©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Two Amino Acid Residues on the Extracellular Domain of the Lutropin/Choriogonadotropin Receptor Important in Signaling (*)

(Received for publication, March 15, 1995; and in revised form, July 31, 1995)

Jianing Huang (§) David Puett (¶)

From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The lutropin/choriogonadotropin receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large extracellular domain. The cDNAs of LH/CG-R wild type and 15 point and double mutants, which encoded residues of opposite charge to that of wild type, were transiently transfected into COS-7 cells. Human choriogonadotropin (hCG) binding was determined, as was hCG-mediated cAMP production. Most of the replacements resulted in no substantive effect on the binding affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production, although the mutants expressed at a lower level than LH/CG-R wild type. The most interesting observation was noted with two point mutants of LH/CG-R, Glu Lys and Asp Lys, which bound hCG but failed to give increased cAMP production. Several of the mutant forms of LH/CG-R that expressed at low levels were further analyzed by soluble binding assays and Western blots. There was no evidence of any significant degree of intracellular trapping of hCG-binding mutant receptors. The expected major (93 kDa) and minor (78 kDa) forms were found for LH/CG-R wild type and several of the mutants. The Lys Asp and Asp Lys mutants exhibited primarily the lower M(r) form, indicating that receptor processing was impaired or that the mutant higher M(r) form was more rapidly degraded than LH/CG-R wild type. These results demonstrate that Glu and Asp, which are located near the first transmembrane helix, are important in receptor activation, while other conserved ionizable residues of LH/CG-R appear important in cell surface expression or stability but not in binding or signaling.


INTRODUCTION

The four glycoprotein hormones act through three homologous G protein-coupled receptors, each characterized by a relatively large glycosylated extracellular domain (Segaloff and Ascoli, 1993). Lutropin and human choriogonadotropin bind to a common receptor (LH/CG-R)(^1), of which cDNAs are available from four species: rat (McFarland et al., 1989), pig (Loosfelt et al., 1989), human (Minegishi et al., 1990), and mouse (Gudermann et al., 1992). Rat LH/CG-R is encoded by 11 exons (Koo et al., 1991; Tsai-Morris et al., 1991), and the extracellular domain contains a number of pseudo-leucine-rich repeats (McFarland et al., 1989; Braun et al., 1991).

Considerable data have been obtained to delineate specific amino acid residues and regions on the hCG alpha and beta subunits that participate in receptor binding and signal transduction. The recent availability of a crystal structure of partially deglycosylated hCG (Lapthorn et al., 1994; Wu et al., 1994) adds a new dimension to the analysis of glycoprotein hormone structure-function relationships. In contrast, relatively little is known about the conformation of the receptor and specific amino acid residues that may be involved in hormone binding and signaling, although mutagenesis has been used to map certain of these regions of LH/CG-R (Xie et al., 1990; Ji and Ji, 1991, 1993; Segaloff and Ascoli, 1993; Ji et al., 1993; Quintana et al., 1993; Wang et al., 1993a, 1993b). A perusal of the available amino acid sequences of LH/CG-R from several species shows the presence of a number of conserved ionizable amino acid residues, many of which are also conserved in the follitropin receptor and the thyrotropin receptor. Such conserved residues may be important in signaling or as hormone contact sites, e.g. to oppositely charged conserved side chains on LH, CG, follitropin, and thyrotropin.

The present study addresses 11 of the ionizable groups on the extracellular portion of rat LH/CG-R: Arg and Arg (encoded by exon 1), Lys (encoded by exon 2), Glu and Glu (encoded by exon 3), Arg and Lys (encoded by exon 5), Glu and Lys (encoded by exon 9), and Glu and Asp (encoded by exon 11). Site-directed mutagenesis was used to prepare single and double mutants where each ionizable side chain was replaced by one of opposite charge. The data suggest that two point mutants of LH/CG-R, Lys and Lys, bind hCG with nearly the same affinity as LH/CG-R wild type but exhibit diminished signaling.


MATERIALS AND METHODS

Supplies

The Transformer site-directed mutagenesis kit was obtained from Clontech Laboratories, Inc, the Sequanase version 2.0 kit was a product of U. S. Biochemical Corp., the plasmid kit was from Qiagen Inc. (Chatsworth, CA), and the Magic Minipreps DNA Purification System was from Promega. Bovine serum albumin, isobutylmethylxanthine, DEAE-dextran, chloroquine, dextran sulfate, dimethyl sulfoxide, and Nonidet P-40 were purchased from Sigma. hCG (CR-121) was provided by National Institutes of Health and by Dr. Steven Birken (Columbia University, NY); [I]hCG (100-150 µCi/µg) was from ICN (Horsham, PA). The cAMP radioimmunoassay kit was from DuPont NEN. Dulbecco's modified Eagle's medium and fetal bovine serum were obtained from Life Technologies, Inc.; NuSerum was from Collaborative Biomedical Products (Bedford, MA).

Mutagenesis of the LH/CG-R cDNA

The cDNA for the rat LH/CG receptor was kindly provided by Dr. William Moyle (Piscataway, NJ) in the expression vector pSVL. The desired 21-base deoxyoligonucleotides were synthesized by Dr. Rudolf Werner (University of Miami, FL) on an Applied Biosystems model 380B DNA synthesizer and by the Molecular Genetics Core Facility of the University of Georgia. In vitro mutagenesis was performed using the Transformer site-directed mutagenesis kit as described (Deng and Nickoloff, 1992), and mutated plasmid clones were identified by double-stranded sequencing.

Expression of LH/CG-R

COS-7 cells (African green monkey kidney cells), obtained from ATCC CRL, were grown at 37 °C in humidified air containing 5% CO(2) in the following medium: Dulbecco's modified Eagle's medium (90%), fetal bovine serum (10%), and 100 units/ml each of penicillin and streptomycin. Cells were transiently transfected at 60-80% confluency by the DEAE-dextran method. Transfection efficiencies were estimated by cotransfecting a pSV-beta-galactosidase plasmid with the pSVL vectors containing the LH/CG-R wild type and mutant cDNAs. 48 h after transfection, the cells were stained in situ for beta-galactosidase. This was accomplished by first washing the cells with PBS five times, then fixing them in a 0.05% (v/v) glutaraldehyde solution in PBS for 10-15 min. After three additional washes with PBS, the cells were incubated in a solution containing 1 mg/ml 5-bromo-4-chloro-3-indoyl beta-D-galactoside 1 mM MgCl(2), 20 mM potassium ferricyanide/ferrocyanide in PBS for at least 1 h at 37 °C. The cells transfected with the pSV-beta-galactosidase control plasmid were readily visualized under the microscope and counted. From the total number of cells in the field, transfection efficiencies were obtained.

[I]hCG Binding to COS-7 Cells

At 48 h post-transfection, the cells were assayed for [I]hCG binding, along with untransfected cells as control. Cells (1 times 10^6 cells/well) were washed twice with serum-free Waymouth's medium containing 1 mg of bovine serum albumin/ml, then 1 ml of this medium was added to each well. For competitive binding assays, [I]hCG (10^5 cpm) and different concentrations of unlabeled hCG were added to each well, while for direct binding assays, cells were incubated with increasing concentrations of [I]hCG. Nonspecific binding was determined by inclusion of a 1000-fold excess of unlabeled hormone. The cells were incubated overnight with gentle shaking at room temperature. On the following day, cells were washed twice with PBS, then trypsinized, collected, and counted in a counter. Specific binding of [I]hCG in the absence of unlabeled hormone gave 4,000-5,000 cpm, and nonspecific binding was about 5%. The results were analyzed by In-Plot (Graphpad Software, San Diego, CA), by Ligand Program (Munson and Rodbard, 1980), and by graphical analysis, the agreement between the various methods being good. Unless indicated otherwise, the data from a typical experiment are shown as mean ± S.E.

[I]hCG Binding to Detergent-solubilized COS-7 Cell Extracts

This procedure is based on that described by others (Xie et al., 1990; Thomas and Segaloff, 1994). Control cells and transfected cells (48 h) were placed on ice and washed twice with cold 0.15 M NaCl, 20 mM Hepes, pH 7.4. They were then scraped into a small volume of the same buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 5 mMN-ethylmaleimide. The cells were centrifuged (1500 times g, 15 min, 4 °C) and lysed by vortexing in a solution containing the same buffer with 1% Nonidet P-40 and 20% glycerol. After incubation on ice for 15 min, the cell lysate was centrifuged (12,000 times g, 15 min, 4 °C), and the supernatant was diluted to 0.1% Nonidet P-40 and incubated overnight with [I]hCG (2-3 times 10^5 cpm) in the presence and absence of a 1000-fold excess of hCG. Then, the binding solution was added to Whatman GF/B filters that had been soaked in 0.3% polyethylenimine (pH 9.1) for at least 1 h (Roche et al., 1985). After filtration and washing five times, the filters were counted in a counter. The specific binding of [I]hCG to solubilized LH/CG-R was between 5 and 15 times 10^3 cpm, and nonspecific binding was about 25% of the total binding.

cAMP Assay

After transfection (48 h), the COS-7 cells were washed twice with Dulbecco's modified Eagle's medium containing 1 mg of bovine serum albumin/ml and incubated in 0.5 ml of the medium containing isobutylmethylxanthine (0.8 mM) at 37 °C for 15 min. Increasing concentrations of hCG were then added, and the incubation was continued for 30 min at 37 °C. After removing the medium, the cells were lysed in 100% ethanol at -20 °C overnight. The extract was collected, dried under flowing nitrogen gas, and resuspended in the buffer of the cAMP assay kit. cAMP concentrations were determined with the [I]cAMP radioimmunoassay kit. Unless indicated otherwise, the results of a typical experiment are given as mean ± S.E.

Western Blot Analysis of Partially Purified LH/CG-R

Following transient transfection (48 h) of COS-7 cells with pSVL plasmids containing the LH/CG-R cDNAs (wild type and mutants), cell lysates were prepared as described above, and LH/CG-R was partially purified using wheat germ agglutinin-agarose chromatography (Rosemblit et al., 1988; Thomas and Segaloff, 1994). LH/CG-R was eluted with 3 mM triacetylchitotriose (Hipkin et al., 1992) in a 20 mM Hepes buffer, pH 7.4, containing 0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide, 5 mM EDTA, 0.1% Nonidet P-40, and 20% glycerol. The partially purified LH/CG-R fractions were concentrated by Centricon-30 spin columns.

An anti-LH/CG-R rabbit antiserum (Rosemblit et al., 1988), kindly provided by Dr. Deborah Segaloff (University of Iowa,), was diluted 4-fold with cold PBS (pH 8.0) and absorbed to a slurry of protein A-Sepharose in a small column at 4 °C. The column was then washed extensively with PBS, and the IgG fraction was eluted by addition of a 50 mM glycine buffer, pH 3.0, containing 0.1 M NaCl, and collected in tubes of 1 ml each containing 75 µl of 0.2 M Tris-HCl and 23.3 µl of 0.85 M Hepes buffer. The most concentrated fractions were pooled and stored at -70 °C.

Western blots were based on the technique of Hipkin et al. (1992) with slight modifications. Prestained M(r) standards (ovalbumin, bovine serum albumin, beta-galactosidase, and myosin from Bio-Rad) and equal amounts of cell lysates in sample buffer, with no boiling and in the absence of reducing agent, were applied to 7% SDS-polyacrylamide gels. An equivalent amount of lysate from untransfected COS-7 cells was used as a negative control. The proteins were electrophoretically transferred to polyvinylidine difluoride membranes, which were then washed twice (5 min each) with 20 mM Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl, and blocked for 2 h at room temperature with PBS containing 5% instant nonfat dry milk, 0.2% Tween 20, and 10% glycerol. Next, the filters were incubated overnight at room temperature with the same blocking solution containing 3 µg/ml rabbit anti-LH/CG-R IgG. The blot was then washed five times (5 min each) with blocking solution, followed by incubation for 1 h at room temperature with a 1:5000-fold dilution of a horseradish peroxidase-labeled donkey anti-rabbit IgG whole antibody fraction in the blocking solution. The second antibody was replaced, and the filters were washed twice (5 min each) with 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl; this was followed by two washes (5 min first wash, 10 min second wash) with the same buffer containing 1% Nonidet P-40. The filters were then washed for 5 min with the 20 mM Tris-HCl, 0.5 M NaCl (pH 7.5) solution, for 10 min with the same buffer containing 1% Nonidet P-40, and finally for 5 min with the buffer solution only. The blots were treated for 1 min with Amersham ECL developer solutions 1 and 2 (combined in equal amounts), then wrapped in Saran wrap and exposed to autoradiography film for about 30 s, followed by immediate development. The apparent M(r) and density of the various bands were obtained after scanning densitometry (PDI, Huntington Station, NY).


RESULTS

The DNA sequences of the rat LH/CG-R wild type and mutants were determined for the mutated and adjacent regions in all clones by dideoxynucleotide sequencing. Following this confirmation of the desired wild type and mutant clones, transfection efficiencies were monitored by transient cotransfection of pSVL-LH/CG-R wild type and mutants into COS-7 cells with pSVL-beta-galactosidase. The cotransfected cells were stained in situ, and transfection efficiencies of 5-10% were estimated.

hCG binding to transfected cells was determined via competition of [I]hCG with hCG and direct binding of [I]hCG. The results from multiple studies using competition and saturation binding with LH/CG-R wild type yielded an apparent K(d) of 0.17 ± 0.02 nM (n = 11) and an average of about 1 ± 0.1 times 10^4 receptors/cell, uncorrected for transfection efficiencies (Table 1). We also found that untransfected COS-7 cells, as expected (Loosfelt et al., 1989), do not contain LH/CG-R (data not shown).



Fig. 1Fig. 2Fig. 3show the results of competition and saturation binding of the LH/CG-R point mutants, and the K(d) values and receptor numbers are given in Table 1. Generally, the K(d) values are within severalfold of that obtained for wild type LH/CG-R, indicating that no major alteration in hormone affinity accompanied the mutations. Several of the receptor point mutants, however, had K(d) values slightly lower than that of wild type, e.g. Lys Arg, Lys Asp, and Lys Asp. In contrast, the number of receptors/cell is, with the exception of the Arg replacements and the Lys Arg replacement, considerably less than that obtained for the wild type receptor. Most of the other LH/CG-R mutants yield on the order of 1-4 times 10^3 receptors/cell, although receptors with the Lys Asp and Asp Lys replacements contained <1 times 10^3 receptors/cell, again uncorrected for transfection efficiencies.


Figure 1: hCG binding to COS-7 cells transfected with pSVL vectors containing the wild type and mutant LH/CG-R cDNAs. Competition binding of [I]hCG and hCG to seven point mutant forms of LH/CG-R with replacements of Arg, Arg, Lys, Glu, and Glu is shown. In each case wild type (Wt) receptor was included for comparison.




Figure 2: hCG binding to COS-7 cells transfected with pSVL vectors containing the wild type and mutant LH/CG-R cDNAs. Competition binding of [I]hCG and hCG to six point mutant forms of LH/CG-R with replacements of Arg, Lys, Glu, Lys, Glu, and Asp is shown. Wild type (Wt) receptor was also included for comparison.




Figure 3: hCG binding to two point mutants of LH/CG-R. Saturation binding of []hCG to COS-7 cells transfected with pSVL vectors containing mutant LH/CG-R cDNAs that produce lysine replacements of Glu and of Asp is shown.



Two double mutants of LH/CG-R, E65K,E68K and E332K,D333K, were also characterized by direct and competitive binding (Fig. 4). The K(d) of the former double mutant was somewhat lower than that of LH/CG-R wild type, and the average number of receptors/cell was 625 ± 106 (n = 3) ( Fig. 4and Table 1). No specific binding was detected with LH/CG-R(Lys, Lys) under the conditions used.


Figure 4: hCG binding to COS-7 cells transfected with pSVL vectors containing mutant LH/CG-R cDNAs. Competition binding of [I]hCG and hCG to two double mutants of LH/CG-R, E65K,E68K (bullet) and E332K,D333K () is shown; wild type (Wt, circle) LH/CG-R was included as control (left panel). Saturation binding of [I]hCG to the double mutant with lysine replacing Glu and Glu is shown (right panel).



Soluble binding assays were performed on several of the point LH/CG-R mutants that exhibited a relatively low number of surface receptors (Table 2). Compared to wild type LH/CG-R, the Lys Asp and Lys Asp mutants yielded only very low levels of specific binding. In contrast, the Glu Lys and the Asp Lys mutants gave specific binding intermediate to that of control and wild type LH/CG-R.



To assess the relative amounts of mutant receptors in cells with a low number of surface receptors, e.g. <25% LH/CG-R wild type, Western blots were obtained. As reported by others (Segaloff and Ascoli, 1993), purified rat LH/CG-R gives an apparent molecular mass of about 93 kDa. Transiently transfected COS-7 cells express a major band of about 93 kDa (Fig. 5), which is presumed to correspond to the 85-kDa form obtained by Hipkin et al. (1992) using stably transfected human embryonic kidney 293 cells. In addition, a minor band of apparent molecular mass of 78 kDa is also detected from LH/CG-R wild type, probably corresponding to the 68-kDa band reported by Hipkin et al.(1992), as are apparent higher and lower M(r) minor bands, which may be nonspecific. Interestingly, the LH/CG-R point mutants, Lys Asp and Glu Lys, as well as the double mutant, E65K,E68K, yield both the 93- and 78-kDa forms in about equal amounts, while the point mutants, Lys Asp, Lys Asp, and Asp Lys, exhibit predominantly the lower M(r) form ( Fig. 5and Table 2). Equal amounts of cell lysate protein were added from cells transfected with wild type and mutant LH/CG-R cDNAs to facilitate comparison of the relative total amount of immunoreactive receptor in each case. This amount of protein, which was necessary to visualize the relatively low levels of mutant receptors, resulted in overloading the gel with wild type LH/CG-R and obscuring the resolution of the major and minor bands. These were resolved, however, by densitometric scanning of the blots and by adding reduced amounts of cell lysate protein (data not shown). Comparable studies on cell lysates that were not purified by lectin chromatography gave similar results (data not shown), indicating that no other non-glycosylated forms of LH/CG-R are present.


Figure 5: Western blots of COS-7 cells transfected with pSVL vectors containing wild type and several mutant LH/CG-R cDNAs. Results are shown for LH/CG-R mutants exhibiting <25% of the receptors/cell relative to wild type LH/CG-R. These include the mutants Lys Asp (K-55-D), Lys Asp (K-121-D), Lys Asp (K-235-D), Glu Lys (E-332-K), Asp Lys (D-333-K), and E65K,E68K (E-65 & 68-K). Results are also shown for control cells (Control) and cells transfected with the wild type LH/CG-R cDNA (Wt). The two arrows denote apparent molecular mass values of 93 kDa (upper) and 78 kDa (lower).



The functionality of each of the mutant receptors was evaluated by cAMP production in response to added hCG. Fig. 6and 7 show typical results for those receptor point mutants that exhibited ED values and maximal cAMP levels similar to LH/CG-R wild type. Interestingly, the LH/CG-R(Asp) mutant, which gave a low level of expression, exhibited an ED and maximal cAMP response like that of wild type receptor. In contrast, the LH/CG-R point mutants Lys and Lys were non-responsive to added hCG (Fig. 8). Likewise, the double mutant, LH/CG-R(Lys, Lys), for which no receptors could be detected, exhibited no hCG-responsive cAMP production. The ED values for cAMP production by hCG in the transiently transfected COS-7 cells are given in Table 1.


Figure 6: Effects of hCG on cAMP production in COS-7 cells transfected with pSVL vectors containing mutant and seven wild type LH/CG-R cDNAs. Results are given for replacements of Arg, Arg, Lys, Arg, and Lys of LH/CG-R. Data of wild type (Wt) LH/CG-R and untransfected (control) cells are shown for comparison.




Figure 8: Lack of an effect of hCG on cAMP production in COS-7 cells transfected with pSVL vectors containing mutant LH/CG-R cDNAs. Results are shown for two point mutants and one double mutant with lysine replacements of Glu and Asp. The data are given as combined means ± S.E. from several independent experiments. The cAMP values at each concentration of hCG are slightly higher than control values (not significant), i.e. in untransfected cells, and significantly lower than values obtained with wild type LH/CG-R (cf. Fig. 6and Fig. 7for typical responses).




Figure 7: Effects of hCG on cAMP production in COS-7 cells transfected with pSVL vectors containing five point mutants and one double mutant of LH/CG-R. Data are presented for lysine replacements of Glu, Glu (single mutants of each and a double mutant), and Glu and for aspartic acid replacement of Lys. Results are also shown for wild type (Wt) LH/CG-R and for untransfected (control) cells. The cAMP response to LH/CG-R(Asp) was lower than that of wild type receptor in this experiment, but this finding was not reproducible.




DISCUSSION

The results presented herein enable us to conclude that replacement of either Glu or Asp with Lys in LH/CG-R yields mutant receptors that bind hCG as well as LH/CG-R wild type but exhibit diminished signaling. Thus, this region of the extracellular domain of the receptor, which is <10 amino acid residues from the first transmembrane helix, appears critical in gonadotropin-mediated receptor activation. While receptor numbers are reduced with these particular mutants relative to LH/CG-R wild type, other single and double receptor mutants with comparable receptor densities, e.g. Asp, Asp, Asp, Lys, and Lys gave cAMP responses like that of LH/CG-R wild type. Thus, the reduced hormone-mediated signaling noted with Lys and Lys is believed to be an intrinsic property of the mutant receptors and not a result of diminished receptor number.

The two M(r) forms of LH/CG-R expressed in COS-7 cells, i.e. 93 and 78 kDa as obtained herein, were first reported by Ascoli and colleagues (Hipkin et al., 1992) who expressed the receptor in stably transfected human embryonic kidney 293 cells, although the molecular mass values they determined were somewhat different than the values we obtained, e.g. 85 and 68 kDa, respectively. They reported data showing that the lower M(r) form was a precursor to the higher M(r) species; in addition, they suggested that the lower M(r) form bound hormone with reduced affinity relative to the major 85-kDa form. Since a variety of M(r) values has been reported for LH/CG-R purified from various tissues/species and expressed in cell lines (cf. Segaloff and Ascoli, 1993), the glycosylation patterns may differ depending upon the source of the receptor. In any case, we find that the lower M(r) form of rat LH/CG-R expressed in COS-7 cells is predominant for most of the mutants that lead to low receptor densities. Since there is no evidence of significant intracellular accumulation of these mutant forms, this observation suggests that the mature mutant receptors have a short half-life relative to LH/CG-R wild type, that processing proceeds less efficiently, or that the mRNA is degraded more rapidly or translated less efficiently.

Single replacements of LH/CG-R at several conserved sites with oppositely charged amino acid residues have no significant effect on the apparent K(d) relative to LH/CG-R wild type. That these particular replacements in LH/CG-R result in little or no change in the apparent K(d) of hCG binding does not necessarily mean that they are not contact sites. For example, Clackson and Wells(1995) replaced each of the 33 side chains on the extracellular domain of human growth hormone receptor, known to be involved in single ligand binding (de Vos et al., 1992), and found that fewer than half of the mutant receptors exhibited any significant loss in binding affinity. The greatest contributions to hormone-receptor binding were attributed to six hydrophobic amino acid residues on the receptor and, to a lesser extent, to five ionizable side chains. These particular 11 contact sites form a contiguous region in which the hydrophobic groups are surrounded by the charged groups. Thus, about two-thirds of the receptor amino acid residues that are rendered solvent-inaccessible upon hormone binding contribute little to binding affinity. Whether a similar relationship will emerge for LH/CG-R remains to be determined.

With the exception of the replacements of Glu and Asp with Lys, the charge reversals we made on LH/CG-R did not alter receptor functionality as judged by cAMP production in response to added hCG. There was, however, a dramatic effect of most of these mutations on receptor density. A reduction in receptor number could result from inefficient translation of the mRNA, instability of the mutant receptor, and/or inefficient assembly into the plasma membrane, as has been noted for several point and deletion mutants of LH/CG-R (Segaloff and Ascoli, 1993).

It is interesting and somewhat surprising that cAMP responsiveness appears independent of receptor density over a fairly wide concentration range. Excluding replacements at positions 332 and 333, LH/CG-R wild type and the various mutants are present at average receptor numbers/cell ranging over 2 orders of magnitude; yet, the maximal amount of cAMP produced at high concentrations of hCG is about the same. These results suggest that the amount of G(s) proteins or adenylate cyclase is limiting with regard to hCG-LH/CG-R-mediated cAMP production.

In summary, Glu and Asp, which are located <10 amino acid residues from the beginning of the first transmembrane helix and are invariant in the known LH/CG, follitropin, and thyrotropin receptors, appear to participate in transmembrane signaling. Thus, these particular amino acid residues may have fundamental roles in signaling for all of the glycoprotein hormone receptors.


FOOTNOTES

*
This research was supported by National Institutes of Health Research Grant DK33973. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Tularik, Inc., South San Francisco, CA 94080.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: LH/CG-R, lutropin/choriogonadotropin receptor; hCG, human choriogonadotropin; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Drs. Steven Birken, William Moyle, and Deborah Segaloff for providing important materials. We also thank Drs. Prema Narayan and Lizette Fernandez for helpful discussions and Dr. K.-C. Peng for technical assistance.


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