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Endocrinology Vol. 140, No. 4 1775-1782
Copyright © 1999 by The Endocrine Society

ARTICLES

Characterization of a Region of the Lutropin Receptor Extracellular Domain Near Transmembrane Helix 1 That Is Important in Ligand-Mediated Signaling1

Carlos A. Alvarez, Prema Narayan, Jianing Huang and David Puett

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bchiris.bmb.uga.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328–337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu332 and Asp333 in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly Gs. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys331 abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro330, Glu332, and Asp333 resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr337 with Ala or Asp, while having no profound change on receptor function, could overcome to some extent limited expression of replacements at positions 332 and/or 333, thus permitting a more definitive analysis of signaling. Replacement of the decapeptide sequence with Gly10 prevents expression, whereas deletion of all 10 residues and deletion of Glu332-Asp333 prevents functional expression at the cell surface. Thus, this invariant sequence in the glycoprotein hormones is required for proper folding, trafficking, and ligand-mediated signaling, but not ligand binding, in LHR. Amino acid residues, Glu332, Asp333, and to a limited extent, Pro330, are important in ligand-mediated signaling but not ligand binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FAMILY of G protein-coupled receptors (GPCR) is now estimated to contain some 1000 members (1, 2). An interesting and unique subset of this family is the group of glycoprotein hormone receptors, including those for LH, CG, FSH, and TSH. These heptahelical receptors contain a relatively large extracellular domain (ECD) of >300 amino acid residues, thus comprising about half of the receptor, and several potential sites of N-glycosylation (3). In the case of the LH receptor (LHR), the ECD is responsible for most (4, 5, 6, 7, 8) but apparently not all of the high-affinity binding of ligand since weak binding has been reported in a truncated receptor devoid of the ECD (9).

Structural data are available for the orientation of the seven transmembrane helices (TMH) of bacteriorhodopsin (10, 11), a light-driven proton pump but not a GPCR, and the GPCR, rhodopsin (12). Also, Baldwin (13) has analyzed over 200 GPCRs and arrived at a generalized helix packing scheme for this class of serpentine receptors.

An understanding of the nature of the ligand-mediated change in the conformation/orientation of the TMHs is crucial in elucidating the mechanism of transmembrane signaling, culminating in activation of the associated G protein(s) (14, 15). The glycoprotein hormone receptors appear to signal primarily via Gs (16); thus, the activation of G{alpha}s increases the catalytic efficiency of adenylyl cyclase leading to an increase in the intracellular concentration of cAMP. It has also been noted that LH and hCG can increase inositol 1,4,5-trisphosphate production (3, 16). This observation was originally interpreted to indicate that Gq was also activated by LHR, although recent data indicate that Gß{gamma}, derived from Gi, may activate phospholipase Cß in L cells (17).

We recently identified two adjacent acidic amino acid residues in the ECD of rat LHR, Glu332 and Asp333, proximal to the exoplasmic region of TMH 1 (Fig. 1Go), that, based upon individual replacements to lysine, appear critical in ligand-mediated signaling but not ligand binding (18). The 10 amino acid residue sequence between the ECD and TMH 1, FNPCEDIMGY, is invariant in the three glycoprotein hormone receptors (3) and may be part of an important switch region responsible for coupling ligand binding to the ECD to the reorientation of one or more TMHs and ultimately altering the interaction with G{alpha} on the cytoplasmic face of the receptor. To better characterize this conserved region of the receptor and to overcome the earlier concerns of low receptor density of LHR mutants with Lys replacements at Glu332 and Asp333, e.g. 10–20% that of wild-type LHR, a series of single, double, triple, and multiple replacements and deletions were made in this region and the mutant LHRs expressed in transformed African green monkey kidney epithelial (COS-7) cells. A novel observation was made in which one functionally silent mutation could be introduced with a functionally sensitive mutation to enhance the level of receptor expression. The results show that Glu332 and Asp333 in rat LHR are important in ligand-mediated signaling, as is Pro330 but to a lesser degree; all replacements of Cys331 interfere with proper plasma membrane expression, although binding of 125I-hCG could be measured in each mutant following solubilization of transfected cells.



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  		Figure 1. Schematic representation of the invariant amino acid sequence proximal to the exoplasmic end of TMH 1 of LHR. In rat LHR, the decapeptide sequence corresponds to amino acid residues 328–337.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis of LHR
The rat LHR cDNA, inserted into the XbaI-BamHI site of the expression vector pSVL, was kindly provided by Dr. William Moyle (Robert Wood Johnson Medical School, Piscataway, NJ), and site-specific mutagenesis for single mutants, double mutants, and deletions was based on the procedure of Deng and Nicholoff (19), mutant clones being identified by double-stranded DNA sequencing (20). Multiple mutations were introduced in the region under study by the use of degenerate primers.

Cell culture and transfection
COS-7 cells, obtained from the American Type Culture Collection (ATCC1651-CRL), were grown and maintained (37 C in a 5% CO2 incubator) in DMEM containing 10% (vol/vol) FBS and 1% of a solution containing penicillin G (10,000 U/ml), streptomycin (10,000 µg/ml), and Amphotericin (25 µg/ml). Lipofectamine (3 µl/µg DNA) (Life Technologies, Inc.) was incubated with DNA in OptiMEM I Reduced Serum Medium (Gibco BRL) for at least 1 h before addition to cells that were approximately 80–90% confluent (5, 10, and 20 µg DNA were added to cells growing in 100 mm dishes, 75 cm2 flasks, and 175 cm2 flasks, respectively). The Lipofectamine-DNA complex was incubated with the cells at 37 C in serum-free DMEM for 5–8 h; then, the transfection medium was replaced with fresh DMEM containing FBS.

Binding assays
Competitive and saturation binding assays to intact cells (approximately 5 x 105 cells/well) were performed 48–60 h after transfection (18). The transfected cells were split into 6-well plates 24 h posttransfection and reached confluence within 12–24 h. For saturation binding, various concentrations of 125I-hCG (DuPont NEN, 70–90 µCi/µg) in the range of 0–0.4 nM were added to the cells. For competitive binding, 0.03 nM of 125I-hCG was added to the cells with various concentrations of hCG (CR127, NIH) from 0–2.6 nM. The binding assay buffer was serum-free Waymouth’s medium containing 1 mg BSA/ml, and incubation was for 16 h at 25 C, nonspecific binding being determined in the presence of 1 µg hCG. The cells were then washed with PBS, lysed with 0.1 M NaOH, and the amount of 125I-hCG bound determined with a {gamma} counter.

LHR mutants that yielded only limited specific binding to intact cells, e.g. <1% of wild-type LHR, were examined by a single-point soluble binding assay (5, 18, 21). Transfected cells were harvested 60 h after transfection, chilled at 4 C, rinsed twice with cold PBS, and, after adding 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, and a mixture of proteinase inhibitors (EDTA, N-ethyl maleimide, and phenylmethylsulfonyl fluoride), pelleted at 4 C. The cells were resuspended in 50 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose and the mixture of proteinase inhibitors, and then homogenized in a Tissuemizer (Tekmar). The homogenate was centrifuged to remove nuclei, mitochondria, and any intact cells, and then the pellet was homogenized again. The pooled supernatants were centrifuged at 100,000 x g for 1 h in a Beckman Coulter, Inc. SW-41 rotor, the pelleted membrane fraction solubilized with 1.5% CHAPS in 50 mM Tris-HCl, pH 7.5, containing proteinase inhibitors, and the protein content estimated from a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Soluble binding assays were performed at 4 C by incubating 50–100 µg of membrane protein in 0.5 ml of 50 mM Tris-HCl, pH 7.5, containing 10% glycerol for 16 h with 0.06 nM 125I-hCG, nonspecific binding being determined in the presence of 1 µg hCG. (Proteinase inhibitors were not added at this step because we determined that they inhibited specific ligand binding.) Receptor-bound radioactivity was recovered by filtration through Whatman GF/B filters that were soaked in 0.3% polyethylenimine in 10 mM Tris-HCl, pH 9.1, for at least 1 h before the assay (22). The filters were then placed in glass tubes and counted in a {gamma} counter.

cAMP determinations
The extracellular concentrations of cAMP were measured about 60 h posttransfection (18). Approximately 24 h after transfection, cells were split into 12-well plates, and confluence (ca. 3 x 105 cells/well) was reached in about 24 h. The cells were preincubated for 15 min with 0.8 mM isobutylmethylxanthine (IBMX) in serum-free DMEM containing 1 mg BSA/ml. Increasing concentrations of hCG from 0–2.6 nM were added to the cells and incubated for 30 min at 37 C. The cells were quickly washed and lysed overnight in ethanol at -20 C. Then, the samples were transferred to glass tubes, dried in a Speed-Vac at room temperature, resuspended in the buffer of the 125I-cAMP RIA kit (DuPont NEN), and assayed as recommended by the manufacturer.

Data analysis
Binding and ligand-mediated cAMP increases were nonlinearly fitted using the Prism program (GraphPad Software, Inc.). Statistical analyses were done using t test procedures (95% confidence limit) to compare averages. Generally, two to four independent experiments, each in duplicate, were performed for binding and cAMP determinations, and the data are presented as mean ± SEM. In some cases (indicated), the results are given as mean ± range of a single experiment, performed in duplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 18 single amino acid residue replacements, 16 multiple replacements, e.g. double, triple, and one 9x replacement, and two deletion mutants were prepared and characterized in the invariant decapeptide sequence (residues 328–337) of rat LHR. Of these 36 mutants, 26 expressed at sufficient levels on the cell surface to permit meaningful ligand binding and ligand-mediated signaling parameters to be assessed using intact cells.

The 26 expressed LHR mutants were characterized by saturation binding studies with 125I-hCG, competitive binding studies using 125I-hCG and hCG, and hCG-mediated cAMP production. Replacements at positions Pro330, Glu332, and Asp333 invariably led to reduced ligand-mediated signaling. Significantly, ED50s for the P330A and P330D replacements were greater than that of wild-type LHR, and the maximal level of cAMP was reduced although basal cAMP levels, expression levels, and binding affinities of these mutants were similar to that of wild-type LHR (Fig. 2Go and Table 1Go). Several noteworthy features emerge from the results on the Glu332 and Asp333 replacements. 1) Ala replacements of these two amino acid residues decreased cell surface expression 4-fold; however, the maximal cAMP levels were reduced 10-fold (Fig. 3Go and Table 1Go). 2) The specificity for Glu and Asp at positions 332 and 333, respectively, is striking. The single mutants E332D and D333E express at reasonable levels and bind hCG with the same affinity as wild-type LHR, but ligand-mediated signaling is low compared with wild-type LHR (Fig. 4Go and Table 1Go). 3) Replacement of Tyr337 with Ala or Asp has only marginal effects on expression and signaling, but inclusion of one of these mutants with replacements at positions Glu332 and/or Asp333, with Y337A or Y337D, can increase expression to almost wild-type levels, presumably by enhancing proper receptor folding or trafficking. However, the signaling ability of the mutant receptors is not restored (Fig. 5Go and Table 2Go). Likewise, addition of a serendipitous T250K replacement greatly increased the expression of an E332D/D333E mutant (Table 2Go).



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  		Figure 2. Competitive binding (upper panels) and dose response of cAMP stimulation (lower panels) for P330A and P330D mutants of LHR in transiently transfected COS-7 cells. The binding data are presented as mean ±SEM of three and two independent experiments for P330A and P330D, respectively, and the signaling data represent a mean ± SEM of two independent experiments.

 

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  		Table 1. Summary of single amino acid residue replacements in the invariant linker region of LHR

 


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  		Figure 3. Saturation binding (A) and dose response of cAMP stimulation (B) for E332A and D333A mutants of LHR in transiently transfected COS-7 cells. The results of a representative saturation binding experiment are shown, and the signaling data are presented as mean ± SEM of three independent experiments.

 


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  		Figure 4. Competitive binding (A) and cAMP production (B) in transiently transfected COS-7 cells expressing wild-type (WT) and the single mutants, E332D and D333E of LHR. The data are presented as mean ± SEM of two independent experiments.

 


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  		Figure 5. Competitive binding (upper panels) and hCG-mediated cAMP production (lower panels) of wild-type LHR and two double mutants, E332D/Y337A and D333E/Y337D. The data are presented as mean ± SEM of two independent experiments.

 

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  		Table 2. Summary of double and triple amino acid residue replacements in the invariant linker region of LHR

 
The important binding and signaling parameters for the other LHR mutants in the invariant amino acid sequence of residues 328–337 are given in Tables 1Go and 2Go for single and double/triple replacements, respectively. Whereas the level of expression, as judged by the amount of ligand binding, varied considerably, there was no significant difference in the IC50s and calculated Kds of the mutants compared with wild-type LHR. Maximal ligand binding, i.e. Bmax or receptor density, can be obtained from competitive binding curves; however, because the Kds of mutant and wild-type LHRs are identical within experimental error, we have reported Bo ratio percentages, i.e. specific binding of each LHR mutant to wild-type LHR at the same concentration of 125I-hCG x 100% (Tables 1Go and 2Go). This approach was validated to yield equivalent ratios of Bmax (mutant:wild-type) in several single and double mutants using saturation binding with 125I-hCG (cf. Fig. 3Go and Tables 1Go and 2Go). The receptor number per cell calculated from three independent binding experiments (uncorrected for transfection efficiency) was 31 ± 9 x 103 for wild-type LHR. In summary, the binding data indicate that each of the LHR mutants that expressed on the cell surface bound hCG with an affinity equivalent to that of wild-type LHR, although the level of expression ranged from 10–114% that of wild-type LHR. Basal values of cAMP in the various mutant LHRs were similar to those of wild-type LHR ( Figs. 2–5GoGoGoGo and data not shown); however, the ED50s and maximal cAMP values varied depending upon the replacements.

Of the 10 mutants that failed to exhibit specific binding of 125I-hCG to intact cells, i.e. 0–1% that of wild-type LHR, eight yielded [(mutant Bo)/(wild-type Bo)] percentages between about 20–50% in a soluble binding assay, whereas two mutant forms of LHR, F328A, and the multiple glycine replacement of residues 328–337, were characterized by about 2% binding relative to wild-type LHR (Table 3Go). Under the conditions used, the reduced binding ratio of these 10 mutants may reflect a reduced receptor number or an increased Kd; however, in view of the results reported in Tables 1Go and 2Go, it seems more likely that these particular replacements and deletions interfere with receptor folding and trafficking to the plasma membrane. Preliminary results were also obtained with two replacements of Asp326, D326A and D326K, another amino acid residue that is invariant in the glycoprotein hormone receptor family. These two point LHR mutants exhibited [(mutant Bo)/(wild-type Bo)] percentages of 21 and 14%, respectively.


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  		Table 3. Summary of Bo values from a soluble binding assay of LHR mutants in the invariant linker region that failed to bind 125I-hCG in intact cells

 
A careful examination of the results in Table 1Go suggests the possibility that receptor density (proportional to Bo expressed as percentage of wild-type LHR under the conditions used) and maximal signaling (Rmax, also expressed as percentage of wild-type LHR) may be roughly correlated in a linear fashion, at least over the range of receptor levels examined. The data are consistent with such a relationship for the replacements Ile334, Met335, and Gly336; these also give ED50 values like that of wild-type LHR. For others, however, the signaling may exceed that expected based on receptor density, e.g. F328Y and N329A, although the ED50s for cAMP production are higher than for wild-type LHR. Replacements of Pro330, Glu332, and Asp333, as discussed above, result in signaling that is invariably less than expected if a linear correlation exists. To visualize these results, the data from Tables 1Go and 2Go were plotted as maximal (ligand-mediated) cAMP production or Rmax, given as a percentage of that elicited by wild-type LHR, vs. receptor density, given as a percentage of wild-type LHR expression, inferred from Bo measurements (Fig. 6Go). Any point on or near the diagonal from 0 (basal cAMP and no receptors)–100% (maximal cAMP elicited by wild-type LHR) could indicate a correlation between signaling and receptor number, e.g. I334V, M335A, G336A, and Y337A. Points in the upper half may reflect mutants that signal more than expected based on receptor density, e.g. F328Y and N329A, while those in the lower half are defined as signaling-deficient mutants, e.g. P330A,D, P330A/Y337D, and all the replacements at positions 332 and/or 333. A plot such as this also emphasizes the ability of Y337A,D and T250K replacements to rescue Glu332 and/or Asp333 replacements by increasing receptor expression, thus enabling a more definitive conclusion to be drawn regarding function.



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  		Figure 6. Plot of maximal (ligand-mediated) cAMP production, expressed as percentage of that elicited by wild-type LHR, vs. receptor density or number of receptors/cell, also expressed as percentage of wild-type LHR. These results, given as means, are taken from Tables 1Go and 2Go and represent all single, double, and triple mutants of LHR examined in this study that expressed at the cell surface. EA/DE/YD, EA/DA/YD, and ED/DE/TK refer to E332A/D333E/Y337D, E332A/D333E/Y337D, and E332D/D333E/T250K, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results obtained in this study demonstrate that the decapeptide sequence in the ECD of rat LHR near TMH 1, invariant in the three glycoprotein hormone receptors, is not involved in any significant way with ligand binding, but several of the amino acid residues are important in ligand-mediated transmembrane signal transduction and proper folding/trafficking of the receptor. Of these, the two acidic residues at positions 332 and 333, Glu and Asp, respectively, are of considerable interest in view of their strict specificity and role in ligand-mediated signaling. For example, replacement of Glu332 with Asp, of Asp333 with Glu, or an interchange of the two to give the double mutant, E332D/D333E, results in mutants that signal poorly. Indeed, based on 13 single, double, and triple mutants at positions 332 and 333, with receptor densities ranging from about 10–90% that of wild-type LHR, the maximal hCG-mediated signaling via cAMP was invariably in the range of 1–25% that elicited by wild-type LHR.

We reported earlier that individual Lys replacements of Glu332 and Asp333 reduced receptor expression to about 20% and 10% that of wild-type LHR, respectively, and abolished ligand-mediated signaling (18). The results reported herein confirm and extend the earlier work and show, for the first time, the striking specificity for Glu at position 332 and for Asp at position 333. We were able to achieve higher levels of receptor expression at these positions by including a Y337A or Y337D replacement, which by itself had negligible effects on receptor expression and signaling but could rescue, in many cases, poorly expressing receptor mutants. The increase in receptor density was particularly noteworthy for mutants E332D (ca. 60%->90% with Y337A), D333E (ca. 40%->70% with Y337D), and E332A/D333A (ca. <1% ->10% with Y337D). In addition, a T250K replacement increased the expression of the mutant LHR, E332D/D333E, from about 30%->100%. Of interest is the report of 46XX and 46XY siblings presenting with hypogonadism who were found to have a E332K mutant (23). From our earlier data (18) and that reported in the present study, we would predict that receptor expression was low in the patients and that the limited number of receptors were deficient in signaling.

In addition to Glu332 and Asp333, Pro330 also appears to be involved in ligand-mediated signaling. Replacements with Ala and Asp had only minor effects on expression levels, but the ED50s were greater than that of wild-type LHR and the maximal level of cAMP production in response to hCG was some 50 ± 10% that elicited by wild-type LHR. Interestingly, while the replacements Y337A and Y337D improve the expression levels of replacements at positions 332 and 333, they seem to reduce the expression of P330A and P330D mutants.

Cys331 may be important in trafficking and may also be involved in a critical disulfide. Position 328 (Phe) apparently requires a hydrophobic side chains, while position 336 (Gly) cannot tolerate a bulky side chain. Wild-type structures are not required at positions 329 (Asn), 335 (Met), and 337 (Tyr) since receptor function is preserved with rather dramatic replacements of each side chain.

No structural information exists on the invariant FNPCEDIMGY sequence in the glycoprotein hormone receptors. There is no obvious propensity for secondary structure formation, and this region of LHR is not part of a leucine-rich repeat that was used for modeling the ECD (24, 25, 26, 27); a major portion of the Cys-rich cluster may, however, adopt a cytokine-like fold (27). Our current working model is that this region of the ECD, and particularly Glu332 and Asp333, functions as part of a conformational switch relaying information of ligand binding to the ECD to the transmembrane portion of the receptor. Studies are currently underway to address this hypothesis.


   Footnotes
 
1 This work was supported by Research Grant DK-33973 from the National Institutes of Health. Back

Received October 1, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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