©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Photoaffinity Labeling of the Lutropin Receptor with Synthetic Peptide for Carboxyl Terminus of the Human Choriogonadotropin Subunit (*)

(Received for publication, January 17, 1996)

Gopal C. Kundu (1)(§) Inhae Ji (1) Daniel J. McCormick (2) Tae H. Ji (1)(¶)

From the  (1)Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944 and (2)Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alphaC-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alphaC-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His to Ser (alpha) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-I-alpha was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-I-alpha exclusively photoaffinity-labeled an 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alphaLys of alpha is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.


INTRODUCTION

hCG (^1)is a placental hormone and is involved in maintenance of the corpus luteum during pregnancy in human females. It is a member of the glycoprotein hormone family which includes LH, FSH, TSH and equine choriogonadotropin. These hormones consist of a common alpha subunit and a distinct beta subunit which are noncovalently associated(1) .

Photoaffinity labeling studies demonstrated that both alpha and beta subunits affinity-labeled the LH/CG receptor(2) . The truncation or substitution of hCGalpha C-terminal amino acid residues reduces the receptor-binding affinity and abolishes cAMP induction(3, 4, 5, 6) . This is consistent with the observation that a dodecamer peptide corresponding to the hCGalpha C-terminal region, alpha, inhibited I-hCG binding to the receptor (7) and that a decamer peptide, alpha, was capable of binding to cells possessing the LH/CG receptor and inducing cAMP synthesis(6) . Also, the crystal structure of HF treated hCG suggests the alphaC-terminal region as part of the potential receptor binding site(8, 9) . Recently, a study using a set of reciprocal mutants of hCG and the LH/CG receptor, hCG


EXPERIMENTAL PROCEDURES

Materials

hCG CR127 was supplied by the National Hormone and Pituitary Program. Peptides were synthesized by Multiple Peptide System (San Diego, CA) and purified as described previously(7) . The N-hydroxysuccinimide ester of 4-azidobenzoylglycine (NHS-ABG) was synthesized, alpha was derivatized with NHS-ABG, and it was radioiodinated as described previously(11) . The resulting ABG-I-alpha was fractionated on a Sephadex G-10 column. It is important to carefully monoiodinate the peptide and preserve its biological activity during the harsh iodination reaction steps.

Binding, cAMP Induction, and Photoaffinity Labeling

Human embryonic kidney 293 cells were transfected with the LH/CG receptor construct as described previously(10) . Cells transiently expressing the LH/CG receptor (293) were incubated with I-alpha, ABG-I-alpha or I-NAc/CONH(2)-alpha in the presence of increasing concentrations of alpha. After washing cells three times until nonspecific binding was insignificant, the radioactivity of bound I-alpha, ABG-I-alpha or I-NAc/CONH(2)-alpha, was determined. Heat denatured fetal calf serum and bovine serum albumin, 15 and 3%, respectively, were used to reduce nonspecific binding. For cAMP assay, 293 cells were incubated with increasing concentrations of alpha, ABG-alpha or NAc/CONH(2)-alpha and intracellular cAMP was determined as described previously(10) . For photoaffinity labeling, 293 cells were incubated with ABG-I-alpha and washed three times. The cells were irradiated with an ACME-LITE model 228A xenon flash lamp (12) . UV light from the flashes are capable of activating UV sensitive groups such as the arylazide of ABG. Each flash lasts 0.2 ms and minimizes random collisional crosslinks and heating of cells(12) . The irradiated cells were solubilized in 3% sodium dodecylsulfate and 100 mM dithiothreitol. The samples were electrophoresed on polyacrylamide gels which were dried on filter paper and exposed to x-ray film.

Affinity Cross-linking of I-NAc/CONH^2-alpha

Transiently transfected 293 cells were incubated with I-NAc/CONH(2)-alpha, and washed to remove nonspecifically bound and unbound peptide as described above. The cells were incubated with 1 mM EDC at room temperature for 20 min for cross-linking, washed, and solubilized for gel electrophoresis. To examine the reaction specificity of EDC, the cells were pretreated with the N-hydroxysuccinimde ester of caproic acid (13) for 30 min or 100 mM of sodium acetate and treated with EDC. Gels were dried and exposed to x-ray film.

Immunoprecipitation

LH/CG receptors on 293 cells were photoaffinity labeled with ABG-I-alpha as described above. They were washed three times on ice with ice cold 150 mM NaCl, 20 mM HEPES, pH 7.4. Cells were scraped in ice cold 150 mM NaCl, 20 mM HEPES (pH 7.4), containing protease inhibitors 30 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 1 mM EDTA. After pelleting cells by centrifugation at 1300 times g for 10 min, pellets were resuspended in 0.5 ml of the buffer containing 1% Triton X-100, 30% glycerol and the protease inhibitors, incubated on ice for 45 min, and diluted with 1.5 ml of the buffer containing 0.1% Triton X-100, 30% glycerol and the protease inhibitors. The mixture was centrifuged at 100,000 times g for 30 min and the supernatant was incubated with normal rabbit sera overnight at 4 °C. The incubation mixture was further incubated with goat anti-rabbit antibody-Sepharose for 2 h and centrifuged 300 times g for 3 min. This precleared supernatant was incubated with rabbit anti-LH/CG receptor antiserum and immunoprecipitated with goat anti-rabbit antibody-Sepharose. The Sepharose was washed several times and boiled in sodium dodecyl sulfate under the reducing condition for 5 min and centrifuged at 300 times g for 3 min. The solubilized supernatant was electrophoresed and the gel was processed for autoradiography. In a control sample, the precleared pellet of goat antirabbit antibody-Sepharose was boiled in sodium dodecyl sulfate for 5 min and centrifuged 300 times g for 3 min. The solubilized supernatant was electrophoresed on polyacrylamide gel.


RESULTS AND DISCUSSION

As shown in Fig. 1, I-alpha, ABG-I-alpha and I-NAc/CONH(2)-alpha bound to the LH/CG receptor on 293 cells with K(d) values of 126 µM (p < 0.05), 264 µM (p < 0.05) and 253 µM (p < 0.05), respectively. They were capable of inducing cAMP synthesis with EC values of 86 µM (p < 0.05), 135 µM (p < 0.05) and 100 µM, respectively. These results demonstrate the bioactivity of the peptide and its derivatives. To determine the identity of their binding molecule, increasing numbers of 293 cells were incubated with I-ABG-alpha, irradiated with xenon flashes, and electrophoresed. The autoradiogram in Fig. 2shows free I-ABG-alpha which was originally bound to 293 cells but dissociated from them during solubilization in sodium dodecylsulfate. In addition to the free I-ABG-alpha band, a 86-kDa band was labeled by I-ABG-alpha. The intensity of bound I-ABG-alpha and the labeled 86-kDa band increased in parallel with increasing numbers of 293 cells. In contrast, the band was absent in the sample lacking 293 cells. In addition to this 293 cell dependence, the formation of the 86-kDa band requires photolysis as it is dependent on the number of xenon flashes and I-ABG-alpha bound to 293 cells failed to produce the 86-kDa band when the sample was not photolyzed. These results suggest that the radioactive 86-kDa band was generated by the photoactivation of I-ABG-alpha and photoaffinity labeling of a 86-kDa molecule. The band was not produced by nonspecific association of I-ABG-alpha with a 86-kDa molecule. Furthermore, the data indicate that I-ABG-alpha exclusively labeled a 86-kDa molecule on 293 cells expressing the LH/CG receptor. As expected, the size of the 86-kDa band material corresponds to the mature 85-86-kDa LH/CG receptor(13, 14) .


Figure 1: Biological activities of alpha, ABG-alpha and NAc/CONH(2)-alpha. For the receptor binding assay, cells expressing the LH/CG receptor (293) were incubated with a constant amount of I-alpha, I-ABG-alpha or I-NAc/CONH(2)-alpha in the presence of increasing concentrations of alpha. After washing cells, the cell-bound radioactivity was measured. The data were used in Scatchard plots to determine K values. For cAMP induction, cells expressing the LH/CG receptor were incubated with increasing concentrations of alpha, ABG-alpha or NAc/CONH(2)-alpha and intracellular cAMP concentrations were determined. These experiments were repeated three times, each in triplicate. The data were analyzed using Student's t test.




Figure 2: Photoaffinity labeling. Increasing concentrations of 293 cells were incubated with I-ABG-alpha and irradiated with xenon flashes. Cells were solubilized and electrophoresed. The gel was exposed to x-ray film. The lower band represents I-ABG-alpha which was originally bound to 293 cells but dissociated during solubilization in sodium dodecyl sulfate. The 86-kDa band represents the photoaffinity labeled band material.



Fig. 3demonstrates the specificity of the photoaffinity labeling. I-ABG-alpha did not bind to mock transfected (293) cells lacking the LH/CG receptor and did not form the 86-kDa band even after UV photolysis. This result indicates the requirement of the LH/CG receptor for I-ABG-alpha binding and labeling the 86-kDa band. Unlabeled hCG or alpha, but not FSH or TSH, blocked I-ABG-alpha binding to 293 cells, another indication of the specificity of I-ABG-alpha binding to the LH/CG receptor. Furthermore, alpha or beta failed to block I-ABG-alpha binding to 293 cells. Since alpha(7) and beta(15) inhibit I-hCG binding to the LH/CG receptor, this result suggests that there is an exclusive and specific binding site for I-ABG-alpha in the LH/CG receptor.


Figure 3: Specificity of photoaffinity labeling. Cells expressing (293) or lacking (293) the LH/CG receptor were incubated with I-ABG-alpha or I-alpha in the presence or absence of 1000 times concentrations of competitors. After irradiation with 15 xenon flashes, cells were solubilized and electrophoresed. The gel was exposed to x-ray film.



Taken together these results clearly indicate I-ABG-alpha binding to the LH/CG receptor. Therefore, they suggest that the 86-kDa band represents photoaffinity labeled LH/CG receptors. To obtain more direct evidence for this conclusion, the photoaffinity labeled band material was immunoprecipitated using rabbit anti-LH/CG receptor antiserum (Fig. 4). When 293 cells were incubated with I-ABG-alpha, photolyzed, solubilized in Triton X-100, and immunoprecipitated with anti-LH/CG receptor antiserum, the 86-kDa-band was precipitated (Fig. 4). However, it was not precipitated when normal rabbit sera was used, when 293 cells were processed in the absence of I-ABG-alpha, or when 293 cells were used instead of 293 cells. These results, along with the photoaffinity labeling data in Fig. 1Fig. 2Fig. 3, positively identify the photoaffinity-labeled 86-kDa band as the LH/CG receptor.


Figure 4: Immunoprecipitation of affinity-labeled LH/CG receptor. Cells, 293 or 293, were incubated with I-ABG-alpha, washed and irradiated with 15 xenon flashes. Cells were treated with Triton X-100 solution and the detergent extracts were incubated with rabbit anti-LH/CG receptor or normal rabbit sera for immunoprecipitation.



I-ABG-alpha has two amino groups which could have been derivatized with ABG. They are the amino group of Lys and the N-terminal amine. Our data cannot specify whether or not the amino group of Lys was involved in the photoaffinity labeling of the receptor. Furthermore, it is difficult to predict the chemical group(s) of the receptor which was labeled as the phenyl nitrene reacts nonspecifically with a variety of functionalities (16) . Since this information is necessary to define the cross-linked point of the peptide and the receptor, we have synthesized I-NAc/CONH(2)-alpha of which the N-terminal amine of alpha was acetylated and the C-terminal carboxylate was amidated. This modified peptide was used for affinity labeling the receptor. Transiently transfected 293 cells were incubated with I-NAc/CONH(2)-alpha and washed to remove unbound peptide. The cells complexed with I-NAc/CONH(2)-alpha were treated with EDC and solubilized for gel electrophoresis. The sample treated with EDC showed the 86-kDa band of the receptor which was labeled with I-NAc/CONH(2)-alpha (Fig. 5). FSH or TSH did not prevent binding and cross-linking of I-NAc/CONH(2)-alpha. On the other hand, hCG or unlabeled alpha blocked binding of I-NAc/CONH(2)-alpha to the cells. Also I-NAc/CONH(2)-alpha did not bind to 293 (data not shown). These results indicate the specificity of I-NAc/CONH(2)-alpha binding to the LH/CG receptor. Carbodiimides including EDC react with carboxyl groups to produce O-acylisoureas which in turn react with amino groups to produce amide bonds(16) . Therefore, the primary reactive group of I-NAc/CONH(2)-alpha with EDC is the amino group of Lys. To verify this reaction specificity the cells complexed with I-NAc/CONH(2)-alpha were treated with the N-hydroxysuccinimde ester of caproic acid which monofunctionally reacts with amino group(13) . As expected, it prevented cross-linking of I-NAc/CONH(2)-alpha to the receptor as did acetate. These data indicate that the cross-linking reaction of EDC involves both amino and carboxyl groups, consistent with the reaction specificity of carbodiimides(16) . Carbodiimides may also react with phenolic groups of Tyr and sulfhydryls of Cys. These reactions are, however, monofunctional and therefore, do not produce cross-linked products(17) . Our data indicate that the amino group of I-NAc/CONH(2)-alpha is cross-linked to a carboxyl group to form an amide. They suggest that the amino group and side chain of hCG alphaLys is in the proximity of a carboxyl group of the receptor in the hormone receptor complex. EDC induced a zero length cross-link between these two counter ions suggests the existence of a salt bridge between them. This conclusion is consistent with the complementarity of hCG alphaLys and LH/CG receptor's Asp(10) .


Figure 5: Affinity cross-linking. Cells, 293, were incubated with I-NAc/CONH(2)-alpha in the presence or absence of 1000 times concentrations of FSH or TSH. After washing cells were treated with EDC, solubilized and electrophoresed. The gel was exposed to x-ray film. As controls, cells complexed with I-NAc/CONH(2)-alpha were treated with EDC in the presence of sodium acetate, a carboxyl specific competitor for EDC induced cross-link, or the N-hydroxysuccinimide of caproic acid, a monofunctional reagent specific to amino groups. The cells did not bind I-NAc/CONH(2)-alpha in the presence of hCG or unlabeled alpha. Also 293 cells did not bind I-NAc/CONH(2)-alpha. Since these sample lanes were blank, the data are not shown.



The binding and labeling site of I-ABG-alpha appears to be specific since it was blocked by alpha, but not by two other peptides, alpha and beta which are known to inhibit I-hCG binding to the receptor. This is consistent with the x-ray crystal structure of HF treated hCG(8, 9) . In the crystal, the alphaC terminus shows a structure up to alphaTyr. The last three residues of the alphaC terminus, His-Lys-Ser does not show a structure, despite their presence in an open 5 nm solvent channel. This result suggests that alphaHis-Lys-Ser are flexible. It is interesting to speculate the alphaC-terminal positions up to alphaTyr, in relationship with alpha and beta. alpha is part of a loop in the hCG crystal and alphaMet, the residue closest to alphaTyr and the alphaC-terminal peptide backbone, is 16.5 Å away from alphaTyr. Therefore, it is not expected for alpha to compete with I-ABG-alpha for receptor binding, consistent with the data in Fig. 3. On the other hand, beta is closer to alphaTyr, betaGln being 8.6 Å away from alphaTyr. Therefore, it may be possible for beta to weakly interfere with, although may not completely block, binding of I-ABG-alpha to the receptor. In fact, the intensity of the photoaffinity labeled 86-kDa-band is somewhat reduced in the presence of beta in Fig. 3.

In this study we demonstrate that the alpha specifically interacts with the LH/CG receptor. In this complex of alpha and the receptor, the amino group of hCG alphaLys is near to a carboxyl group of the receptor to form an ion pair. This interaction between the peptide and the receptor is sufficient to activate the receptor to induce cAMP synthesis as does the hormone itself.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD-18702. 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.

§
Current address: Heritable Disorder Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892-1830.

To whom correspondence and requests for reprints should be addressed: Dept. of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944. Tel.: 307-766-6272; Fax: 307-766-5098; Ji{at}uwyo.edu.

(^1)
The abbreviations used are: hCG, human choriogonadotropin; CG, choriogonadotropin; LH, luteinizing hormone; FSH, follicle-stimulating hormone; TSH, thyroid-stimulating hormone; ABG, 4-azidobenzoylglycine; NHS, N-hydroxysuccinimide; NAc/CONH(2)-alpha, alpha which is acetylated at the N terminus and amidated at the C terminus; EDC, 1-ethyl-3-(1-dimethylaminopropyl)-carodiimide.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.