Thus, the reduced response of the mutant receptor is likely to be a consequence of the inherent reduced ligand binding capacity of the receptor.

The binding data suggest that the mutant (I114F) receptor binds hCG about 5% as effectively as WT receptor, yet the maximal cAMP response with the mutant receptor is about 25% that of WT receptor. There are two potential explanations for this phenomenon. Ligand binding involves a single step, whereas ligand-responsive cAMP production involves multiple steps and multiple molecules. Therefore, it is not surprising to observe nonlinear correlation between ligand binding and cAMP production. It is also possible that hCG stimulation of the WT receptor saturated the signaling capacity of the cAMP pathway in HEK293 cells but not for the mutated receptor.

To the best of our knowledge, mutations similar to I114F have not been reported by others. A number of LRR mutants of hLHR have been generated (3, 5, 29). However, these studies focused on how the surface amino acids interact with hormone ligands rather than amino acids in the hydrophobic core. Furthermore, these mutagenesis studies concentrated on the ß-strand regions, whereas I114F is outside the ß-strand region. Song et al. (30, 31) also generated a lot of hLHR mutants, some of which involved conserved hydrophobic amino acid residues, but we do not find in their published work any mutations of Ile at position 114.

Our functional data are insufficient to establish the effect of the mutation on the structure of the receptor. However, the recent determination of a 2.9-Å resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular domain of hFSHR (2) provides a better template for homology modeling of hLHR than had been available previously (32, 33). The current model of hLHR necessarily assumes the topology of the hFSHR structure, with N-terminal LRRs that form an essentially flat surface and C-terminal LRRs that exhibit a horseshoe-like curvature.

The cores of LRR proteins are densely packed with hydrophobic side chains, including those of Leu, Ile, and Phe. Some side chains of polar residues (e.g. Asn) also pack inward so as to stabilize turns by forming buried hydrogen bonds. The strain caused by the disruption of the packing within the hLHR core when Ile114 is replaced by Phe may well be propagated to the protein surface, displacing the side chains involved in intermolecular contacts. Thus, the reduced activity observed in the I114F mutant may reflect distortion of the LH {alpha}-chain binding surface that results when the larger Phe side chain is packed in the pocket normally occupied by Ile114 (Fig. 6BGo).

Of the mutant side chains studied, those of Val and Leu are most similar to that of Ile in size, shape, and chemical composition. By contrast, Gly is too small to fill the pocket occupied by Ile, whereas Phe and Trp are presumably too large to be readily accommodated. Clearly, the charged Asp side chain is not well suited for packing in the hydrophobic pocket. Consequently, it is not surprising that the receptor activities measured for the I114V and I114L mutants are closer to that of WT than are the activities measured for the other mutants.

Interestingly, Ile114 is preceded in the LHR sequence by a conserved Tyr residue that accounts for most of the carbon-carbon contacts seen at the FSHR-FSHR dimer interface in the crystal structure of Fan and Hendrickson (2, 6). Even though there is solid evidence supporting the self-association of the LHR (34, 35, 36), the functional relevance of LHR-LHR dimers is not well established. Nonetheless, if Tyr113 does participate in the weak association of LHR hormone-binding domains, the Phe that replaces Ile114 very likely weakens this association by displacing Tyr113 and other nearby residues at the LHR-dimer interface, thus affecting signal transduction by the mutated receptor.

LRRs are present in a variety of proteins with diverse functions (37, 38, 39, 40, 41, 42, 43, 44), and mutations in LRR proteins can lead to disease. For example, the bleeding disorder Bernard-Soulier syndrome is associated with mutations in the LRR proteins GPIb{alpha} and GPIbß (45). We have identified a heterozygous inactivating mutation of hLHR in a patient with LCH. This mutation changes a conserved hydrophobic amino acid of the LRR. Site-directed mutagenesis showed that mutations that alter the size, shape, and/or charge of the side chain of amino acid 114 will result in reduced or abolished hormone-receptor interaction. Given the overall topology that is shared by LRR proteins, it is likely that other mutations of conserved hydrophobic residues in LRR proteins will be seen to alter function.

Patient
The subject was a previously healthy phenotypic female infant who was noted at 9 months of age to have an enlarged clitoris with a prominent hood, bilateral palpable gonads in the labia majora, separate vaginal and urethral openings, and no posterior fusion. The patient is a 46,XY female with an elevated LH of 16 IU/liter and an inappropriately low testosterone of 72 ng/dl for the LH value. The testosterone, the dihydrotestosterone (20 ng/dl), androstenedione (86 ng/dl), and inhibin B (790 pg/ml) levels were elevated for a male of 9 months. FSH (1.1 IU/liter) and Mullerian inhibiting substance (43 ng/ml) were normal for a 9-month male infant. Ultrasound of gonads demonstrated a left gonad 1.4 x 1.2 x 1.0 cm and a right gonad 1.8 x 1.0 x 0.9 cm in the labia majora. Voiding cystourethrogram revealed the presence of the lower one third of a vagina and an elongated posterior urethra. Results of an ACTH stimulation test using 250 µg Cortrosyn performed at 9 months revealed no defect in cortisol or testosterone biosynthesis. An hCG stimulation test using Ovidrel (recombinant hCG) (40 µg/m2, sc) on d 1 and 3 was done at 23 months and showed a decreased Leydig cell response with a testosterone value of 92 ng/dl. Laboratory tests were performed by Esoterix Endocrinology (Calabasas Hill, CA), and results of the ACTH and the hCG stimulation tests are summarized in Table 1Go. In addition, the androgen receptor was sequenced; no mutations in any of the eight exons were demonstrated (results not shown). Gonadectomy was performed shortly after the hCG stimulation test. This revealed normal testicular histology for a 2-yr-old male. Informed consent for this study was obtained and approved by the Institutional Review Board of Stanford University and the National Institutes of Health.


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  		Table 1. Results of ACTH Stimulation Test and hCG Stimulation Test

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
PCR Amplification and Sequencing of the Exons of the hLHR
Genomic DNA was extracted from peripheral blood of the propositus and her parents. The exons of the hLHR gene were amplified by PCR. A total of 750 ng of genomic DNA were used as template for PCR using 10 U Taq2000 DNA polymerase (Stratagene, La Jolla, CA) for every 50-µl reaction. Two microliters of each 5' and 3' primer at 10 µM were added per PCR. A 30 cycle PCR profile of denaturation at 95 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec were used for the amplification of the exons except exon 11. The extension time for the amplification of exon 11 was 1.5 min. The PCR products were recovered for cyclo-sequencing with Big Dye Terminator, version 3.0 (Applied Biosystems, Hayward, CA).

Generation of hLHR-I114F Construct
The pcDNA3-hLHR-WT cDNA recombinant plasmid was provided by Dr. Aaron J. W. Hsueh (Stanford University, Palo Alto, CA). The pcDNA3-hLHR-I114F expression cassette was generated by PCR-based site-directed mutagenesis. A total of 250 ng of the pcDNA3-hLHR-WT cDNA recombinant plasmid were the template, and primers 5KpnLHR (5'-GGTACCGCCCATGAAGCAGCGGTTCTCGGCG-3') and 3A340TLHR (5'-GATTTATAAATGCTCCGGGCTCAAAGTATCTCAGATTTTTGGTGTTC-3') each of 1 µl were used in the first PCR. Mutated nucleotide is in bold in the primer sequence. Seven units of Taq2000 DNA polymerase and 3.5 U Pfu DNA polymerase (Stratagene) were added to the PCR mixture of 50-µl final volume. The PCR product was recovered and was used as a 5' megaprimer in second PCR. One microliter of primer 3XbaLHR (5'-TGCCATCTTTCTAGAGTGATGAC-3') at 2 µg/µl was used to amplify the hLHR full-length sequence. A 30-cycle PCR profile of denaturation at 95 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec were used for both first PCR and second PCR, except the extension time was 1.5 min for second PCR. The second PCR product of 1410 bp long was double digested with KpnI and XbaI (New England BioLabs, Beverly, MA). The PCR fragment was ligated to the large fragment of KpnI- and XbaI-digested pcDNA3-hLHR-WT. Mutation was confirmed by nucleotide sequencing. A similar approach was used to generate hLHR with Ile at position 114 substituted by Gly, Val, Leu, Trp, and Asp.

hCG-Stimulated cAMP Accumulation Assay
HEK293 cells were plated onto 96-well plate (2 x 104 cells per well), grown overnight, and then transfected with 0.18 µg of expression cassette and 0.02 µg of vector that expresses ß-galactosidase by Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Two days after transfection, cells were stimulated with hCG (Sigma, St. Louis, MO) of various concentrations and 0.25 mM 3-isobutyl-1-methyl-xanthine for 2 h. The cAMP level of stimulated cells was determined by cAMP Direct EIA kit (Amersham Biosciences, Piscataway, NJ). Cell transfection efficiency was normalized by monitoring ß-galactosidase activity of the transfectants as described previously (13). Triplicate experiments were done and the mean and SD values were reported. Significance of the difference in maximum cAMP production was calculated by Student’s t test.

Receptor Binding Assays
Slight modification of the method of Lloyd and Ascoli (46) was adopted for the cell cytoplasmic surface receptor binding assay. HEK293 cells were grown overnight in 24-well plate (2 x 105 cells/well), and then cotransfected with 0.76 µg either pcDNA3-hLHR-WT/I114F or empty vector together with 0.04 µg ß-galactosidase expression plasmid. After 48 h of transfection, transfectants were equilibrated at 4 C for 20 min before 2 ng 125I-labeled-hCG (DuPont NEN, Boston, MA) and unlabeled hCG were added. The cells were equilibrated with the hCG for 24 h at 4 C. A total of 200 µl medium was removed for counting. The cells were then washed with 92.5% DMEM-F12 (Invitrogen) containing 200 mM HEPES, 2.5% BSA, and 7.5% horse serum (Invitrogen) (pH 7.5). Washed cells were lysed with alkaline solution (0.2 N NaOH, 150 mM NaCl) and counted with a Cobra II {gamma}-counter (PerkinElmer Life Sciences, Boston, MA). The method of Ascoli (27) was used for glycerol/detergent-solubilized cell membrane preparation and receptor binding. Triplicate experiments were done and the mean and SD values are reported.

EGFP-hLHR Fusion Gene Construction
The C-terminal end of the first 42 amino acids of hLHR-WT was joined in-frame to the N-terminal of EGFP, and the C terminus of EGFP was joined in-frame to the N terminus of the 26th amino acid of hLHR-WT/I114F. Plasmid pcDNA3-hLHR-WT was first linearized with EagI followed by fill-in reaction (1 µg DNA, 0.5 U Klenow fragment; 20 min on ice), and then digested with HindIII to release a 150-bp fragment, which contained the putative signal sequence of hLHR. Plasmid pEGFP-N2 (BD Clontech, Palo Alto, CA) was linearized with PstI, and then filled in as described above. The blunt-ended pEGFP-N2 was digested with HindIII. The 150-bp DNA fragment was ligated to the HindIII-digested pEGFP-N2 so that the hLHR putative signal sequence was joined in-frame to the 5' end of the EGFP open reading frame to generate the plasmid pSEGFP. The hLHR coding sequence was amplified by using the primer 5BsrGLHR (5'-GAGCTGTACATTATGGGCTATGACTTCCTTAGGGTCCTG-3') and 3NotLHR (5'-TATGCGGCCGCAGTTACTGATGTAACAGTTAACACTC-3'). Twenty nanograms of the pcDNA3-hLHR-WT or pcDNA3-hLHR-I114F plasmids were used as the template for PCR using a mixture of 1 U Pfu DNA polymerase and 5 U Taq2000 DNA polymerase for every 100-µl reaction. A 30-cycle PCR profile of denaturation at 96 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 2 min were used. The PCR product and pSEGFP were double digested with BsrGI and NotI. The digested product and pSEGFP were ligated to generate pSEGFP-hLHR-WT and pSEGFP-hLHR-I114F (Fig. 4AGo).

Flow Cytometry-Based Quantitation of Cell Surface Receptor Expression
HEK293 cells grown to about 70% confluency were transfected with pcDNA3, pcDNA3-WT, pcDNA3-I114F, pSEGFP-WT, and pSEGFP-I114F by Lipofectamine 2000 (Invitrogen) in 60-mm dishes. At 48 h after transfection, cells were recovered from the culture dish by washing once with PBS, and then incubated with PBS containing 50 mM EGTA, 50 mM EDTA, and 300 mg/ml glucose (EGTA/glucose) at room temperature for 3 min. After incubation, EGTA/glucose was aspirated, and the treated cells were incubated at room temperature for another 3 min. Treated cells were resuspended in ice-cold PBS containing 5% goat serum (PBSS). The cell concentration was adjusted to 1 x 107 cells/ml, and 100 µl of cell suspension were used for the flow cytometer study. Cells suspension was first incubated with 1:100 rabbit anti-GFP polyclonal antibodies (Abcam, Cambridge, MA) in PBSS on ice for 1 h. Antibody-bound cells were washed three times with PBSS and then equilibrated with 1:100 second antibody; rhodamine-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Inc., Rockford, IL) for 1 h on ice. Second antibody equilibrated cells were washed three times with PBSS before analysis by Guava EastCyte flow cytometer (Guava Technologies, Hayward, CA). Isotype-matched control was done by equilibrating pSEGFP-WT- or pSEGFP-I114F-transfected cells with second antibody alone.

Proteasome Susceptibility Assay and Membrane Insertion
For receptor stability study, pSEGFP-hLHR-WT/mutant of 0.2 µg was transfected into HEK293 cells in 96-well plate (1 x 104 cells/well). Twenty-four hours after transfection, ALLN (Calbiochem, San Diego, CA) was added to 10 µM for 18 h, and the fluorescence of treated and untreated cells was recorded with a Carl Zeiss Axiovert 200 inverted fluorescence microscope (Carl Zeiss MicroImaging, Thornwood, NY). For the membrane insertion study, transfected cells were examined at high-power magnification with a x100 oil immersion lens at 72 h after transfection.

Molecular Modeling of LRR of hLHR
Residues 50–263 of the hLHR (accession no. P22888) were modeled by homology, using the crystal structure of the hormone-binding domain of the hFSHR (2) (PDB code, 1xwd, chain C) as a template. The SegMod algorithm (28) implemented in the program GeneMine was used to build the LHR model. For each residue in the model, the nearest residue in the template was determined based on the distance between {alpha}-carbons. The nearest residue in the template was identical to the modeled residue for 105 of the 214 residues modeled (49%). The {alpha}-carbons of these 105 conserved residues are within a root mean square distance of 0.72 Å from the corresponding atoms in the template. The LHR model is shown in Fig. 6Go, A and B, which were prepared with the programs MOLSCRIPT (47) and Raster3D (48).


   ACKNOWLEDGMENTS
 
We thank Dr. Aaron J. W. Hsueh for giving us the pcDNA3-hLHR-WT plasmid.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

M.Y.-K.L., P.J.S., D.B., V.B., P.Y.F., O.M.R., and W.-Y.C. have nothing to declare.

First Published Online May 18, 2006

Abbreviations: ALLN, N-Acetyl-Leu-Leu-Nle-CHO; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; hCG, human chorionic gonadotropin; HEK, human embryonic kidney; hFSHR, human FSH receptor; hLHR, human LH/chorionic gonadotropin receptor; LCH, Leydig cell hypoplasia; LHR, LH/chorionic gonadotropin receptor; LRR, leucine-rich repeat; WT, wild type.

Received for publication December 13, 2005. Accepted for publication May 8, 2006.


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