©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Lys in the Third Extracellular Loop of the Lutropin/Choriogonadotropin Receptor Is Critical for Signaling (*)

(Received for publication, October 10, 1995)

Lizette M. Fernandez (§) David Puett (¶)

From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602 and Department of Biochemistry and Molecular Biology, REPSCEND Laboratories, University of Miami, Miami, Florida 33101

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The lutropin/choriogonadotropin receptor (LH/CG-R) contains a relatively large extracellular domain, in addition to the seven transmembrane helices (TMH), three extracellular loops (ECL), and three intracellular loops typical of G protein-coupled receptors. While high affinity ligand binding has been attributed to the N-terminal extracellular domain, there is evidence that portions of the three ECLs may function in ligand binding and transmembrane signaling. We have investigated the role of several ionizable amino acid residues of rat LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP production. COS-7 cells were transfected with the pSVL expression vector containing cDNAs of either wild-type or mutant rat LH/CG-R. Several point mutants of Lys, located at the interface of ECL III and TMH VII, bound hCG like wild-type receptor but exhibited greatly diminished ligand-mediated signaling. Neither the point mutant, Lys Asp (ECL I), nor the double mutant, Asp Lys/Lys Asp (ECLs I and III, respectively), showed significant hCG binding to intact cells; in detergent-solubilized cells, only the double mutant bound hCG. The mutants Arg Glu (interface of the extracellular domain and TMH I) and Lys Glu (ECL II) proved to be similar to wild-type receptor in binding and signaling. Our results establish that Lys is important in signaling but not ligand binding. Its location on the opposite side of the membrane from G(s) precludes a direct interaction, thus emphasizing the importance of a conformational change in the receptor and suggesting that ligand binding to receptor and ligand-mediated receptor activation are dissociable phenomena.


INTRODUCTION

The LH/CG-R (^1)(1) is expressed on several types of gonadal cells (2) and has a crucial role in reproductive processes. Upon binding to their common receptor, LH and CG increase adenylate cyclase activity. Although cAMP appears to be the principal mediator of the actions of gonadotropins on most gonadal cells, there is evidence to support activation of the phospholipase C pathway as well, which results in the formation of inositol 1,4,5-trisphosphate and increased [Ca] levels (3, 4, 5) .

The LH/CG-R, FSH-R, and TSH-R are members of the glycoprotein hormone receptor family, characterized by a relatively large extracellular N-terminal region and a membrane-embedded C-terminal region containing seven TMHs. This C-terminal region is homologous to the small ligand binding members of the G protein-coupled receptor superfamily. However, unlike the small ligand binding receptors, where binding occurs in a cleft formed by the TMHs, the glycoprotein hormone receptors utilize their extracellular domain as the high affinity binding site for the heterodimeric glycoprotein hormones with molecular masses of 30-37 kDa (6, 7, 8, 9, 10) . This structural difference classifies these receptors as a distinct subfamily of the G protein-coupled receptor superfamily (11) .

The N- and C-terminal domains of the rat LH/CG-R each contain over 300 amino acid residues, the latter distributed over three ECLs, seven TMHs, three intracellular loops, and a cytoplasmic tail(1) . If the LH/CG-R spans the membrane similar to the small ligand binding G protein-coupled receptors, one would expect the seven putative TMHs to form a pocket like that of the bacteriorhodopsin and rhodopsin receptors(12, 13) ; the six hydrophilic connecting loops are essential in maintaining this conformation. Additionally, although the high affinity binding site of the LH/CG-R is located in the ECD, there is evidence to support the presence of a lower affinity binding site in the C-terminal domain of the receptor(14, 15) . Therefore, the ECLs represent potential hormone contact sites.

In an attempt to define the role of the LH/CG-R ECLs in hormone binding and signaling, several Arg and Lys residues were replaced: Arg (at the boundary between the ECD and TMH I), Lys (ECL I), Lys (ECL II) and Lys (ECL III). Additionally, a reciprocal mutation of Asp and Lys was characterized. The relative positions of these amino acid residues are shown in Fig. 1. These particular residues are invariant at the homologous positions in the LH/CG-R and the FSH-R of all known species; in the TSH-R, the positions equivalent to residues 401 and 583 are His and Gly, respectively, and the other residues are invariant. Our results revealed that Lys is not involved in hormone binding but is essential for full receptor activation.


Figure 1: Schematic representation of the major portion of the C-terminal region of LH/CG-R, including the seven putative transmembrane helices, the three exoplasmic loops, and the three cytoplasmic loops. The single mutations of the full-length LH/CG-R prepared and characterized in this study are indicated: Arg Glu (R341E), Lys Asp (K401D), Lys Glu (K488E), Lys Glu(K583E), Lys Gln (K583Q), Lys Arg (K583R), and Lys Pro (K583P) LH/CG-R. In addition, a reciprocal mutation was investigated: Asp Lys/Lys Asp (D397K, K583D) LH/CG-R.




EXPERIMENTAL PROCEDURES

Materials

I-hCG (100-150 µCi/µg) was purchased from ICN Biochemicals Inc. (Costa Mesa, CA) and DuPont NEN. [alpha-S]dATP (1000-1500 Ci/mmol) and the I-cAMP radioimmunoassay kit were products of DuPont NEN. hCG was a gift from Dr. Steven Birken (Columbia University, New York, NY). The Transformer(TM) mutagenesis system was obtained from Clontech (Palo Alto, CA), the Sequenase version 2.0 kit was purchased from United States Biochemical Corp., and the Wizard Minipreps DNA purification system was a product of Promega (Madison, WI). The plasmid Maxiprep DNA purification kit was obtained from Qiagen, Inc. (Chatsworth, CA). Lipofectamine, fetal bovine serum, trypsin-EDTA, Waymouth's medium, gentamicin, and penicillin-streptomycin were from Life Technologies, Inc. DEAE-dextran, isobutylmethylxanthine, chloroquine, triacetylchitotriose, BSA, protein molecular size markers, and Nonidet P-40 were purchased from Sigma. The Enhanced Chemiluminescence Western blotting analysis system was from Amersham Life Sciences, and the BCA protein assay system was purchased from Pierce. Agarose-bound wheat germ agglutinin was a product of Vector Laboratories (Burlingame, CA), and Centricon-30 columns were obtained from Amicon (Beverly, MA). Immobilon P transfer membrane was purchased from Millipore (Bedford, MA), and glass fiber filters were from Whatman (Maidstone, UK). Polyclonal anti-LH/CG-R was kindly provided by Dr. Deborah Segaloff (University of Iowa, Iowa City, IA). Most other reagents were purchased from Sigma, Life Technologies, Inc., or Fisher.

Mutant cDNAs of the Rat LH/CG-R

The cDNA for the rat LH/CG-R, inserted into the XbaI-BamHI site of the expression vector pSVL, was the generous gift of Dr. William Moyle (Robert Wood Johnson Medical School, Piscataway, NJ). The 21-base deoxyoligonucleotides coding for the appropriate codon changes were synthesized by Dr. Rudolf Werner (University of Miami, Miami, FL) and by the Molecular Genetics Instrumentation Facility at the University of Georgia. In vitro mutagenesis was performed (16) and mutant clones identified by dideoxy sequencing(17) . Mutant cDNAs were amplified and DNA was obtained using the Qiagen plasmid Maxiprep kit.

Expression of the Rat LH/CG-R

COS-7 cells were kindly provided by Dr. Nevis Fregien (University of Miami, Miami, FL) and also purchased from the American Type Culture Collection (Rockville, MD). The cells, maintained at 37 °C in humidified air containing 5% CO(2) in 90% DMEM and 10% fetal bovine serum, with 100 units/ml each penicillin and streptomycin, were transiently transfected with the eukaryotic expression plasmid pSVL-LH/CG-R, wild-type and mutants, using the DEAE-dextran method (18) or the Lipofectamine method as recommended by Life Technologies, Inc. For DEAE-dextran transfection, 20 µg each of pSVL-LH/CG-R and pSV-beta-gal, to estimate transfection efficiencies, were added to a 10-ml solution of 90% DMEM, 10% NuSerum transfection medium containing a 0.4 mg/ml DEAE-dextran and 0.1 mM chloroquine, and the washed cells (4 times 10^6) were incubated for 3.5-4 h at 37 °C. After removal of the transfection medium, the cells were shocked for 2 min with 10% Me(2)SO; then COS-7 growth medium was added to the washed cells. For transfections with Lipofectamine, a 7.85-ml solution of serum-free DMEM, containing Lipofectamine (57 µl/ml), 10 µg each of the pSVL-LH/CG-R construct and the pSV-beta-gal plasmid and 10% (v/v) Opti-MEM media, was incubated with cells (2-3 times 10^6) for 5 h at 37 °C. The transfection medium was removed, the cells were washed, and COS-7 growth medium was added. With both types of transfection, the COS-7 cells were incubated overnight at 37 °C in humidified air with 5% CO(2). 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside staining of gluteraldehyde-fixed cells was used to estimate transfection efficiencies, which were roughly 10% for DEAE-dextran and 40% for Lipofectamine.

I-hCG Cell-surface Binding to Transfected Cells

The COS-7 cells were maintained for 16 h after transfection and then replated (5 times 10^5 cells/well, six-well tissue culture plates). Some 48-51 h post-transfection, the cells were about 70% confluent. Cells were then washed twice with serum-free Waymouth's medium containing 1 mg of BSA/ml, and 1 ml of this media was added to each well. Increasing concentrations of unlabeled hCG were then added to each well, followed by addition of 25 pMI-hCG (approximately 10^5 cpm). Total and nonspecific binding were determined by addition of I-hCG in the absence and presence of excess unlabeled hCG (54 nM). The plates were incubated at 25 °C for 16-18 h with gentle shaking. The cells were washed twice with cold phosphate-buffered saline, then trypsinized, collected, and counted in a counter. All determinations were performed in duplicate. Binding affinities and maximal binding capacities were calculated using the Ligand program (19) .

I-hCG Binding to Detergent-soluble Extracts

The transfected cells were maintained for 16 h after transfection and then replated (2-2.5 times 10^6 cells/dish, 10-cm tissue culture dishes). The protocol is adapted from that described by others(6, 20, 21) . About 48 h post-transfection, the transfected cells were placed on ice for 15 min and then washed twice with 5 ml of ice-cold 0.15 M NaCl, 20 mM Hepes, pH 7.4 (buffer A). Cells were scraped into 2 ml of cold buffer A containing 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 5 mMN-ethylmaleimide and pelleted by centrifugation at 2000 times g for 20 min at 4 °C. The pellet was resuspended in 0.25 ml of 1% Nonidet P-40, 20% glycerol in buffer A containing protease inhibitors and incubated on ice for 15 min. This mixture was centrifuged at 16,000 times g for 15 min at 4 °C. The supernatant was diluted with 2.25 ml of 20% glycerol in buffer A; 0.5 ml of the extract was incubated for 16-18 h at 4 °C with 50 pMI-hCG (10^5 cpm). Total and nonspecific binding were determined in the presence and absence of excess unlabeled hCG, respectively. Bound radioactivity was separated from unbound by filtration through Whatman GF/B filters that were previously soaked in 0.3% polyethylenimine in 10 mM Tris-HCl, pH 9.1 (22) . The filters were washed five times with 0.1 M NaCl, 10 mM NaN(3), 1 mg of BSA/ml in phosphate-buffered saline and counted in a counter. All determinations were performed in duplicate.

Intracellular cAMP Assay

Some 16-18 h after transfection, the transfected cells were replated (1 times 10^5 cells/well, 12-well tissue culture plates). At 48-51 h post-transfection, the cells were washed twice with DMEM containing 1 mg of BSA/ml and incubated in 0.5 ml of this medium with 0.8 mM isobutylmethylxanthine for 15 min at 37 °C. Increasing concentrations of hCG were then added and the incubation was continued for 30 min at 37 °C. The cells were washed twice with fresh medium without isobutylmethylxanthine and then lysed in 100% ethanol at -20 °C overnight. The extract was collected, dried under nitrogen gas, and resuspended in the buffer of the I-cAMP assay kit, and cAMP concentrations were determined by radioimmunoassay. All measurements were performed in duplicate; means and standard errors were calculated using the Prism program.

Partial Purification of the LH/CG-R

Transfected cells were maintained for 16-20 h after transfection and then replated (2-2.5 times 10^6 cells/dish, 10-cm tissue culture dishes). Some 48-51 h post-transfection, detergent-soluble extracts were prepared as described above. The procedures for cell lysis and partial purification of wild-type and mutant LH/CG-Rs were based on reports from other laboratories(21, 23, 24) . Following cell lysis with 1% Nonidet P-40 and centrifugation as described above, the supernatant was diluted 2-fold with 10% glycerol in buffer A, then loaded onto a small agarose-bound wheat germ agglutinin column equilibrated with 0.1% Nonidet P-40 and 10% glycerol in buffer A, and rotated at 4 °C overnight. Following extensive washing with this buffer, the LH/CG-R was eluted with one column volume of 3 mM triacetylchitotriose (24) containing 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 5 mMN-ethylmaleimide. The eluted material was concentrated to approximately 0.1 ml in a Centricon-30 spin column, and the protein concentration was determined using the BCA assay.

SDS-PAGE and Western Blots

Equal amounts of purified cell lysates in sample buffer with no reducing agent were applied to a 7% SDS-polyacrylamide gel without boiling. After the electrophoresis, the proteins were electrophoretically transferred to polyvinylidene difluoride membranes, which were then washed twice in Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl) and blocked for 2 h at room temperature in blocking solution (10% glycerol, 5% instant nonfat dry milk, 0.2% Tween 20 in phosphate-buffered saline). The filters were incubated overnight at room temperature with the same blocking solution containing 3 µg/ml rabbit anti-LH/CG-R IgGs, obtained by protein A-Sepharose purification of anti-LH/CG-R antiserum(23) . The membranes were washed five times for 5 min each with blocking solution and then incubated for 1 h at room temperature with a 1:5000 dilution of a horseradish peroxidase-labeled donkey anti-rabbit IgG whole antibody in blocking solution. Next, the membranes were washed as follows: twice with TBS, twice with 1% Nonidet P-40 in TBS, then once each with TBS, 1% Nonidet P-40 in TBS, and TBS alone. The membranes were exposed to an Amersham Enhanced Chemiluminescence developer solution for 1 min, wrapped in Saran Wrap, and exposed to Kodak XAR-5 film for 1 min. The films were densitometrically scanned (PDI System, Huntington, NY).


RESULTS

The mean K(d) from multiple independent transfections (n = 9) for hCG binding to wild-type LH/CG-R on transfected intact COS-7 cells was 0.14 nM, with a range of 0.07-0.25 nM (Table 1), there being no difference between transfections with DEAE-dextran and Lipofectamine. On the other hand, receptor numbers/cell (uncorrected for transfection efficiencies) were greater with Lipofectamine transfection (mean of 2 times 10^4) than with DEAE-dextran transfection (mean of 0.5 times 10^4). The difference in receptor number/cell, however, had no significant impact on the maximal cAMP production elicited by hCG at 100 ng/ml in COS-7 cells transfected with the cDNA to wild-type LH/CG-R; the mean was 20.6 (range = 12.3-31.7) pmol of cAMP/10^5 cells under the conditions used and was independent of receptor density over the range investigated (Table 1). Likewise, the effective mean concentration of hCG necessary to increase the intracellular cAMP concentration from basal values to 50% of the maximal value in COS-7 cells expressing wild-type LH/CG-R was 0.19 nM (range = 0.05-0.4 nM) (Table 1); this too was independent of receptor density. For ease in comparing the different experiments, the maximal hCG-mediated cAMP production over basal is normalized to 100% for each wild-type LH/CG-R control, and the value obtained with the various receptor mutants, also corrected for basal, is expressed as a percentage of that of wild-type. There was no evidence for a functional endogenous LH/CG-R in nontransfected COS-7 cells: the mean (range) of the basal cAMP production was 3.9 (1.6-6.7) pmol cAMP/10^5 cells and that of control cells in the presence of 100 ng/ml hCG was 4.6(3.8-5.2) pmol cAMP/10^5 cells.



The Lys Glu mutation resulted in a LH/CG-R that specifically bound I-hCG, and this binding was inhibited by unlabeled hCG in a concentration-dependent manner (Fig. 2A). Although the estimated number of receptors varied in the transfected cells, e.g. 2 times 10^3 for wild-type and 1 times 10^3 for mutant LH/CG-R (uncorrected for transfection efficiency), the mutant LH/CG-R yielded a binding affinity equivalent to that of wild-type LH/CG-R (Table 1). The Lys Glu replacement resulted in markedly decreased production of cAMP in response to added hCG, e.g. 15% that of wild-type LH/CG-R at 100 ng/ml hCG (Fig. 3A, Table 1). Since this result could be attributed to the decreased number of cell surface receptors, this mutant LH/CG-R was also transfected using Lipofectamine to obtain higher efficiency and increased receptor expression. The mutant receptor bound hCG with an affinity comparable to wild-type LH/CG-R (Table 1). There was an increase in cell surface expression, e.g. 2 times 10^4 receptors/cells (uncorrected for transfection efficiency), for both wild-type and mutant LH/CG-R, but the mutant receptor again stimulated cAMP production only 15% that of wild-type LH/CG-R at 100 ng/ml hCG (Table 1).


Figure 2: hCG binding to COS-7 cells transfected with wild-type and mutant cDNAs to LH/CG-R. Competition binding of I-hCG and hCG to wild-type LH/CG-R and six single mutants of LH/CG-R are given in panels A-D. The binding of I-hCG with no added hCG was normalized to 100% in each case.




Figure 3: cAMP levels in COS-7 cells transfected with wild-type and mutant cDNAs to LH/CG-R in the presence and absence of hCG. Panels A-D give data on six single mutant forms of LH/CG-R; in each case, results are provided for cells expressing wild-type LH/CG-R and for control cells.



To investigate the side-chain specificity of Lys, replacements were also made with Arg, Gln, and Pro. In each case the mutations yielded LH/CG-Rs that bound I-hCG with affinities comparable to that of wild-type LH/CG-R (Fig. 2B, Table 1), but these mutant receptors also resulted in cAMP accumulation <30% that of wild-type LH/CG-R upon addition of hCG to transfected cells (Fig. 3B, Table 1).

Competitive binding assays were unable to detect significant cell surface binding of I-hCG to cells transfected with cDNAs to the Lys Asp single mutation and the Asp Lys/Lys Asp reciprocal mutation (Table 2). I-hCG binding to detergent-soluble extracts of transfected cells was found for the reciprocal mutant but not the point mutant (Table 2). However, Western blot analysis indicated that the (Lys Asp) LH/CG-R was expressed (Fig. 4). Wild-type and mutant LH/CG-Rs were about equally distributed among three bands of apparent molecular mass 101, 93, and 82 kDa; the total protein in the mutant LH/CG-R was much less than that of wild-type LH/CG-R.




Figure 4: Western blots of lysates from COS-7 cells transfected with cDNAs to wild-type LH/CG-R (WT) and (Lys Asp)LH/CG-R (K401D). Equivalent amounts of cellular protein were analyzed in each case; control cells, i.e. nontransfected, exhibited no immunostaining bands (data not shown). The apparent molecular mass values of the three immunoreactive bands are indicated; these were based on comparisons of the mobilities with those of prestained standards (rabbit muscle myosin, 205 kDa; Escherichia coli beta-galactosidase, 116 kDa; BSA, 66 kDa; chicken egg ovalbumin, 45 kDa; bovine erythrocyte carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 20 kDa; bovine milk alpha-lactalbumin, 14.2 kDa; bovine milk aprotinin, 6.5 kDa).



Arg and Lys were each replaced with Glu; both mutations yielded LH/CG-Rs that bound I-hCG with affinities comparable to that of wild-type LH/CG-R (Fig. 2, C and D, Table 1). These mutant LH/CG-Rs were capable of stimulating cAMP production in a dose-dependent manner, quite similar to wild-type LH/CG-R (Fig. 3, C and D, Table 1).


DISCUSSION

Our results on the substitution of Lys of the rat LH/CG-R with 4 amino acid residues, positively charged Arg, negatively charged Glu, polar but nonionizable Gln, and nonpolar Pro (an imino acid), show that the mutant LH/CG-Rs bind hCG as well as wild-type receptor, but coupling to adenylate cyclase is greatly diminished. These findings are particularly intriguing since Lys is located extracellularly, at the boundary of ECL III and TMH VII, and is unable to interact directly with G(s). Thus, one can conclude that ligand-mediated transmembrane signaling involves a conformational change of the LH/CG-R in which Lys participates in some manner. Furthermore, this observation provides additional evidence that receptor activation/signaling can be dissociated from high affinity hormone binding in LH/CG-R. A similar conclusion was recently reached in reference to another portion of the rat LH/CG-R, namely the extracellular region just prior to TMH I(25) . In that study we showed that individual replacements of Glu and Asp with Lys yielded mutant receptors that also bound hCG like wild-type LH/CG-R but exhibited greatly diminished signaling. Interestingly, recent results with FSH indicate that different sites are involved in receptor binding and signal transduction(26) .

The finding that the [Arg]LH/CG-R mutant, in which one positively charged side chain is replaced with another, does not signal effectively indicates a stringent specificity for the lysine side chain. Interestingly, a charged residue at the interface of ECL III and TMH VII is not required for proper membrane localization since the replacements of Lys with Gln and Pro yielded mutants that gave levels of cell surface expression comparable to that of wild-type receptor.

There is no information on the type of interaction between Lys of LH/CG-R and either the ligand or another portion of the receptor. Since replacements of Lys have no appreciable effect on hCG binding, one would conclude that any such interaction between the hormone and Lys makes a negligible contribution to the overall free energy of binding. However, high affinity hormone binding may occur to the ECD, followed by a conformational change that ``presents'' the hormone to Lys in ECL III where low affinity binding may occur. Using overlapping synthetic peptides, Roche et al.(15) found that a peptide based on the sequence of rat ECL III inhibited the binding of I-hCG to rat luteal membranes with an IC of 0.2 mM. The most obvious interaction of Lys would be electrostatic, e.g. an ion-dipole or an ion-ion bond between the positively charged amino group and a negatively charged carboxyl group on hCG or on the receptor ECD or ECLs. Alternatively, a more hydrophobic environment could result in an abnormal pK of Lys, which could lead to the formation of a strong hydrogen bond. Since our results suggest some form of ligand-mediated conformational change of the receptor, it is reasonable to expect that Lys of the LH/CG-R may flip from one type of interaction to another concomitant with hormone binding and that this change accompanies, or perhaps even triggers, a change in the conformation or relative interhelical position of TMH VII.

Ji et al.(27) reported that Asp of the LH/CG-R, located at the boundary of TMH II and ECL I, formed an ion pair with Lys of the alpha-subunit of hCG. They postulated that ligand binding may alter the conformation of the unoccupied receptor by reorienting TMH II (28) and concluded that this residue was important in signaling but was not essential for hormone binding(29) , i.e. as we found for Lys.

Whatever the exact role of Asp in LH/CG-R function, we asked whether it and Lys could participate in an ion-ion interaction. This was based in part on the close proximity of TMHs II and VII suggested by projection maps of rhodopsin (13) and by reciprocal ion-pair mutations in the gonadotropin-releasing hormone receptor(30) . Moreover, interhelical interactions of TMH I, in proximity to II and VII, have been shown for the adrenergic receptors(31) . We found that the reciprocal mutation failed to express in a functional form at the cell surface, although hormone binding could be detected in detergent-solubilized cells. Thus, Asp and Lys can be individually replaced with oppositely charged amino acid residues and cell surface expression is obtained, but replacing both residues results in intracellular trapping.

Lys is invariant in the gonadotropin receptors, i.e. LH/CG-R and FSH-R, but in TSH-R the equivalent position is occupied by Gly(2) . In a revealing study, ECL III of the TSH-R was replaced with that from the beta(2)-adrenergic receptor; it was found that the mutant TSH-R bound TSH with high affinity, but the sensitivity of TSH-stimulated cAMP production and the maximal level of TSH-mediated cAMP production were significantly diminished in the mutant TSH-R(32) . Coupled with our studies on Lys in ECL III of the LH/CG-R, these results indicate an important role of ECL III in glycoprotein hormone signaling and a possible region of specificity delineating gonadotropin receptors from the TSH-R.

Two other positively charged residues of LH/CG-R, which are invariant in the glycoprotein hormone receptors, Arg (located in the ECD at the interface with TMH I) and Lys (ECL II) were each replaced with Glu with no observable effect on hCG binding or receptor activation. Lys (ECL I) was replaced with Asp, and the mutant receptor failed to exhibit significant hCG binding to intact or detergent-solubilized cells, suggesting that this amino acid residue may be involved in hormone binding. However, binding may occur but is difficult to detect if there is a low level of mutant receptor expression, if the receptor is rapidly degraded or if the K(d) is significantly increased. Lys is present at this position in LH/CG-R and FSH-R, while in TSH-R it is occupied by His; depending upon the pK of the His, the potential exists for retention of a positive charge at this location in ECL I of all glycoprotein hormone receptors.

Our results on Western analysis of wild-type LH/CG-R and (Lys Asp) LH/CG-R revealed the presence of three bands of apparent molecular mass 101, 93, and 82 kDa. The original Western blot experiments on expressed LH/CG-R were performed using human 293 cells stably transfected with the wild-type LH/CG-R cDNA(24) . A major 85-kDa form, considered the mature receptor, and a minor 68-kDa form, which appeared to be the incompletely glycosylated form, were found. The apparent 93-kDa band observed in transiently transfected COS-7 cells corresponds to the M(r) of purified rat ovarian LH/CG-R(2) , although wide variations have been reported in the M(r) of LH/CG-R from various sources. In another study from our laboratory using a slightly different set of protein standards, we found major and minor bands of apparent molecular mass 93 kDa (80%) and 78 kDa for expressed wild-type LH/CG-R(25) . The greater amount of protein loaded in that study could easily prevent resolution of the bands of 93 and 101 kDa reported herein. Interestingly, the Lys Asp LH/CG-R mutant also gave the same apparent M(r) forms as wild-type LH/CG-R, but at much lower levels.

In summary, these results enable us to conclude that Lys of the rat LH/CG-R is critical in receptor activation following hormone binding. Since Lys (ECL III) is located on the opposite side of the membrane from G proteins and does not appear to be involved in hormone binding, these findings offer considerable weight to the concept that receptor binding and activation are dissociable phenomena. Of interest are other observations we recently made that certain amino acid residues in TMH VII are also critical for signal transduction (33) . Thus, this region of the receptor appears to be important in transmembrane signaling subsequent to hormone binding.


FOOTNOTES

*
This research was supported in part by Research Grant DK33973 from the National Institutes of Health. 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.

§
Recipient of a Predoctoral Fellowship DK33973 from the National Institutes of Health.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens, GA 30602. Fax: 706-542-0182; puett@bchiris.biochem.uga.edu.

(^1)
The abbreviations used are: LH/CG-R, lutropin/choriogonadotropin receptor; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; ECD, extracellular domain; ECL, extracellular loop; FSH, follitropin; FSH-R, follitropin receptor; hCG, human choriogonadotropin; LH, lutropin; TBS, Tris-buffered saline; TMH, transmembrane helix; TSH, thyrotropin; TSH-R, thyrotropin receptor.


ACKNOWLEDGEMENTS

We thank Drs. Jianing Huang and Prema Narayan for their interest in this work and for helpful discussions.


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