The ß-Subunit of Human Choriogonadotropin Interacts with the Exodomain of the Luteinizing Hormone/Choriogonadotropin Receptor and Changes Its Interaction with the {alpha}-Subunit

SoHee Hong, InHae Ji and Tae H. Ji

Department of Molecular Biology University of Wyoming Laramie, Wyoming 82071-3944


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human CG (hCG) consists of a common {alpha}-subunit and a hormone-specific ß-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific ß-subunit in hCG with the receptor’s exodomain. A photoactivable hCG derivative consisting of the wild-type {alpha}-subunit and a photoactivable ß-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the ß-subunit in hCG makes the direct contact with the exodomain.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nearly 2000 G protein-coupled receptors have been classified into more than 100 subfamilies according to the sequence homology, ligand structure, and receptor function (1). A substantial degree of amino acid homology is found among members of a particular subfamily, but comparisons between subfamilies show significantly less or no similarity. This indicates the overall diversity occurring in the G protein-coupled receptor superfamily. The most striking difference has been observed in the sites and modes of ligand binding and signal generation. In general, several distinct modes have emerged for high-affinity ligand binding to the transmembrane core exclusively (photon, biogenic amines, nucleosides, eicosanoids, and moieties (lysophosphatidic acid and sphingosine-1-phosphate) of lipids, to both the core and exoloops (peptides <=40 amino acids), to exoloops and N-terminal exodomain (polypeptides <=90 amino acids), or exclusively to the exodomain (glycoprotein hormones >= 30 kDa) (1). The distinction between ligand binding and receptor activation is supported by the existence of antagonists that competitively inhibit agonist binding. However, it is experimentally difficult to distinguish ligand binding from receptor activation. Fortunately, a few receptor systems such as glycoprotein hormone receptors and protease-activated receptors are available as good models to distinguish ligand binding from receptor activation and, thus, investigate the transition between these two important steps.

hCG is a heterodimeric glycoprotein hormone, consisting of a common {alpha}-subunit and a hormone-specific ß-subunit (2). It binds to the LH receptor (LHR) that comprises two equal halves, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (3, 4). The exodomain is approximately 350 amino acids long, which alone is capable of high-affinity hormone binding (5, 6, 7, 8) with hormone selectivity (9, 10, 11) but without hormone action (7, 12). Mutational analysis of the exodomain also implicates its involvement in hormone binding (13, 14, 15). On the other hand, receptor activation occurs in the endodomain (16), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (1). The existing evidence suggests that hCG initially binds to the exodomain with high affinity and hormone specificity, and then the resulting hormone/exodomain complex is thought to interact with the endodomain (16). This secondary interaction is considered to generate hormone signals (1, 15, 16). Sometime between the initial interaction and the signal generation, hCG undergoes a crucial structural change involving the intersubunit interaction (17). Although the structural change is an important part of the hormone-receptor interaction and signal generation, it is unknown when and where it takes place. This study was launched to determine whether the ß-subunit in hCG contacts the exodomain of the receptor. There are numerous mutations of hCGß, which resulted in the binding-defective hCG. Such results are consistent with the view that the mutated amino acid residues are important for hCG binding to the receptor: they cannot distinguish whether the mutated amino acids were indeed involved in interacting the receptor or the mutations might have caused a (global) conformational change in the hormone (which prevented receptor binding). Our study gets around this ambiguous point and proves the direct interaction between hCGß and the exodomain.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To study the interaction of the ß-subunit of hCG with the exodomain of the LHR, we have generated two exodomains, Arg1-Tyr295 (LHR295) and Arg1-Gly336 (LHR336), that lack the endodomain. In addition, the ß-subunit of hCG was derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoic acid (AB), an amino-specific UV activable agent, and radioiodinated. The labeled hCGß is free of hCG{alpha} on autoradiographs as will be shown later in a number of figures. There are five amino groups at the N terminus, Lys2, Lys20, Lys104, and Lys122, of the hCG ß-subunit (18) that may react with the reagent. The resulting AB-125I-ß was reconstituted with untreated {alpha} to produce {alpha}/AB-125I-ß. Because both the photoactivable group and radioactive tracer are attached only to the ß-subunit of the derivatized hCG, radioactive labeling of receptors would indicate the labeling by the ß-subunit. However, for the photoaffinity labeling to be biospecific, {alpha}/AB-125I-ß should be bioactive and specific to the LHR and its exodomains. Therefore, {alpha}/AB-125I-ß was tested for binding to the cells expressing the LHR, LHR295, or LHR336 in the presence of increasing concentrations of unlabeled hCG. As shown by the displacement assay and corresponding Scatchard plot in Fig. 1Go, intact cells expressing the LHR bound {alpha}/AB-125I-ß. However, {alpha}/AB-125I-ß did not bind the cells expressing LHR295 or LHR336 because the exodomains were trapped in the cells and not transported to the cell surface (6, 7). When the cells expressing the LHR, LHR295 or LHR336 were solubilized in NP-40 and assayed, {alpha}/AB-125I-ß bound to solubilized exodomains and wild-type receptor. The binding affinity of {alpha}/AB-125I-ß was comparable to the affinity of 125I-hCG. These results indicate that {alpha}/AB-125I-ß is capable of binding the LHR, LHR295, and LHR336 with high affinity.



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Figure 1. {alpha}/AB-125I-ß Binding to LHR295, LHR336, and Wild-Type LHR

Cells expressing LHR295, LHR336, or wild-type LHR (LHRwt) were assayed, with or without solubilization in NP-40, for {alpha}/AB-125I-ß binding in the presence of increasing concentrations of unlabeled hCG. The results are presented in displacement of 125I-hCG binding (A) and Scatchard plot (B). Experiments were repeated three times in duplicate, and means and SDs were calculated. Untransfected cells did not show specific binding of {alpha}/AB-125I-ß and 125I-hCG. In addition, AB-125I-ß did not bind to the wild-type LHR. Open symbols represent [125I]hCG binding, and solid symbols represent {alpha}/AB-125I-ß binding as indicated in the table of Fig. 1Go. The different symbols represent the LHRwt, LHR295, and LHR336, as indicated in the table.

 
Photoactivation of {alpha}/AB-125I-ß and Cross-Linking of the {alpha}- and ß-Subunits of hCG
To test the covalent derivatization and activity of the UV activable AB, {alpha}/AB-125I-ß was irradiated with UV for increasing periods (Fig. 2Go). The intensity of the cross-linked {alpha}ß-dimer band increased, after longer UV irradiation and reached a plateau after irradiation for more than 1 min. The maximum level of the cross-linked {alpha}ß-dimer band was significant: approximately 39% of the total activity (the sum of the ß-band and {alpha}ß-band). This result indicates that the majority of {alpha}/AB-125I-ß was uniformly derivatized with AB and effective in reacting with the {alpha}-subunit. Two minor radioactive bands had an electrophoretic mobility significantly faster than the gel mobility of the ß-subunit. They are the proteolytic products of the ß-subunit that were produced during derivatization, radioiodination, reconstitution with hCG{alpha}, fractionation, and storage. To reduce proteolysis, protease inhibitors were included in the reconstitution mixture and, without the inhibitors, the proteolysis was more extensive. The intensity of the two bands increased in parallel to the storage time and, therefore, experiments were performed soon after {alpha}/AB-125I-ß was prepared. Interestingly, the intensity of the proteolytic product bands diminished after more than 1 min of UV irradiation and nearly disappeared after UV irradiation for >=3 min. We suspect that the proteolytic products could have adhered to the glass tubes, as peptides often adsorb to glass upon UV irradiation.



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Figure 2. UV Irradiation of Derivatized hCG ({alpha}/AB-125I-ß)

AB-125I-ß (lane 1), 125I-hCG (lane 2), and {alpha}/AB-125I-ß (lanes 3–8) were, with or without prior UV irradiation, solubilized in SDS and DTT, electrophoresed on polyacrylamide gel, and autoradiographed. The band intensity was analyzed using Molecular Imager Analysis System as described in Materials and Methods. The percent radioactivities of the hCG{alpha}ß-dimer band as shown in the inset bar graph were calculated by dividing the intensity of the hCG{alpha}ß-dimer band with the corresponding total band intensity of each gel lane. The total activity is the sum of the ß-band and {alpha}ß-dimer band activities and excludes the activity of the two lower bands of proteolytic products. The band positions of hCG{alpha}, hCGß, and hCG{alpha}ß were determined using 125I-{alpha} in 125I-hCG, AB-125I-ß, and 125I-{alpha} cross-linked to ß in [125I]hCG by bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone as the respective markers as described previously (22 ).

 
Photoaffinity Labeling of the LHR, LHR295, and LHR336
To photoaffinity label solubilized LHR295, LHR336, and the LHR, they were incubated with {alpha}/AB-125I-ß, irradiated with UV, solubilized in SDS and dithiothreitol, and electrophoresed. When the exodomains and receptor are labeled with {alpha}/AB-125I-ß, the labeled complexes are expected to appear as bands of higher molecular masses than the {alpha}- and {alpha}ß-bands in autoradiographs. On the autoradiograph (Fig. 3Go) of the gel, the LHR295 sample showed the 80-kDa and 100-kDa bands in addition to the 30-kDa hCGß and 50-kDa hCG{alpha}ß bands. Although the 100-kDa band was faint, it became conspicuous when the autoradiograph was overexposed. Similarly, the LHR336 sample showed the 82-kDa and faint 102-kDa bands in addition to the 30-kDa hCGß and 50-kDa hCG{alpha}ß bands. These results are comparable with the result of the LHR sample that produced the 116-kDa and faint 136-kDa bands. Therefore, the 80-kDa and 100-kDa bands of the LHR295 sample and the 82-kDa and 102-kDa bands of the LHR336 sample are likely to correspond to the 116-kDa and 136-kDa bands of the LHR sample. The composition of these bands will be discussed later.



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Figure 3. Autoradiograph of Photoaffinity-Labeled LHR295, LHR336, and Wild-Type LHR

Lower Panel (lane 1), AB-125I-ß was electrophoresed as described in the legend to Fig. 2Go. Lane 2, 125I-hCG was cross-linked with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone and electrophoresed as described previously (22 ). The sample shows the [125I]hCG{alpha} (20 kDa) band and the faint, cross-linked [125I]hCG{alpha}ß dimer (50 kDa) band. Lanes 3–5, Cells expressing LHR295, LHR336 or LHRwt were solubilized in NP-40, incubated with {alpha}/AB-125I-ß, irradiated with UV, solubilized in SDS under reducing conditions, and electrophoresed. Approximately 66 pmol of LHR295, LHR336, and wild-type LHR were used in the experiment. The gel was dried and exposed to x-ray film for autoradiograph. Molecular mass estimates of radioactive bands are presented in parentheses, and the apparent composition of band materials is indicated with arrows. Upper Panel, The overexposure of the photoaffinity-labeled complex bands. The bands of photoaffinity-labeled LHR295, LHR336, and wild-type LHR were faint as shown in the lower panel autoradiograph. Therefore, the upper portion of the autoradiograph showing the photoaffinity- labeled complexes was overexposed and shown.

 
UV-Dependent and Stepwise Photoaffinity Labeling of LHR336
To determine the relationship of the labeling with UV irradiation, LHR295 was incubated with {alpha}/AB-125I-ß, irradiated with UV for increasing times, solubilized in SDS and dithiothreitol (DTT), and electrophoresed. The autoradiograph of the gel (Fig. 4AGo) shows the UV-dependent appearance of two conspicuous bands (the 50-kDa hCG{alpha}ß dimer band and the 80-kDa band). Their band intensity was undetectable without UV irradiation and increased in parallel to increasing UV irradiation. These results indicate that UV irradiation is necessary for the band formation.



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Figure 4. Conditions for Photoaffinity Labeling of LHR295

A, UV-dependent photoaffinity labeled AB-125I-ß and cross-linked [125I]hCG (lanes 1 and 2) were electrophoresed as described in the legend to Fig. 3Go. Lanes 3–8, LHR295 solubilized in NP-40 was incubated with {alpha}/AB-125I-ß, irradiated with UV for increasing periods, and processed as described in the legend to Fig. 3Go. The inset of the autoradiograph shows the overexposed portion of the photoaffinity-labeled complex bands around the 100-kDa band. The intensities of the ß/LHR295 and {alpha}ß/LHR295 bands were estimated and divided by the corresponding total band intensity of each gel lane to calculate their percentages. The results are presented in the upper bar graph in Figs. 4–6GoGoGo. The percent radioactivities of the hormone/exodomain complexes are significantly low in this and later figures, This is because the total radioactivities in these gel lanes include unbound [125I]hCG present in the samples of solubilized exodomains. B, {alpha}/AB-125I-ß concentration-dependent photoaffinity labeling. Lanes 1 and 2, AB-125I-ß and cross-linked ]125I]hCG were electrophoresed as described in the legend to Fig. 3Go. Lanes 3–8, A constant amount of LHR295 solubilized in NP-40 was photoaffinity labeled with increasing amounts of {alpha}/AB-125I-ß as described to the legend to Fig. 3Go. C, LHR295 concentration-dependent photoaffinity labeling. Lanes 1 and 2, AB-125I-ß and cross-linked [125I]hCG were electrophoresed as described in the legend to Fig. 3Go. Lanes 3–8, Increasing amounts of LHR295 solubilized in NP-40 were incubated with a constant amount of {alpha}/AB-125I-ß and photoaffinity labeled as described to the legend to Fig. 3Go. The amount of solubilized LHR295 was determined by Scatchard plots as shown in Fig. 1Go.

 
In addition to the two bands, there was the faint 100-kDa band that was somewhat more obvious in the original autoradiograph. It is clear that both the 80-kDa and 100-kDa bands contain AB-125I-ß. According to our unpublished observation, the 100-kDa band contains 125I-{alpha}. These results indicate that AB-125I-ß (30 kDa) photoaffinity labeled a 50-kDa material to produce the 80-kDa band. In addition, when AB-125I-ß in {alpha}/AB-125I-ß labeled both the hCG{alpha} (20 kDa) and the 50-kDa material, it produced the 100-kDa (20 kDa + 30 kDa + 50 kDa) band. Because each hCGß has five amino groups that can be derivatized with AB, AB-125I-ß could carry up to five AB and photoaffinity label both hCG{alpha} and the 50-kDa material. This interpretation is also consistent with the sequential appearance of the photoaffinity-labeled 82-kDa and 102-kDa complexes. For example, the 80-kDa {alpha}ß-band preceded the appearance of the 100-kDa band. Therefore, the 50-kDa component corresponds to LHR296, which has an apparent molecular mass of approximately 50 kDa including six chains of N-oligosaccharides (19) with terminal sialic acids (20). The percent radioactivities of the hormone/exodomain complexes are low in this and later figures. This is because the total radioactivities in these gel lanes include unbound [125I]hCG present in the samples of solubilized exodomains. Therefore, we suspect that the actual percentage of labeled hormone/exodomain complexes among total hormone/exodomain complexes is significantly higher, being in the range of 20%.

Dependence of Photoaffinity Labeling on the Concentration of {alpha}/AB-125I-ß and LHR295
When a constant concentration of LHR295 was incubated with increasing concentrations of {alpha}/AB-125I-ß and irradiated with UV, the intensity of the 80-kDa band increased (Fig. 4BGo). In addition, the faint 100-kDa band also appeared at high {alpha}/AB-125I-ß concentrations. The result indicates that the labeling is dependent on the concentration of AB-125I-ß. The intensity of the 80-kDa band also increased when increasing concentrations of LHR295 were photoaffinity labeled with a constant concentration of {alpha}/AB-125I-ß (Fig. 4CGo), indicating the dependence of the photoaffinity labeling on the LHR295 concentration.

Hormone Specificity of Photoaffinity Labeling LHR295
Glycoprotein hormones consist of a common {alpha}-subunit and a hormone-specific ß-subunit. Despite this structural similarity, they have cognate receptors and only cross-activity. To examine the hormone specificity of the photoaffinity labeling, LHR295 was photoaffinity labeled with {alpha}/AB-125I-ß in the presence of an excess amount of unlabeled hCG, denatured hCG, FSH, and TSH (Fig. 5Go). Untreated hCG completely blocked the photoaffinity labeling of LHR295. However, denatured hCG, FSH, and TSH did not prevent the photoaffinity labeling although FSH and TSH slightly reduced the affinity labeling. It is clear that the photoaffinity labeling is specific to active hCG. The results are also consistent with the low-affinity cross-activity of FSH and TSH with hCG.



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Figure 5. Hormone Specificity of Photoaffinity Labeling of LHR295

Lanes 1 and 2, AB-125I-ß and cross-linked [125I]hCG were electrophoresed as described in the legend to Fig. 3Go. Lanes 3–8, {alpha}/AB-125I-ß (150,000 cpm) and LHR295 were incubated in the presence of a 100-fold higher concentration of unlabeled hCG, hCG denatured (de-hCG) by boiling in urea, FSH, or TSH. The incubation mixtures were irradiated with UV and processed as described in the legend to Fig. 3Go.

 
Immunological Identification of the Photoaffinity-Labeled LHR295
To verify the identity of the photoaffinity-labeled LHR295, we used Flag-LHR295, Flag-LHR336, and Flag-LHRwt that have an N-terminal Flag sequence. The N-terminal Flag epitope did not interfere with the activity and expression of the Flag-LHR (21). Flag-LHR295 solubilized in NP-40 was photoaffinity labeled with {alpha}/AB-125I-ß, immunoprecipitated with anti-Flag antibody and Protein-G Sepharose, solubilized in SDS and DTT, and electrophoresed on polyacrylamide gel. The autoradiograph of the gel (Fig. 6AGo, gel lane 7) shows the 80-kDa ß/LHR295 and 100-kDa {alpha}ß/LHR295 bands, indicating that the band materials contain immunospecific Flag-LHR295 and radioactive AB-125I-ß. The bands were not visible if any of {alpha}/AB-125I-ß, Flag-LHR295, UV irradiation, anti-Flag antibody, or protein-G Sepharose was omitted in photoaffinity labeling and immunoprecipitation. More specifically, the sample that lacked only the UV irradiation showed the AB-125I-ß band but not the 80-kDa ß/LHR295 and 100-kDa {alpha}ß/LHR295 bands. These results indicate that {alpha}/AB-125I-ß was associated and immunoprecipitated with Flag-LHR295 but was not covalently linked to it. Normal mouse sera could not substitute anti-Flag antibody for immunoprecipitating the 80-kDa ß/LHR295 and 100-kDa {alpha}ß/LHR295 bands. Furthermore, LHR295 lacking the Flag epitope could not be immunoprecipitated even if all of the necessary factors, except Flag-LHR295, were included in photoaffinity labeling and immunoprecipitation (data not shown). These results indicate that the 80-kDa ß/LHR295 and 100-kDa {alpha}ß/LHR295 bands were properly and specifically immunoprecipitated with AB-125I-ß and that Flag-LHR295 was present in the bands.



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Figure 6. Immunoprecipitation of Photoaffinity-Labeled Complexes

A, Immunoprecipitation of {alpha}/AB-125I-ß complexed to Flag-LHR295 and LHR295. Lanes 1 and 2, AB-125I-ß and cross-linked [125I]hCG were electrophoresed as described in the legend to Fig. 3Go. Lanes 3–8, {alpha}/AB-125I-ß was incubated with Flag-LHR295, irradiated with UV, and immunoprecipitated using monoclonal antiFlag antibody (antiFlag) and protein G-Sepharose (protein G) as described in Materials and Methods. As controls, {alpha}/AB-125I-ß, Flag-LHR295, UV treatment, antiFlag antibody, or protein G-Sepharose was omitted in some samples as indicated by -. B, Immunoprecipitation of {alpha}/AB-125I-ß complexed to Flag-LHR336. Experiments were the same as those shown in Fig. 8 except that Flag-LHR336 was used instead of Flag-LHR295. C, Immunoprecipitation of {alpha}/AB-125I-ß complexed to Flag-LHRwt and LHRwt. Experiments were the same as that shown in Fig. 8 except that Flag-LHRwt was used instead of Flag-LHR295.

 
Since Flag-LHR295 was immunoprecipitated, photoaffinity labeled Flag-LHR336 should also be immunoprecipitated. Therefore, Flag-LHR336 was photoaffinity labeled with {alpha}/AB-125I-ß and processed for immunoprecipitation. The 82-kDa ß/LHR336 and 102 kDa {alpha}ß/LHR336 bands were precipitated (Fig. 6BGo). They were not immunoprecipitated when any of {alpha}/AB-125I-ß, Flag-LHR295, UV irradiation, anti-Flag antibody, or protein-G Sepharose was omitted. In addition, normal mouse sera could not replace anti-Flag antibody in the immunoprecipitation, an indication of the specificity for anti-Flag epitope. Another positive control (Flag-LHRwt) was successfully and specifically immunoprecipitated, whereas the corresponding negative control lacking the Flag sequence (LHRwt) was not immunoprecipitated (Fig. 6CGo). Taken together, our results unequivocally demonstrate that the immunoprecipitation was specific for the Flag epitope and that the material (~50 kDa) photoaffinity labeled with {alpha}/AB-125I-ß was LHR295.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In a series of experiments involving photoaffinity labeling and immunoprecipitation, we have shown that {alpha}/AB-125I-ß specifically interacted to LHR295 with a high affinity and photoaffinity labeled it. In a similar manner, {alpha}/AB-125I-ß also photoaffinity labeled LHR336 and LHRwt. These results indicate the direct contact of the ß-subunit in hCG with LHR295 and therefore, the exodomain of the LHR. This result is consistent with our earlier observation that the peptide mimic corresponding to an exodomain sequence affinity labeled the ß-subunit of hCG (22).

It has been shown that hCG undergoes a structural change involving the interaction between the two subunits after it binds the LHR on intact cells (17). However, it was unclear when the change occurs, e.g. when hCG initially interacts with the exodomain of the receptor or when the hCG/exodomain complex makes the secondary contact with the endodomain and generates a signal (1). Another question concerns where the structural changes in hCG occur. These are questions pertinent to understanding the mechanisms of the two-step hCG-receptor interactions and signal generation. Our observations in the present study shed some light on these issues. However, before discussing them, it is helpful to review the structure of {alpha}/AB-125I-ß. NHS-AB can react to the five amino groups at the N terminus, Lys2, Lys20, Lys104, and Lys122 of the hCG ß-subunit (18). Based on the crystal structure of hCG (23), it is possible to predict the potential cross-linking activity of AB attached to each of the amino groups. The reagents attached to the N-terminus and Lys2 could cross-link the ß-subunit to the N-terminal region of the {alpha}-subunit since these two groups are adjacent (Fig. 7Go). The two N-terminal regions are, however, located at the rear side of the putative receptor-binding phase of the hormone. ßLys20 is in the ß-loop 1 that is part of the putative receptor binding site and therefore, the AB attached to ßLys20 could label the receptor. However, ßLys20 is beyond the maximum labeling distance (7 Å) from the {alpha}-subunit, and its side chain projects away from the {alpha}-subunit. ßLys104 is in the seat belt that is near {alpha}-loop 2 and part of the putative receptor-binding site. This is consistent with the results of the mutational analysis of ßLys104 (24) and chemical modification (25). The AB coupled to ßLys104 could label the {alpha}-subunit or the receptor. ßLys122 is in the C-terminal region, which is unnecessary for the interaction with the {alpha}-subunit and receptor. Therefore, our data narrow the potential photoaffinity labels for the receptor to those attached to ßLys20 and ßLys104. On the other hand, the potential intersubunit labels are the reagents attached to the ßN-terminus, ßLys2, and ßLys104. Overall, our results are consistent with previously reports on amino group labeling, accessibility, and cross-linking as summarized by Gordon and Ward (26).



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Figure 7. Structure of hCG and Potential Sites for Derivatized AB

The crystal structure of hCG {alpha}ß-subunits (23 ) and the potential sites for derivatization with AB are projected in three different orientations. The hCG {alpha}-subunit is shown in purple, the ß-subunit in green, and the potential AB-derivatization site of the ß Lys residues in red.

 
Change in the Intersubunit Interaction after hCG Binding to the Exodomain
In an earlier report (17) we showed that the efficiency of the cross-link between the {alpha}- and ß-subunits of a UV activable derivative of hCG is 3-fold higher when the hCG derivative is free in solution than bound to the receptor. In that study, hCG{alpha} was derivatized and reassociated with untreated hCGß. This suggests that the interaction between the hCG subunits changes after binding to the receptor. With regard to the structural change of hCG upon binding to the receptor, the {alpha}ß-intersubunit cross-link was 3-fold higher when hCG was free than it was bound to the receptor (17). This indicates that the interaction between the two subunits of hCG changed after hCG bound to the receptor. However, it was unclear when the change takes place, whether during the initial hCG contact with the exodomain or during the secondary contact of the hCG/exodomain complex with the endodomain. The affinity labeling data in this study (Table 1Go and Figs. 2Go and 4Go) show that the change occurred during the initial interaction of hCG with the exodomain. For example, up to 39% of {alpha}/AB-125I-ß that was in solution and not bound to receptor was cross-linked to produce the {alpha}ß-dimer (Table 1Go and Fig. 2Go). Therefore, the ratio of cross-linked {alpha}ß and uncross-linked ß is 0.64. In contrast to these significant intersubunit cross-links of free hCG, the intersubunit cross-link of {alpha}/AB-125I-ß that was bound to LHR295 was significantly low. For example, the ratios of cross-linked {alpha}ß and uncross-linked ß of {alpha}/AB-125I-ß bound to LHR295 were 0.07–0.1 (Table 1Go). These results indicate a significant difference in the interaction between the two subunits before and after binding to LHR295. Therefore, there was a change in the intersubunit interaction upon {alpha}/AB-125I-ß binding to LHR295. Furthermore, this change took place when hCG bound the exodomain, i.e. before the secondary interaction of the hCG/exodomain complex with the endodomain and generation of a hormone signal.


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Table 1. Efficiency of Intersubunit Cross-Link of Free and Exodomain-Bound {alpha}/AB-125I-ß

 
In conclusion, the ß-subunit of hCG bound to the LHR makes contact with the exodomain, probably involving ßLys20 and ßLys104. The interaction leads to a structural change of hCG before the secondary interaction of the hCG/exodomain complex with the endodomain. This change impacts the interaction between the hCG {alpha}ß-subunits.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutagenesis, Expression, and Assay of the Exodomain of the LH/CG Receptors
Exodomains, Arg1-Tyr295 (LHR295) and Arg1-Gly336 (LHR336), of the rat LH/CG receptor were produced by converting either Ser296 or Tyr337 to a stop codon, respectively. The exodomain cDNAs were produced in pSELECT vector containing the LH/CG receptor cDNA using the Altered Sites Mutagenesis System (Promega Corp., Madison, WI), sequenced, and subcloned into pcDNA3 (Invitrogen, San Diego, CA) as described (27), and sequenced again to verify mutation sequences. This procedure does not involve PCR and, therefore, unintended mutation is unlikely to occur during mutagenesis. The wild-type receptor and exodomain plasmids were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in MEM containing 10% horse serum and 500 µg/ml of G-418. Since the exodomains were trapped in the transfected cells and not secreted, the cells were solubilized in NP-40, which was assayed for [125I]hCG binding as previously described (21) and also used for affinity labeling. All assays were carried out in duplicate and repeated four to five times. Means ± SD were calculated. hCG (CR 127), hCG{alpha}, hCGß, FSH, and TSH were supplied by the National Hormone and Pituitary Program (Baltimore, MD). Denatured hCG was prepared by boiling in 8 M urea, 2.7 M guanidine hydrochloride, and 100 mM DTT for 20 min.

Derivatization and Radioiodination of hCGß and Reconstitution of Derivatized hCG ({alpha}/AB-125I-ß 4055)
hCGß was derivatized with 4-azidobenzoic acid (AB) using the N-hydroxysuccinimide ester of AB, radioiodinated, purified, and reconstituted with untreated hCG{alpha} as described previously (28). Briefly, 33 µg of hCGß were dissolved in 40 µl of 10 mM Na2HPO4 (pH 7.4) and incubated with 5.3 µl of 16.5 mM NHS-AB at 25 C for 60 min. The resulting AB-ß was radioiodinated and fractionated on Sephadex G-50 superfine after which the AB-125I-ß fraction (~1 x 108 cpm) was incubated with 30 µg of hCG{alpha} and one tablet of protease inhibitor cocktail (Roche Molecular Biochemicals, Nutley, NJ) with gentle mixing. The resulting {alpha}/AB-125I-ß was fractionated on Sephadex G-100 superfine column (0.7 x 50 cm). The resulting hCG derivative ({alpha}/AB-125I-ß) was capable of binding the LHR as untreated hCG did (28) and was used for photoaffinity labeling of the LHR and LHR exodomains. Hormone binding to intact cells expressing the wild-type LHR as well as solubilized LHR and exodomains was carried out, and the data were analyzed as described above. Based on the binding data and Scatchard analyses, receptor concentrations were determined.

Photoaffinity Labeling of LHR Exodomains with {alpha}/AB-125I-ß
Solubilized LHR295, LHR336, and wild-type LHR (120 µl) were separately placed in siliconized glass tubes, incubated with 150,000 cpm of {alpha}/AB-125I-ß at 4 C for 16 h, irradiated with a MineralightR-52 UV lamp, solubilized in 2% SDS and 100 mM DTT, and electrophoresed. Gels were dried on filter paper, autoradiographed, and, in addition, phosphoimaged on a model GS-525 Molecular Imager System Scanner (Bio-Rad Laboratories, Inc., Richmond, CA) as described previously (22).

Immunoprecipitation of Photoaffinity-Labeled Flag Exodomains
For immunoprecipitation of photoaffinity-labeled LHR295 and LHR336, the Flag epitope was inserted at the N terminus of mature exodomains (21). The resulting Flag-LHR295 and Flag-LHR336 were incubated with {alpha}/AB-125I-ß and immunoprecipitated using monoclonal anti-Flag antibody as described previously (21).


    FOOTNOTES
 
Address requests for reprints to: Dr. Tae H. Ji, Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944.

This work was supported by NIH Grants HD-18702 and DK-51469.

Received for publication November 25, 1998. Revision received May 6, 1999. Accepted for publication May 13, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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