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Endocrinology Vol. 140, No. 6 2486-2493
Copyright © 1999 by The Endocrine Society

ARTICLES

The {alpha}-Subunit of Human Choriogonadotropin Interacts with the Exodomain of the Luteinizing Hormone/Choriogonadotropin Receptor1

SOHee Hong, InHae Ji and Tae H. Ji

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

Address all correspondence and requests for reprints to: Dr. Tae H. Ji, Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944. E-mail: ji{at}uwyo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The LH/CG receptor, a G protein-coupled receptor, consists of two parts, the N-terminal extracellular segment (exodomain) and the membrane-associated C-terminal segment (endodomain). hCG initially binds the exodomain of the receptor and then, the hormone/exodomain complex is thought to make the secondary contact with the endodomain of the receptor and generate a hormone signal. However, little direct evidence is available about which hormone subunits ({alpha} or ß) interact with which domains of the receptor.

To determine whether the {alpha}-subunit contacts the exodomain of its receptor, hCG containing [125I]{alpha} and truncated exodomain lacking the endodomain were prepared. They were chemically cross-linked, and the resulting cross-linked complexes were solubilized and electrophoresed. The results indicate that the {alpha}-subunit of hCG was directly and specifically cross-linked to the exodomain. To verify the cross-linked exodomain by the independent method, the Flag epitope was inserted between the signal sequence and the mature exodomain. hCG containing [125I]{alpha} was cross-linked to the Flag exodomain, and the resulting cross-linked hCG/Flag exodomain complexes were immunoprecipitated with anti-Flag antibody. The results show that the material cross-linked to hCG containing [125I]{alpha} is indeed the exodomain. In conclusion, our results show the direct interaction of the {alpha}-subunit with the exodomain and, therefore, its crucial role in the hormone-receptor interaction in addition to its involvement in signal generation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
hCG, LH, FSH, and TSH are members of the glycoprotein hormone family and consist of two dissimilar subunits, {alpha} and ß (1). The {alpha}-subunit is common to all glycoprotein hormones, whereas the ß-subunit is hormone specific and, therefore, possesses the hormone specificity. LH and hCG recognize the same receptor (LHR). On the other hand, FSH and TSH have their own cognate receptors. In addition to the structural similarities, these hormones activate the same effectors, adenylyl cyclase and phospholipase C. Because the {alpha}-subunit and the hormone action are shared by all glycoprotein hormones, the {alpha}-subunit in hCG may in part be involved in the hormone action (2, 3).

The comparison of the structures of the hormones with the structures of their receptors is enlightening. The LHR and other glycoprotein hormone receptors belong to a structurally unique subfamily of G protein-coupled receptors. Unlike other receptor subfamily members, the glycoprotein hormone receptors comprise two structural and functional halves, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (4, 5, 6, 7). The exodomain is about 350 amino acids long, which alone is capable of high affinity hormone binding (8, 9, 10, 11) with hormone selectivity (12, 13, 14) but without hormone action (10, 15). Mutational analysis of the exodomain also shows its involvement in hormone binding (16, 17, 18). Receptor activation occurs in the endodomain (19), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (20). Existing evidence suggests that glycoprotein hormones initially bind to the exodomain with high affinity and hormone specificity, and then the resulting hormone/exodomain complex is thought to interact with the endodomain (19). This secondary interaction is considered to generate hormone signals (18, 19, 20).

Therefore, the contact sites of the initial and subsequent interactions are important, but they remain unclear. To investigate these issues, we launched a series of studies to determine which hormone subunits interact with which domains of the receptor. In an earlier article, we reported that a synthetic peptide corresponding to a sequence near the N-terminal region of the LHR exodomain was cross-linked to both the {alpha}- and ß-subunits of hCG (21). Although the result indicates the interaction of the peptide with hCG, the in vivo interaction of such sites was difficult to prove. The major problem was the lack of a suitable technology to show the physical interactions of each hormone subunit’s interaction with the exodomain or the endodomain. As a step to resolve the issue, the LHR exodomain lacking the endodomain was expressed and affinity labeled with the {alpha}-subunit in hCG in this work. This is an extension of our previous affinity labeling work, demonstrating that both of the hCG subunits interact with the receptor (22, 23). However, the previous work was unable to show whether the two hCG subunits interacted with the LHR exodomain, endodomain, or both.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis and expression of the exodomain of the LH/CG receptor
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 complementary (cDNAs) were produced in pSELECT vector containing the LH/CG receptor cDNA using the Altered Sites Mutagenesis System (Promega Corp., Madison, WI), sequenced, subcloned into pcDNA3 (Invitrogen, San Diego, CA) as described previously (24), 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 (HEK) 293 cells by the calcium phosphate method. Stable cell lines were established in minimum essential medium containing 10% horse serum and 500 µg/ml G-418. As the exodomains were trapped in the transfected cells and not secreted, the cells were solubilized in Nonidet P-40, which was assayed for [125I]hCG binding as previously described (25) and also used for affinity cross-linking. All assays were carried out in duplicate and repeated four or five times. The means ± SD were calculated. hCG (CR 127), hCG{alpha}, hCGß, FSH, and TSH were supplied by the National Hormone and Pituitary Program. Denatured hCG was prepared by boiling in 8 M urea, 2.7 M guanidine hydrochloride, and 100 mM dithiothreitol (DTT) for 20 min.

[125I]hCG binding to solubilized exodomains and LH/CG receptor
Transfected cells were washed twice with ice-cold 150 mM NaCl and 20 mM HEPES, pH 7.4 (buffer A). Cells were scraped on ice, collected in buffer A containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 5 mM N-ethylmaleimide, and 10 mM EDTA), and pelleted by centrifugation at 1300 x g for 10 min. Cells from a 10-cm plate were resuspended in 0.6 ml buffer A containing 1% Nonidet P-40, 20% glycerol, and the above protease inhibitors (buffer B); incubated on ice for 15 min; and diluted with 5.4 ml buffer A containing 20% glycerol plus the protease inhibitors (buffer C). The mixture was centrifuged at 100,000 x g for 60 min. The supernatant (500 µl) was mixed with 150,000 cpm [125I]hCG, 6.5 µl 0.9% NaCl, and 10 mM Na2HPO4 at pH 7.4 containing increasing concentrations of unlabeled hCG. After incubation at 4 C for 16 h, the solution was thoroughly mixed with 250 µl buffer A containing bovine {gamma}-globulin (5 µg/ml) and 750 µl buffer A containing 20% polyethylene glycol 8,000. After incubation at 4 C for 10 min, samples were pelleted at 1,300 x g for 30 min, and supernatants were removed. Pellets were resuspended in 1.5 ml buffer A, centrifuged, and counted for radioactivity. hCG was radioiodinated as described previously (23), and the specific activity was 2–4 x 108 cpm/8 µg hCG.

Affinity cross-linking of [125I]hCG to the LH/CG receptor
HEK 293 cells (120 µl) stably expressing the wild-type LHR were incubated with 150,000 cpm [125I]hCG at 37 C for 90 min. Then, 2.4 µl 50 mM bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (SES) in DMSO were added to each tube and incubated at room temperature for 20 min. SES is a homobifunctional cross-linking reagent (26) that has been used to cross-link hCG{alpha} to the LHR at the reagent concentration of 0.3 mM or more (27). The cross-linking reaction was terminated by adding 7.2 µl 100 mM glycine in PBS. Samples were boiled for 2 min in 2% SDS and 100 mM DTT to cleave disulfides and dissociate interacting components of complexes. The solubilized samples were electrophoresed on 8–10% polyacrylamide gradient gels to separate solubilized subunits, molecules, and cross-linked complexes. Gels were dried on filter paper and exposed to x-ray film for autoradiograph.

Affinity cross-linking of [125I]hCG to solubilized LH/CG receptor
Disposable glass tubes were siliconized under dimethyldichlorosilane vapor overnight and autoclaved. In each siliconized tube, 120 µl solubilized receptors were mixed with 150,000 cpm [125I]hCG and incubated at 4 C for 16 h. Then, 2.4 µl 50 mM SES in DMSO were added to each tube and incubated at room temperature for 20 min. The cross-linking reaction was terminated by adding 7.2 µl 100 mM glycine in PBS. Samples were boiled for 5 min in 2% SDS and 100 mM DTT. The solubilized samples were electrophoresed on 8–10% polyacrylamide gradient gels. Gels were dried on filter paper and exposed to a molecular imaging screen (Bio-Rad Laboratories, Inc., Richmond, CA) overnight. The imaging screen was scanned on a model GS-525 Molecular Imager System Scanner (Bio-Rad Laboratories, Inc.), and radioactive band profile was analyzed using Image Analysis Systems version 2.1 (Bio-Rad Laboratories, Inc.). Gels were exposed to X-Omat x-ray film (Eastman Kodak Co., Rochester, NY) at -75 C for about 4 days. Each experiment was repeated three to five times. Apparent molecular masses of the hCG subunits, cross-linked hCG{alpha}ß dimer, and labeled hormone/receptor complexes were estimated on the basis of the relative electrophoretic mobility of markers on various acrylamide gradient gels as described previously (27). This method predicts molecular masses more realistically than the usual one-gel method (27).

Immunoprecipitation of labeled Flag exodomains
For immunoprecipitation of exodomains, the Flag epitope, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, was inserted between the C-terminus (Ser26) of the signal sequence and the N-terminus (Arg27) of mature exodomain and receptor (28). Cells expressing Flag exodomains and Flag-LHR were solubilized as described above, mixed with 150,000 cpm [125I]hCG, and incubated at 4 C for 16 h. After incubation, affinity cross-linking was performed as described above. The samples were incubated with 25 µg/ml anti-Flag M2 antibody (Eastman Kodak Co.) for 4 h at 4 C and then incubated with 30 µl protein G-Sepharose on a rotating wheel at 4 C for 2 h. Resulting immune complexes were pelleted at 16,000 x g for 30 sec at 4 C. The pellets were washed twice with 1.5 ml buffer containing 1% Triton X-100, 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, and 1 mM CaCl2. Immune complexes were dissociated in 30 µl sample buffer containing 50 mM Tris base, 1 mM EDTA, 4% SDS, 100 mM DTT, 0.05% bromophenol blue, and 12% glycerol by boiling for 10 min. After pelleting protein G-Sepharose beads, the supernatant was electrophoresed on an 8–11% polyacrylamide gradient gel. Gels were dried and exposed to X-Omat x-ray film at -75 C for about 5 days.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
When hCG is radioiodinated, the {alpha}-subunit is labeled with [125I]iodine, but the ß-subunit is not, as shown in Fig. 1Go and earlier articles (23). Therefore, [125I]hCG has been routinely used for the affinity labeling of the LHR with the {alpha}-subunit of hCG. Cross-linking [125I]hCG bound to the LHR on the surface of intact cells or the exodomains solubilized in nonionic detergent solutions is termed affinity labeling of the receptor or exodomain with hCG. It requires cross-linking reagents. SES is a homobifunctional cross-linking reagent (26), containing two identical reactive groups, N-hydroxysuccinimides. These reactive groups of one reagent molecule can react with two free amino groups, and thus cross-link them, intramolecularly or intermolecularly. Such a chemically cross-linked complex cannot be separated by solubilization in SDS and, therefore, appears as a single band on gel electrophoresis (27). Conversely, the cross-linking-dependent appearance of new high mol wt bands on gel is an indication of the formation of cross-linked complexes.



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  		Figure 1. Cross-linking of [125I]hCG to the LHR expressed on intact cells. Human embryonic kidney 293 cells expressing the wild-type LHR were incubated with [125I]hCG, washed to remove unbound [125I]hCG, treated with increasing concentrations of SES, solubilized in SDS and DTT, and electrophoresed on polyacrylamide gel. Dried gel was autoradiographed, and the band intensity was analyzed using the Molecular Imager Analysis System as described inMaterials and Methods. Mol wt standards were determined as described in Materials and Methods. The percent radioactivities of the hCG{alpha}/LHRwt (106-kDa) complex and hCG{alpha}ß/LHRwt (136-kDa) complex were calculated by dividing the band intensity with the total band intensity of each gel lane. These percent radioactivities were plotted against the SES concentration as shown in the bar graph (top panel).

 
As samples are treated with increasing concentrations of SES, the extent of cross-linking increases. Among many amino groups, the closest pairs are most readily cross-linked, and distant pairs are slowly cross-linked. Consequently, the closest pairs of amino groups are cross-linked first, and distant pairs are cross-linked later. The extent of cross-links can usually be controlled by varying the reagent concentration or the cross-linking period (26). The latter is less efficient, however. Therefore, when hCG and the receptor are treated with increasing concentrations of SES, the {alpha}- and ß-subunits of hCG are cross-linked first to produce the hCG{alpha}ß dimer. Next, either the hCG{alpha} subunit or the hCGß subunit is cross-linked to the receptor. Finally, both of the two hCG subunits are cross-linked to the receptor to produce the trimeric complex. This incremental (stepwise) cross-link has been frequently reported for hCG and the LHR (27). Therefore, it has been a powerful tool to analyze the interaction of hCG and its receptor as well as interactions between other molecules and their subunits (26). In this article, the method was used, for the first time, to examine the interaction of hCG with the LHR exodomain.

Affinity labeling of the LHR with [125I]hCG
The cells expressing the LHR were incubated with [125I]hCG, treated with increasing concentrations of SES to cross-link [125I]hCG to the receptor, and solubilized in SDS and DTT to cleave disulfide bonds and dissociate noncovalently associated components of complexes. Solubilized samples were electrophoresed on polyacrylamide gel, which was dried and autoradiographed by exposure to x-ray film. When the cells expressing the LHR were incubated with [125I]hCG, solubilized in SDS and DTT without cross-linking in the absence of SES, and electrophoresed, the only radioactive band on the autoradiograph was the 20-kDa [125I]{alpha}-band (Fig. 1Go, gel lane 1). The radioactive band was previously proven to be [125I]hCG (23, 27, 29), indicating that only the hCG{alpha} subunit was radioiodinated. As [125I]hCG complexed to the cells was treated with increasing concentrations of SES, new bands appeared on the autoradiograph. At 0.03 mM SES, [125I]hCG{alpha} was cross-linked to [125I]hCGß to produce the 50-kDa [125I]{alpha}ß-dimer band. At 0.1 mM SES, [125I]hCG{alpha} was cross-linked to hCGß to produce the 50-kDa [125I]{alpha}ß-dimer band and also cross-linked to the 86-kDa receptor to produce the 106-kDa {alpha}/LHR complex. At 0.3 mM SES [125I]hCG{alpha} and hCGß were cross-linked to the 86-kDa receptor to produce the 136-kDa {alpha}ß/LHR complex in addition to the 50-kDa [125I]{alpha}ß-dimer band and the 106-kDa {alpha}/LHR band. During these incremental cross-links, the 50-kDa [125I]{alpha}ß-dimer band appeared first, whereas the intensity of the [125I]{alpha}-band decreased. Next, the 106-kDa band and the 136-kDa band appeared in order. The sequence of the appearance and disappearance of the latter two bands was interesting. As the SES concentration increased, the 106-kDa band appeared, and then the 136-kDa band appeared while the 106-kDa band faded. This pattern of cross-linking is a characteristic of the incremental cross-linking of interacting components. For example, [125I]{alpha} (20 kDa) is first cross-linked to the LHR (86 kDa) to produce the 106-kDa (20-kDa plus 86-kDa) band, and then hCGß (30 kDa) is cross-linked to the [125I]{alpha}/LHR complex (106 kDa) to produce the 136-kDa (20-kDa plus 30-kDa plus 86-kDa) band (27). The mol wt of hCG{alpha}, hCGß, and hCG{alpha}ß are 15, 23, and 38 kDa, respectively, but they appear as 20, 30, and 50 kDa on SDS-polyacrylamide gel due to glycosylation (27).

Binding and affinity labeling of truncated exodomains
To study the interaction of hCG with the exodomain of the LHR, two exodomains encompassing Arg1-Tyr295 (LHR295) and Arg1-Gly336 (LHR336) were expressed in HEK293 cells. These truncated exodomains were not transported to the cell surface or secreted into the cell culture medium. Therefore, the cells expressing them were solubilized in Nonidet P-40, and solubilized exodomains were used for [125I]hCG binding and affinity cross-linking. Solubilized LHR295 and LHR336 bound [125I]hCG, and their binding affinities were comparable to the binding affinity of the solubilized LHR (Fig. 2Go). This result indicates that the exodomains contain the high affinity hCG-binding site. To determine whether the {alpha}-subunit of hCG interacts with LHR336 and LHR295 and can be cross-linked to them, solubilized LHR295 and LHR336 as well as solubilized wild-type LHR were incubated with [125I]hCG. The incubation mixtures were treated with SES, then with SDS and DTT, and electrophoresed. On the autoradiograph (Fig. 3Go) of the gel, the LHR336 sample showed the 72- and 102-kDa bands in addition to the 20-kDa hCG{alpha} and 50-kDa hCG{alpha}ß bands. Similarly, the LHR295 sample showed the 70- and 100-kDa bands in addition to the 20-kDa hCG{alpha} and 50-kDa hCG{alpha}ß bands. Solubilized wild-type receptor was also labeled to produce the 106-kDa (hCG{alpha}/LHRwt) band and the 136-kDa (hCG{alpha}ß/LHRwt) band. However, the intensities of these bands were markedly less than the intensities of the same bands when wild-type receptors on intact cells were labeled (as shown in Fig. 1Go). In fact, the 136-kDa band is faint in the original autoradiograph and is not obvious in Fig. 3Go. These results underscore the difficulties in affinity labeling solubilized receptors. Despite the difficulties, the number of labeled bands is comparable with the result of [125I]hCG labeling of the wild-type LHR on intact cells. Therefore, the 72- and 102-kDa bands of the LHR336 sample and the 70- and 100-kDa bands of the LHR295 sample appear to correspond to the 106- and 136-kDa bands of the wild-type LHR sample.



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  		Figure 2. [125I]hCG binding to truncated exodomains. The exodomains lacking the endodomain (LHR295 and LHR336) were separately expressed in 293 cells, solubilized in Nonidet P-40, and assayed for [125I]hCG binding in the presence of increasing concentrations of unlabeled hCG. In addition, the wild-type receptor expressed in 293 cells were solubilized in Nonidet P-40 and assayed for [125I]hCG binding. The results are presented as the displacement of [125I]hCG binding (A) and a Scatchard plot (B). Experiments were repeated three times in duplicate, and means and SDs were calculated. Untransfected cells did not show specific binding of hCG.

 


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  		Figure 3. Autoradiograph of [125I]hCG cross-linked to LHR295, LHR336, and wild-type LHR. Cells expressing LHR295, LHR336, or wild-type LHR (LHRwt) and untransfected cells were solubilized in Nonidet P-40, incubated with [125I]hCG, treated with 1 mM SES, solubilized in SDS under reducing conditions, and electrophoresed. The gel was exposed to x-ray film and autoradiographed. Mol wt estimates of radioactive bands are presented in parentheses, and the apparent compositions of band materials are indicated with arrows.

 
There are bands between the hCG{alpha} band and the hCG{alpha}ß bands in Fig. 3Go and later figures. They are the degradation products of the cross-linked hormone-receptor complexes that occur in the detergent solution. They do not represent hCGß, although they are near the hCGß position, because the ß-subunit in hCG is not readily radioiodinated (23). Such proteolytic degradation is one of technical difficulties we have encountered in this work and can be partially overcome using inhibitors of proteolytic enzymes. The intensity of these bands increased significantly when the affinity-labeled samples were not immediately electrophoresed.

Composition of affinity-labeled [125I]hCG/LHR336complexes and dependence of labeling LHR336 on the concentrations of LHR336 and [125I]hCG
Next, we decided to analyze the composition of cross-linked [125I]hCG/LHR336 complexes. As alluded to, the progress of stepwise cross-linking can predict the composition of cross-linked complexes, and this can be accomplished by cross-linking samples with increasing concentrations of cross-linking reagents. For that purpose, LHR336 was incubated with [125I]hCG, treated with increasing concentrations of SES, solubilized in SDS and DTT, and electrophoresed. The autoradiograph of the gel (Fig. 4AGo) shows four major bands (the 20-kDa hCG{alpha}, 50-kDa hCG{alpha}ß, and 72- and 102-kDa bands). The appearance of the 50-kDa {alpha}ß and 72- and 102-kDa bands was stepwise, and the band intensities were distinct. The hCG{alpha} band was the only major band below 0.1 mM SES. In contrast, the hCG{alpha}ß band appeared at 0.3 mM SES, increased, and eventually became the predominant band at 0.1 mM SES or more. This result indicates that the hCG{alpha} band diminished as cross-links progressed, and the cross-linked hCG{alpha}ß dimer increased. This pattern is similar to the profile of cross-linking [125I]hCG to the LHR (Fig. 1Go). Next, the 72-kDa band appeared, followed by the 102-kDa band. As the 102-kDa band increased, the 72-kDa band diminished (Fig. 4AGo, bar graph). As [125I]hCG{alpha}, hCGß, and LHR336 are involved in this cross-linking, a simple explanation of the results is that [125I]{alpha} (20 kDa) was first cross-linked to a 52-kDa component to produce the 72-kDa (20- plus 52-kDa) band. Then, hCGß (30 kDa) was cross-linked to the 72-kDa complex of [125I]{alpha}/52-kDa component and/or hCG{alpha}ß (50 kDa) was cross-linked to the 52-kDa component, which produced the 102 kDa (20- plus 30- plus 52-kDa) band. Therefore, the 52-kDa component corresponds to LHR336, which has an apparent molecular mass of about 53.6 kDa including six chains of N-oligosaccharides (30) with terminal sialic acids (31). At SES concentrations of 3 mM or more, the radioactivity accumulated at top of the gel lanes, which probably represents [125I]hCG/LHR complex cross-linked to another exodomains (32). Such molecular complexes do not necessarily dissociate in nonionic detergent solutions. Although the percent intensities of the 72-kDa band and the 102-kDa band were relatively low, the labeling efficiency of hormone bound to LHR336 was 3- to 5-fold higher than the percent intensities indicate. This is because the radiolabeled hormone in the gel lanes includes unbound hormone.



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  		Figure 4. Cross-links of [125I]hCG to LHR336. A, SES concentration-dependent cross-linking. LHR336 solubilized in Nonidet P-40 was incubated with [125I]hCG, treated with increasing concentrations of SES, solubilized in SDS under reducing conditions, and electrophoresed on polyacrylamide gel. Dried gel was autoradiographed, and the band intensity was analyzed using the Molecular Imager Analysis System as described in Materials and Methods. The percent radioactivities of the hCG{alpha}/LHR336 complex and the hCG{alpha}ß/LHR336 complex were calculated by dividing the band intensity with the total band intensity of each gel lane. The percent radioactivities of the hormone/exodomain complexes are significantly lower in this and later figures compared with the percent radioactivities of the hormone/receptor complexes shown in Fig. 1Go, This is because the total radioactivities in these gel lanes include unbound [125I]hCG present in the samples of solubilized exodomains. In contrast, unbound [125I]hCG was removed from intact cells before electrophoresis in the experiment shown in Fig. 1Go. B, LHR336 concentration-dependent cross-links of [125I]hCG to LHR336. A constant amount of [125I]hCG was cross-linked to increasing amounts of LHR336 solubilized in Nonidet P-40 as described above. The amount of solubilized LHR336 was determined by Scatchard plots as shown in Fig. 2Go. C, [125I]hCG concentration-dependent cross-links. Increasing amounts of [125I]hCG were cross-linked to a constant amount of LHR336 solubilized in Nonidet P-40 as described above.

 
To determine whether the cross-links are specific for [125I]hCG and LHR336, they were cross-linked under increasing concentrations of LHR336 while maintaining the hCG concentration constant (Fig. 4BGo). The intensity of the hCG{alpha}/LHR336 and the hCG{alpha}ß/LHR336 bands increased at higher concentrations of LHR336. This result indicates that the band formation was dependent on the LHR336 concentration. Conversely, [125I]hCG and LHR336 were cross-linked at increasing concentrations of [125I]hCG and a constant concentration of LHR336 (Fig. 4CGo). Again, the intensities of the two complex bands increased after increasing concentrations of [125I]hCG. The result suggests that the formation of the hCG{alpha}/LHR336 and hCG{alpha}ß/LHR336 complexes was also dependent on the concentration of [125I]hCG. The percentage of the hCG{alpha}ß/LHR336 band peaked at 3.9 ng hCG and declined at concentrations over 3.9 ng hCG. Therefore, the LHR336 concentration appeared to become a limiting factor in the complex formation, because the complex formation peaked and declined even if [125I]hCG was in excess.

Hormone specificity of affinity-labeled LHR336
hCG binds the LHR with high affinity. However, FSH and TSH recognize the LHR with very low affinities, although these glycoprotein hormones share the common {alpha}-subunit, are glycosylated, and have similar mol wt. Therefore, to test the hormone specificity of the affinity labeling, LHR336 was labeled with [125I]hCG in the presence of an excess amount of unlabeled hCG, denatured hCG, FSH, or TSH (Fig. 5Go). Untreated hCG completely abrogated the affinity labeling of LHR336. However, denatured hCG, FSH, and TSH did not prevent the affinity labeling. The fact that FSH and TSH were able to somewhat attenuate the affinity labeling is consistent with the low affinity cross-activity of FSH with hCG. These results show that active hCG, but not denatured hCG, specifically labeled LHR336.



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  		Figure 5. Hormone specificity of [125I]hCG cross-linking to LHR336. [125I]hCG (150,000 cpm) and LHR336 were incubated in the presence of a 100-fold higher concentration of unlabeled hCG, denatured hCG (de-hCG), or FSH. The incubation mixtures were treated with SES and processed as described in Fig. 4Go.

 
Identification of the [125I]hCG-labeled materials by immunoprecipitation
To date, our data are consistent with the view that LHR336 was affinity labeled with [125I]{alpha}-subunit of hCG. However, it is desirable to positively identify the affinity-labeled materials. For this purpose, we decided to use highly specific monoclonal antibody prepared against the LHR. Because all antibodies generated against the LHR show some nonspecificity, the Flag epitope (33), Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, was inserted between the C-terminus of the signal sequence and the N-terminus of mature receptors. The resulting receptors were Flag-wild-type LHR (Flag-LHRwt), Flag-LHR336, and Flag-LHR295. In a previous article, we demonstrated that the N-terminal Flag epitope did not interfere with the activity or expression of the LHR (25).

In this work, the Flag-LHRs were affinity labeled with [125I]hCG and immunoprecipitated with high affinity monoclonal anti-Flag antibody. Flag-LHR336 was incubated with [125I]hCG, treated with SES, and immunoprecipitated with anti-Flag antibody using protein G conjugated to Sepharose. After extensively washing the Sepharose, the immunoprecipitated material was solubilized in SDS and DTT and electrophoresed. The autoradiograph of the gel (Fig. 6AGo, lane 6) shows the 72- and 102-kDa bands, indicating that the band materials comprise Flag-LHR336 and [125I]hCG{alpha}. The immunoprecipitation of the 72- and 102-kDa bands requires [125I]hCG, Flag-LHR336, SES, anti-Flag antibody, and protein G. The bands could not be immunoprecipitated if any one of them was omitted. Furthermore, LHR336 lacking the Flag epitope was not immunoprecipitated, although all the factors except Flag-LHR336 were present (Fig. 6BGo). This result unequivocally demonstrates that the immunoprecipitation was specific for the Flag epitope and that [125I]hCG{alpha} was cross-linked to the Flag carrying material for immunoprecipitation. As a positive control, Flag-LHRwt was successfully immunoprecipitated (Fig. 7AGo). The 106-kDa hCG{alpha}/Flag-LHR336 and the 136-kDa hCG{alpha}ß/Flag-LHR336 bands appeared only when [125I]hCG, Flag-LHRwt, SES, anti-Flag antibody, and protein G were all included in the immunoprecipitation. Again, the LHRwt lacking the Flag epitope was not immunoprecipitated (Fig. 7BGo). Flag-LHR295 was also immunoprecipitated (Fig. 7CGo). These results of the immunoprecipitation experiments including extensive positive and negative controls indicate that the immunoprecipitated 72- and 102-kDa band materials shown in Fig. 6AGo consist of [125I]hCG{alpha} and LHR336. Therefore, the 72-kDa complex has to be composed of [125I]hCG{alpha} (20 kDa) and LHR336 (52 kDa). Similarly, the 102-kDa complex comprises [125I]hCG{alpha}ß dimer (50 kDa) and LHR336 (52 kDa). This conclusion is also in accord with the composition of the affinity-labeled complexes of slightly smaller LHR295 (50 kDa).



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  		Figure 6. Immunoprecipitation of [125I]hCG and Flag-LHR336 complexes. A, Lane 1, [125I]hCG in the absence of LHR336 and Flag-LHR336 was treated with SES, solubilized, electrophoresed, and processed as described in Fig. 4Go. Lane 7, [125I]hCG and Flag-LHR336 were incubated, treated with SES, solubilized, electrophoresed without immunoprecipitation, and processed as described in Fig. 4Go. Lanes 2–6, [125I]hCG and Flag-LHR336 were incubated, treated with SES, and immunoprecipitated using monoclonal anti-Flag antibody (anti-Flag) and protein G-Sepharose (protein G) as described in Materials and Methods. In control samples, [125I]hCG, Flag-LHR336, SES, anti-Flag antibody, or protein G-Sepharose was omitted, as indicated by a dash. B, Lane 1, [125I]hCG in the absence of LHR336 and Flag-LHR336 was treated with SES, solubilized, electrophoresed, and processed as described in Fig. 4Go. Lanes 2–6, [125I]hCG and LHR336 were immunoprecipitated as described in Fig. 6AGo.

 


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  		Figure 7. Immunoprecipitation of [125I]hCG complexed to Flag-LHRwt and Flag-LHR295. Experiments were the same as those shown in Fig. 6Go, except that Flag-LHRwt, LHRwt, and Flag-LHR295 were used instead of Flag-LHR336 in A and LHR336 in B. A, Lane 1, [125I]hCG was treated with SES, solubilized, electrophoresed, and processed as described in Fig. 6Go. Lane 7, [125I]hCG and Flag-LHRwt were incubated, treated with SES, solubilized, electrophoresed without immunoprecipitation, and processed as described in Fig. 4Go. Lanes 2–6, [125I]hCG and Flag-LHRwt were incubated, treated with SES, and immunoprecipitated using monoclonal anti-Flag antibody (anti-Flag) and protein G-Sepharose (protein G) as described in Materials and Methods. In control samples, [125I]hCG, Flag-LHRwt, SES, anti-Flag antibody, or protein G-Sepharose was omitted, as indicated by a dash. B, Lane 1, [125I]hCG was treated with SES, solubilized, electrophoresed, and processed as described in Fig. 4Go. Lane 7, [125I]hCG and LHRwt were incubated, treated with SES, solubilized, electrophoresed, and processed as described in Fig. 4Go. Lanes 2–6, [125I]hCG and LHRwt were immunoprecipitated as described in Fig. 6AGo. C, Lane 1, [125I]hCG was treated with SES, solubilized, electrophoresed, and processed as described in Fig. 6Go. Lane 7, [125I]hCG and Flag-LHR295 were incubated, treated with SES, solubilized, electrophoresed, and processed as described in Fig. 4Go. Lanes 2–6, [125I]hCG and Flag-LHR295 were incubated, treated with SES, and immunoprecipitated using monoclonal anti-Flag antibody (anti-Flag) and protein G-Sepharose (protein G) as described in Materials and Methods. In control samples, [125I]hCG, Flag-LHR295, SES, anti-Flag antibody, or protein G-Sepharose was omitted, as indicated by a dash.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of affinity-labeling and immunoprecipitation experiments show that [125I]hCG specifically interacted with and affinity-labeled LHR336. The labeling was hormone specific and saturable. It required functionally active hCG and cross-linking reagent. Furthermore, the affinity labeling indicates the direct contact of the [125I]{alpha}-subunit of hCG with LHR336.

Amino acids of hCG{alpha} involved in cross-linking to the exodomain
There are seven amino groups in hCG{alpha} and five in hCGß that can be cross-linked by the amino-specific reagent, SES (Table 1Go). In hCG{alpha}, the amino groups are present at the N-terminus, Lys44, Lys45, Lys51, Lys63, Lys75, and the penultimate C-terminal Lys91 (34). The crystal structure of hCG shows that the {alpha}-subunit comprises three loops formed by Cys knots and the N- and C-terminal segments (35). The N-terminal segment and {alpha}-loop 1 are in the rear of the putative receptor-binding phase. Therefore, the N-terminal amino group is unlikely to be cross-linked to the receptor. On the other hand, {alpha}-loops 2 and 3 and the {alpha}C-terminal segment are in the putative receptor-binding phase. In particular, the {alpha}C-terminal segment and {alpha}-loop 2 are at the center of the receptor-binding site. In fact, there is evidence that the {alpha}C-terminal segment interacts with an undefined part of the receptor (36). Among Lys44, Lys45, and Lys51 in the {alpha}-loop 2, the side-chains and their amino groups of Lys45 and Lys51 are exposed to the surface of the putative receptor binding site, whereas the side chain of Lys44 projects away from the site. This is consistent with previous reports on amino group labeling, accessibility, and cross-linking, as summarized by Grodon and Ward (37). Therefore, Lys45 and Lys51 are likely to be cross-linked to the exodomain, whereas Lys44 is less likely to be cross-linked to the exodomain. Interestingly, three tandem hydrophobic amino acids (Met47-Leu48-Val49) are present between Lys45 and Lys51, and all three are exposed to the surface of the receptor-binding phase. The surface exposure of such a hydrophobic peptide segment suggests its involvement in the protein-protein interaction, i.e. the interaction with the receptor in this case. Lys63 and Lys75 in {alpha}-loop 3 are distant from the putative receptor-binding site, being in the periphery of the putative receptor-binding phase. Therefore, the most likely candidates for the {alpha}Lys residues cross-linked to the exodomain are Lys45 and Lys51 in {alpha}-loop 2 and the penultimate C-terminal Lys91. It has been speculated that a primary hCG-binding site of the exodomain is the putative crescent structure (16, 38, 39), which is comprised of Leu-rich motifs (40). It will be interesting to determine which of Lys45, Lys51, and Lys91 are involved in binding to the exodomain, perhaps cross-linking them to the exodomain.


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  		Table 1. Distances between amino groups of hCG{alpha} and hCGß

 
Intersubunit cross-linking
The interaction and intersubunit cross-link between the hCG{alpha} and hCGß subunits has an important implication in the relationship of the hCG structure with the initial and secondary interactions of hormone receptor and signal generation. Therefore, it will be examined here. As described above, hCG{alpha} has seven amino groups that can react with SES. On the other hand, hCGß has five amino groups at the ßN-terminus, ßLys20, ßLys104, and ßLys122. ßLys122 is in the C-terminal extension of hCGß that is not involved in the hormone’s function. It is also unnecessary for the interactions between the two subunits of hCG and between hCG and the receptor. The {alpha}N-terminus is near the ßN-terminus and ßLys2 and, therefore, is likely to be involved in the intersubunit cross-link. {alpha}Lys44, {alpha}Lys45, and {alpha}Lys51 in {alpha}-loop 2 are within a cross-linkable distance from ßLys104, but could not be cross-linked to it because other groups fill the spaces between them. The three Lys residues are too far away from the ßN-terminus and ßLys2 to be cross-linked. {alpha}Lys44 and {alpha}Lys45 are within the cross-linkable distance from ßLys20. By contrast, {alpha}Lys63 and {alpha}Lys75 in {alpha}-loop 3 and {alpha}Lys91 are beyond the cross-linkable distance from ßLys20. Therefore, intersubunit cross-links will be limited from {alpha}Lys44 and {alpha}Lys45 to ßLys2 and from the {alpha}N-terminus to the ßN-terminus and ßLys2 (Table 1Go). In hCG{alpha}, there are pairs of amino groups that are within the cross-linkable distance. They are {alpha}Lys44 and {alpha}Lys45 as well as {alpha}Lys44 and {alpha}Lys51. Although the position of {alpha}Lys91 is not defined in the crystal structure, {alpha}Lys91 might be cross-linked to {alpha}Lys51 and {alpha}Lys63 if {alpha}Lys91 is freely mobile as suspected. These conclusions are consistent with the results of previous chemical modification studies (37, 41, 42).


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

Received October 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pierce JG, Parson TS 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Chang K-W, Glazer AN, Pierce JG 1973 the effects of modification of the COOH terminal regions of bovine thyrotropin and its subunits. J Biol Chem 248:7930–7937[Abstract/Free Full Text]
  3. Yoo J, Zeng H, Ji I, Murdoch WJ, Ji TH 1993 COOH-terminal amino acids of the {alpha} subunit play common and different roles in human choriogonadotropin and follitropin. J Biol Chem 268:13034–13042[Abstract/Free Full Text]
  4. McFarland K, Sprengel R, Phillips H, et al 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494–499[Abstract/Free Full Text]
  5. Loosfelt H, Misrahi M, Atger M, et al 1989 Cloning and sequencing of porcine LH-CG receptor cDNA: variants lackings transmembrane domain. Science 245:525–528[Abstract/Free Full Text]
  6. Nagayama Y, Kaufman KD, Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184–1190[CrossRef][Medline]
  7. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol 4:525–530[Abstract]
  8. Tsai-Morris CH, Buczko E, Wang W, Dufau ML 1990 Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity. J Biol Chem 265:19385–19388[Abstract/Free Full Text]
  9. Xie YB, Wang H, Segaloff DL 1990 Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265:21411–21414[Abstract/Free Full Text]
  10. Ji I, Ji TH 1991 Exons 1–10 of the rat LH receptor encode a high affinity hormone binding site and exon 11 encodes G-protein modulation and a potential second hormone binding site. Endocrinology 128:2648–2650[Abstract]
  11. Davis D, Liu X, Segaloff D 1995 Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function. Mol Endocrinol 9:159–170[Abstract]
  12. Braun T, Schofield PR, Sprengel R 1991 Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity. EMBO J 10:1885–1190[Medline]
  13. Moyle WR, Campbell RK, Myers RV, Bernard MP, Han Y, Wang X 1994 Co-evolution of ligand-receptor pairs. Nature 368:251–255[CrossRef][Medline]
  14. Liu X, DePasquale JA, Griswold MD, Dias JA 1994 Accessibility of rat and human follitropin receptor primary sequence (R265–S296) in situ. Endocrinology 135:682–691[Abstract]
  15. Remy JJ, Bozon V, Couture L, Goxe B, Salesse R, Garnier J 1993 Reconstitution of a high-affinity functional lutropin receptor by coexpression of its extracellular and membrane domains. Biochem Biophys Res Commun 193:1023–1030[CrossRef][Medline]
  16. Bhowmick N, Huang J, Puett D, Isaacs NW, Lapthorn AJ 1996 Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. Mol Endocrinol 10:1147–1159[Abstract]
  17. Thomas D, Rozell T, Liu X, Segaloff D 1996 Mutational anlayses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repeats 1–6 are involved in hormone binding. Mol Endocrinol 10:760–768[Abstract]
  18. Dufau ML 1998 The luteinizing hormone receptor. Annu Rev Physiol 60:461–496[CrossRef][Medline]
  19. Ji TH, Murdoch WJ, Ji I 1995 Activation of membrane receptors. Endocrine 3:187–194
  20. Ji TH, Grossmann M, Ji I 1998 G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem 273:17299–17302[Free Full Text]
  21. Phang T, Kundu G, Hong S, Ji I, Ji TH 1998 The amino-terminal region of the lutropin/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. II. Photoaffinity labeling. J Biol Chem 273:138401–13847
  22. Ji I, Ji TH 1980 Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells. Proc Natl Acad Sci USA 77:7167–7170[Abstract/Free Full Text]
  23. Ji I, Ji T 1981 Both {alpha} and ß subunits of human choriogonadotropin photoaffinity label the hormone receptor. Proc Natl Acad Sci USA 78:5465–5469[Abstract/Free Full Text]
  24. Ji I, Ji TH 1993 Receptor activation is distinct from hormone binding in intact lutropin-choriogonadotropin receptors and Asp397 is important for receptor activation. J Biol Chem 268:20851–20854[Abstract/Free Full Text]
  25. Hong S, Phang T, Ji I, Ji TH 1998 The amino-terminal region of the lutropin/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. I. Mutational analysis. J Biol Chem 273:13835–13840[Abstract/Free Full Text]
  26. Ji TH 1983 Bifunctional reagents. Methods Enzymol 91:580–609[Medline]
  27. Ji I, Bock J, Ji TH 1985 Composition and peptide maps of cross-linked human choriogonadotropin-receptor complexes on porcine granulosa cells. J Biol Chem 260:12815–12821[Abstract/Free Full Text]
  28. Hong S, Ryu K-S, Oh M-O, Ji I, Ji TH 1997 Roles of transmembrane prolines and proline-induced kinks of the lutropin/choriogonadotropin receptor. J Biol Chem 272:4166–4171[Abstract/Free Full Text]
  29. Ji I, Yoo B, Kaltenbach C, Ji T 1981 Structure of the lutropin receptor on granulosa cells: photoaffinity labeling with the {alpha} subunit in human choriogonadotropin. J Biol Chem 256:10853–10858[Abstract/Free Full Text]
  30. Davis DP, Rozell TG, Liu X, Segaloff DL 1997 The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not absolutely required for correct folding, cell surface expression, hormone binding, or signal transduction. Mol Endocrinol 11:550–562[Abstract/Free Full Text]
  31. Okada Y, Ji I, Nishimura R, Ji TH 1988 Carbohydrates of the lutropin receptor. In: Mochizuki M, Hussa R (eds) Placental Protein Hormones. Excerpta Medica, New York, pp 269–278
  32. Osuga Y, Hayashi M, Kudo M, Conti M, Kobilka B, Hsueh A 1997 Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation. J Biol Chem 272:25006–25012[Abstract/Free Full Text]
  33. Hopp T, Prikett K, Price V, et al 1988 A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6:1205–1210
  34. Morgan F, Birken S, Canfield R 1975 The amino acid sequence of human chorionic gonadotropin; the {alpha} subunit and ß subunit. J Biol Chem 250:5247–5258[Abstract/Free Full Text]
  35. Lapthorn JP, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human chorionic gonadotropin. Nature 369:455–461[CrossRef][Medline]
  36. Kundu GC, Ji I, McCormick DJ, Ji TH 1996 Photoaffinity labeling of the lutropin receptor with synthetic peptide for the carboxyl terminal of the human choriogonadotropin {alpha} subunit. J Biol Chem 271:11063–11066[Abstract/Free Full Text]
  37. Gordon WL, Ward DN 1985 Structural aspects of luteinizing hormone actions. In: Ascoli M (ed) Luteinizing Hormone Action and Receptors. CRC Press, Boca Raton, pp 173–197
  38. Jiang X, Dreno M, Buckler D, et al 1995 Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure 3:1341–1353[Medline]
  39. Couture L, Naharisoa H, Grebert D, et al 1996 Peptide and immunochemical mapping of the ectodomain of the porcine LH. J Mol Endocrinol 16:15–25[Abstract]
  40. Kobe B, Deisenhofer, J 1994 The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19:415–421[CrossRef][Medline]
  41. Yadav SP, Brew K, Puett D 1994 Holoprotein formation of human chorionic gonadotropin; differential trace labeling with acetic anhydride. Mol Endocrinol 8:1547–1558[Abstract]
  42. Yadav SP, Brew K, Majercik MH, Puett D 1994 A label selection approach to assess the role of individual amino groups in human chorionic gonadotropin receptor binding. J Biol Chem 269:3991–3998[Abstract/Free Full Text]



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