©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Model of Human Chorionic Gonadotropin and Lutropin Receptor Interaction That Explains Signal Transduction of the Glycoprotein Hormones (*)

(Received for publication, April 17, 1995; and in revised form, May 12, 1995)

William R. Moyle (§) Robert K. Campbell (¶) S. N. Venkateswara Rao Nagi G. Ayad Michael P. Bernard Yi Han Yanhong Wang

From the Department of Obstetrics/Gynecology, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The goal of these studies was to devise a model that explains how human chorionic gonadotropin (hCG) interacts with lutropin (LH) receptors to elicit a hormone signal. Here we show that alpha-subunit residues near the N terminus, the exposed surface of the cysteine knot, and portions of the first and third loops most distant from the beta-subunit interface were recognized by antibodies that bound to hCG-receptor complexes. These observations were combined with similar data obtained for the beta-subunit (Cosowsky, L., Rao, S. N. V., Macdonald, G. J., Papkoff, H., Campbell, R. K., and Moyle, W. R.(1995) J. Biol. Chem. 270, 20011-20019), information on residues of hCG that can be changed without disrupting hormone function, the crystal structure of deglycosylated hCG, and the crystal structure of a leucine-repeat protein to devise a model of hCG-receptor interaction. This model suggests that the extracellular domain of the LH receptor is ``U-'' or ``J''-shaped and makes several contacts with the transmembrane domain. High affinity hormone binding results from interactions between residues in the curved portion of the extracellular domain of the receptor and the groove in the hormone formed by the apposition of the second alpha-subunit loop and the first and third beta-subunit loops. Most of the remainder of the hormone is found in the large space between the arms of the extracellular domain and makes few, if any, additional specific contacts with the receptor needed for high affinity binding. Signal transduction is caused by steric or other influences of the hormone on the distance between the arms of the extracellular domain, an effect augmented by the oligosaccharides. Because the extracellular domain is coupled at multiple sites to the transmembrane domain, the change in conformation of the extracellular domain is relayed to the transmembrane domain and subsequently to the cytoplasmic surface of the plasma membrane. While the model does not require the hormone to contact the transmembrane domain to initiate signal transduction, small portions of both subunits may be near the transmembrane domain and assist in initiating the hormonal signal. This is the first model that is consistent with all known information on the activity of the gonadotropins including the amounts of the hormone that are exposed in the hormone-receptor complex, the apparent lack of specific contacts between much of the hormone and the receptor, and the roles of the oligosaccharides in signal transduction. This model differs from existing models of hormone action in that the extracellular domain has a much larger role in hormone action than serving as a high affinity hormone trap.


INTRODUCTION

The glycoprotein hormones form a family of structurally related trophic factors that regulate the gonads and the thyroid(1) . In humans these include the placental hormone hCG (^1)and the pituitary hormones hLH, hFSH, and human thyroid-stimulating hormone. Each is an alphabeta heterodimer containing a common alpha-subunit and a hormone-specific beta-subunit. Hormone function is initiated by binding of the hormones to a plasma membrane receptor that contains a large extracellular domain and a plasma membrane domain composed of seven hydrophobic alpha-helices(2, 3) . This leads to activation of G-proteins and subsequent production of second messengers.

The mechanism by which hormone-receptor interaction leads to signal transduction is not known. In a widely assumed model of hCG-receptor interaction that we term the ``tether'' model, hCG binds with high affinity to the extracellular domain of the LH receptor(4) . Because the extracellular domain is tethered to the transmembrane domain, this brings the hormone close to the transmembrane domain. Signal transduction starts when a second site on the hormone binds to the transmembrane domain. Support for this model is based on the observations that the extracellular domain has high affinity for hCG (4, 5, 6, 7) and the report that hCG can elicit signal transduction from an LH receptor analog containing only the transmembrane domain(7, 8) . The tether model predicts that one large or two smaller regions of hCG on at least two faces of the protein would interact with the receptor. The portions of hCG that interact with the LH receptor are unknown.

One way to test the model would be to identify portions of the hormone that contact specific parts of the receptor. Most putative contact sites have been identified by scanning the hormone to find amino acid substitutions that cause a loss in hormone function or by measuring the abilities of synthetic hormone peptides to block hormone activity (9, 10, 11) . As noted in a companion study(53) , the complexity of these hormones may confound interpreting the results of these studies. For example, truncation of the alpha-subunit at residue 87 leads to a hormone analog with very low affinity for the LH receptor, suggesting that this region may be involved in receptor contacts(1, 12) . However, as will be shown here, this analog is recognized better than hCG by antibodies that have higher affinities for the free alpha-subunit than for hCG. This implies that the mutation has altered the interaction between the subunits and that it will be impossible to distinguish effects on hormone conformation from those on receptor binding without using high resolution techniques such as x-ray crystallography or NMR spectroscopy.

Another way to test the tether model is to identify portions of the hormone that are exposed in the hormone-receptor complex. We have used this approach because it does not depend on loss of function mutants and provides data that are more easily interpreted. The tether model predicts that more than one surface of hCG would be hidden in the hormone-receptor complex. Thus, it can be tested by scanning the surface of the hormone to find regions that are exposed or that are hidden in the hormone-receptor complex. This can be accomplished by identifying hormone epitopes that are recognized by monoclonal antibodies after hCG binds to LH receptors. Since antibodies are much larger molecules than hCG, they will bind to only those surfaces of hCG that do not contact the receptor or other nearby proteins.

In a companion study(53) , we showed that a large portion of the hormone beta-subunit is exposed in the hormone-receptor complex and that the conformation of the region most likely to make high affinity contacts with the receptor is changed following hormone binding. Here we report that a similar large portion of the alpha-subunit can also be detected in the hormone-receptor complex. The crystal structure of deglycosylated hCG (13) shows that the alpha-subunit is formed from three large loops held together by a cysteine knot (Fig. 1). Loops one and three are adjacent, and loop two is found at the other end of the protein. Loop two is the most conserved part of the alpha-subunit and is located near beta-subunit loops one and three. To identify alpha-subunit residues involved in alpha-subunit antibody binding sites, we measured the abilities of the antibodies to bind heterodimers composed of hCG beta-subunit and bovine/human alpha-subunit chimeras. Because bovine alpha-subunit had low affinity for many antibodies made against the human alpha-subunit, chimeras having human alpha-subunit residues at an antibody binding site would be expected to bind an antibody much better than chimeras with bovine alpha-subunit residues at these same sites. By comparing the sequences of analogs that bound antibodies with those that did not, we identified key residues likely to be involved in binding of a large panel of anti-alpha-subunit antibodies. The relative locations of these residues determined in epitope maps were consistent with the crystal structure of deglycosylated hCG(13) . The alpha-subunit residues that are exposed in the hormone-receptor complex include residues in the N terminus and portions of the first and third loops. In combination with data for the beta-subunit in a companion study (53) and knowledge of residues that can be changed without disrupting hormone function, these observations enabled us to devise a model that explains how hCG interacts with LH receptors and initiates signal transduction.


Figure 1: Ribbon diagram of the alpha-subunit. This figure illustrates the location of the three alpha-subunit loops (dark blue) and the two oligosaccharide chains (red sticks). In the heterodimer, loop two of the beta-subunit is located near the front of loops one and three of the alpha-subunit(13) . The beta-subunit seat belt loop that surrounds the second alpha-subunit loop passes over the second loop midway down its length. The oligosaccharide shown at the bottom (Asn), but not the top (Asn) is essential for full signal transduction. Residues noted on the figure illustrate the relative locations of key antibody binding sites summarized in Table 2. Red, green, blue, and orange labels refer to some of the key residues in the epitopes of type I, II, IV, and V antibodies, respectively.






MATERIALS AND METHODS

Antibodies and Hormones

Urinary hCG preparation CR121 (13450 IU/mg), a tryptic fragment of the alpha-subunit(14) , and antibodies A102, A109, A201, A202, A407, A401, A402, A407, B105, and B109 (15, 16, 17, 18, 19) were provided by Drs. Robert Canfield, Alex Krichevsky, and Steven Birken (Columbia University, New York, NY). Antibodies A110, A111, and A112 were provided by Drs. Richard Krogsrud and S. Berube (BioMega Diagnostics, Montreal, Canada). Antibodies A113 and B112 were provided by Drs. Glenn Armstrong and Robert Wolfert (Hybritech Corp., San Diego, CA). Antibodies E501 and E502 were supplied by Dr. Robert Ryan (Mayo Clinic, Rochester, MN) and have been described(20, 21, 22) . Antibodies were radiolabeled using IODO-GEN (23) to a final specific activity of 30-50 µCi/µg.

Mutagenesis

Plasmids pSVL-hCGalpha, pSVL-bLHalpha, and pSVL-hCGbeta` containing cDNAs for hCG alpha-subunit, a synthetic bovine LH alpha-subunit, and hCG beta-subunit, respectively, have been described previously(24, 25) . The amino acid sequences of the chimeric alpha-subunits and the relative locations of restriction enzyme recognition sites used to assemble the mutant hormones are illustrated schematically in Fig. 2. Each bovine/human alpha-subunit chimera is named according to the unique human alpha-subunit residues that have been substituted into the bovine sequence. DNA constructs encoding the chimeric alpha-subunits were assembled by cassette mutagenesis or by exchanging fragments located between common endonuclease restriction sites in the human alpha-subunit cDNA and the synthetic gene encoding the bovine alpha-subunit (25) to give the constructs outlined in Fig. 2. pSVL-alphaBH11-17 was assembled by introducing a synthetic DNA cassette encoding human alpha-subunit residues 11-17 between the BsmI and FspII sites in the synthetic gene encoding the bovine alpha-subunit. pSVL-alphaBH64-68 was assembled by introducing a synthetic DNA cassette encoding human alpha-subunit residues 64-68 between the BalI and BstXI sites in the synthetic gene encoding the bovine alpha-subunit. Vectors expressing analogs alphaBH11-26/50-53, alphaBH11-26/81, alphaBH11-26/73-75, and alphaBH11-26/64-68 were made by cassette mutagenesis of pSVL-alphaBH11-26. pSVL-alphaBH11-26/50-53 was made by introducing a synthetic DNA cassette encoding human alpha-subunit residues 50-53 between the BglII and SpeI sites of pSVL-alphaBH11-26. To make the remaining chimeras, the XbaI-PstI fragment of pSVL-alphaBH11-26 was cloned into the XbaI-PstI sites of pIBI31 (IBI, New Haven, CT). Cassettes encoding human alpha-subunit residues 81, 73-75, and 64-68 were cloned into the BstXI-PstI, BalI-BstXI, and BglII-BalI sites of this vector. The XbaI-PstI fragments of these constructs were then cloned into the XbaI-PstI sites of pSVL-alphaBH11-26 to make pSVL-alphaBH11-26/81, pSVL-alphaBH11-26/73-75, and pSVL-alphaBH11-26/64-68. pSVL-alpha88, an analog lacking residues 88-92 was made by cassette mutagenesis between the PstI and BamHI restriction sites. All mutations and the coding sequences that were subjected to polymerase chain reactions were confirmed by double-stranded dideoxy DNA sequencing.


Figure 2: Diagram of the chimeras and analogs used in these studies. These analogs were produced by a combination of cassette and polymerase chain reaction mutagenesis as outlined in the text. The nomenclature refers to the locations of residues derived from the human alpha-subunit. The amino acid sequence of the bovine and human alpha-subunits are FPDGEFTMQGCPECKLKENKYFSKPDAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNVRVENHTECHCSTCYYHKS and APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS, respectively. Solid bars are residues derived from bovine alpha-subunit and stippled bars are from human alpha-subunit.



Production and Characterization of Recombinant alpha-Subunit Chimeras

Plasmids encoding chimeric alpha-subunit DNAs were purified by ultracentrifugation in CsCl gradients and co-transfected with pSVL-hCGbeta into COS-7 cells as described(24, 26) . Three days after transfection, the alphabeta heterodimers in the media were measured using a sandwich immunoassay (18) except that antibodies B105 or B112 were used to capture the analogs and radiolabeled B109 was used to detect the analogs bound to B105 or B112. We also monitored the abilities of these analogs to bind to rat luteal membrane receptors using the biological receptor-based immunoradiometric assay(27) .

Receptor Binding Studies

Procedures for measuring the abilities of hormone analogs to bind to LH receptors by competition with I-hCG have been described elsewhere(18) . Procedures for measuring the abilities of the antibodies to inhibit binding of radioiodinated hCG to cells expressing LH receptors or membranes from rat corpora lutea have also been described earlier(18, 27, 28) . Briefly, the antibody was incubated with hCG for 30-60 min prior to addition of cells. Then, after further incubation at 37 °C for 60 min, the cells or membranes were collected by centrifugation, and the supernatant was aspirated. Radiolabeled hCG in the cell pellet was measured in a gamma counter. As a control, we monitored the ability of unlabeled hCG to block the binding of the radiolabeled hCG. Methods for measuring binding of radioiodinated antibodies to hormone-receptor complexes have also been described previously(18, 28) . Briefly, cells or luteal membranes were incubated with hCG (1 µg, 60 min at 37 °C), washed, and then incubated with radiolabeled antibody (60 min at 37 °C). Bound and free radiolabel was separated by centrifugation, and the radiolabel in the pellet was measured in a counter.

Model of hCG-Antibody and hCG-Receptor Complexes

A model of glycosylated hCG was prepared as described in a companion study(53) . This structure was used to recreate an epitope map based on the binding sites of the antibodies identified using the chimeras and other analogs. The map was built by visually placing the portion of the antibody Fab fragment that contacts lysozyme adjacent to residues determined to participate in antibody binding sites. This was accomplished by superimposing the residues of lysozyme recognized by the antibody over the residues of hCG identified in an antibody binding site. The remainder of the Fab was oriented perpendicular to the surface of hCG. Finally, the lysozyme residues were undisplayed leaving the Fab molecules docked next to hCG. The coordinates for the heavy and light chains used in this process were obtained from a crystal structure of lysozyme-antibody complex (29) and correspond to the structure pdb3hfm.ent in the Protein Data Bank.

Modeling of hCG-receptor complexes will be described in detail elsewhere. Briefly, we modeled the LH receptor on the crystal structure of of porcine ribonuclease inhibitor (30) obtained from the Protein Data Bank (i.e. 1bnh.ent). We aligned the sequences of the leucine repeats in the LH receptor with those of the crystal structure of ribonuclease inhibitor. In this alignment residues encoded by the intron-exon junctions of the receptor become located in solvent exposed loops furthest from the portion of the extracellular domain that contacts the transmembrane domain. Five of the six glycosylation signals found in the LH receptor are also located in these loops. After adding oligosaccharides, we subjected the structure to energy minimization and molecular dynamics at 300 K using the modeling package Sybyl (Tripos, St. Louis, MO). The crystal structure of hCG was ``docked'' to this model of the extracellular domain of the receptor using biological information on the parts of the hormone that are exposed in the hormone-receptor complex reported in this and in a companion study(53) . The groove of the hormone between the alpha- and beta-subunits thought to make the high affinity contacts identified in the companion study (53) was placed over receptor residues 93-170 which have been shown to control LH receptor binding specificity(31) . The hormone was rotated slightly around this putative contact until the exposed and hidden surfaces of the hormone were in positions that agreed sterically with the observations on antibody binding made in this and a companion study(53) . The fully glycosylated complex was then subjected to energy minimization and molecular dynamics until it reached a stable minimum energy. Finally, to illustrate the transmembrane domain, we visually docked the structure of rhodopsin (i.e. 1bac.ent from the Protein Data Bank) to the plasma membrane side of the hormone-receptor complex. During docking, the N-terminal end of the first alpha-helix was placed beneath the C-terminal end of the extracellular domain. The structure of rhodopsin was then rotated until the helices were perpendicular to the central cavity of the extracellular domain. In this configuration helices 1-5 of rhodopsin made a direct contact with the extracellular domain of the receptor while helices 6 and 7 were under the open space created by the ``U'' shape of the extracellular domain.


RESULTS

A Portion of the alpha-Subunit Is Exposed after hCG Binds to Membrane LH Receptors

The goal of these studies was to identify portions of the hCG alpha-subunit that do not contact the LH receptor. Therefore, we monitored the abilities of the alpha-subunit antibodies in our panel to bind to hCG-receptor complexes and found two that did (cf. Table 1for data for A105 and A407, remainder not shown). Most alpha-subunit antibodies blocked hCG binding to receptors (Fig. 3). This included one of those (i.e. A105) that bound to hCG-receptor complexes. Like many other antibodies that bind to hCG-receptor complexes, the inhibition caused by A105 was only partial.




Figure 3: Ability of antibodies to inhibit binding of I-hCG to LH receptors. Increasing amounts of antibodies as shown on the abscissa were incubated with radiolabeled hCG for 30 min at 37 °C. After the antibody had bound to the hormone, the mixture was added to homogenates of ovarian corpora lutea and the binding of I-hCG to LH receptors was measured as described in the text. Radiolabel that became bound to the membrane receptors is illustrated here. Values are means of closely agreeing duplicates (all antibodies except A101) or triplicates (A101).



Previous studies including those using antisera to hCG alpha-subunit have failed to detect exposed residues of the alpha-subunit and led to models of hormone binding in which the entire alpha-subunit is close to the receptor(32) . The observations that two alpha-subunit antibodies bound to hormone-receptor complexes show that these models are incorrect. However, we have also found that hCG can bind to membranes and other surfaces ``nonspecifically''(23) . Binding of A407 was readily detected with virtually every preparation of radiolabeled antibody; binding of A105 to receptor complexes was seen only with freshly iodinated preparations. To be certain that we were not observing the binding of A105 to hCG that became bound ``nonspecifically'' to the membranes, we repeated the studies using analogs of hCG that had related structures but different abilities to bind to LH receptors. A105 bound to receptor complexes prepared by incubating membrane receptors with hCG analogs having lutropin activity. It did not bind receptor preparations that had been incubated with similar amounts of closely related analogs that have low LH activity (Table 1). This showed that A105 recognized an exposed portion of the alpha-subunit of hCG after the hormone was specifically bound to LH receptors.

Epitope Maps Show That Two Different Regions of the alpha-Subunit Are Exposed in hCG-Receptor Complexes

To learn if more than one alpha-subunit epitope region was exposed in hCG-receptor complexes, we mapped the relative binding sites of the alpha-subunit antibodies. These maps showed that alpha-subunit antibodies could be classified into five types (16, 17, 18) (Fig. 4, Table 2) and that two non-overlapping epitopes were exposed in the hCG-receptor complex. Type I antibodies bound to hCG at the same time as type II, IV, and V antibodies and could be divided into two subcategories; those that had approximately the same affinity for hCG and the alpha-subunit (type Ia) and those that had much higher affinity for the free alpha-subunit than hCG (type Ib). None of the type I antibodies bound to hCG at the same time as antibodies that recognized epitopes on the second loop of the beta-subunit (i.e. B101, B107, and B109). This indicated that the epitopes in the type I region were near the subunit interface. Type II antibodies bound to hCG at the same time as those in types I, IV, and V. The only type III antibody in our panel did not bind to hCG at the same time as antibodies from types Ia, Ib, and II. Because the type III epitope overlaps the epitopes for both those of the type I and II antibodies, the type I and II epitopes appear to be adjacent. A407 and A105 were the only examples of types IV and V antibodies and each bound to hCG at the same time as all other antibodies to the alpha- and beta-subunits. Both these antibodies bound to hCG-receptor complexes, an indication that a substantial portion of the alpha-subunit was not involved in receptor contacts. The observation that there was only a single example of each of these types suggested that these epitopes are weakly antigenic. This would account for the inability of most hCG antibodies to recognize the alpha-subunit in hCG-receptor complexes.


Figure 4: Epitope map describing the binding sites of the antibodies used in these studies. These maps illustrate the relative properties of the antibodies to bind to hCG as discussed in the text. They were determined by measuring the abilities of the antibodies to bind to hCG at the same time.



Identification of Key Residues in the Antibody Binding Sites

A summary of the key alpha-subunit residues recognized by each antibody is illustrated in Table 2and is based on the studies described below. (These positions of these residues are also noted on Fig. 1.) Our strategy to identify antibody binding sites involved the use of hormone analogs that were made in mammalian cells. Cells transfected with cDNA for the alpha- and beta-subunits secreted free subunits in addition to the alphabeta heterodimer. Because it was not practical to purify the microgram quantities of analogs made following transient transfections, we devised a procedure to trap a known amount of heterodimeric form of each analog to a solid phase so that we could quantify its binding to antibodies. This was required because binding of antibodies to some analogs was intermediate between that of human and bovine alpha-subunit. B112 was used to capture the heterodimers because it binds to a site on hCG beta-subunit that does not interfere with binding of antibodies to the alpha-subunit(26) . B112 also captured the free beta-subunit but not the free alpha-subunit made by the cells. We measured the alphabeta heterodimers bound to B112 using antibody B109. B109 has high affinity for a conformation of the beta-subunit created when either human or bovine alpha-subunits combine with the hCG beta-subunit(25, 26) . B109 did not recognize free hCG beta-subunit bound to B112. Self-displacement assays in which unlabeled B109 was used to inhibit binding of radiolabeled B109 to heterodimers containing human, bovine, or chimera alpha-subunit showed that the affinity of B109 for all the heterodimers was indistinguishible (not shown).

Type Ia Antibodies Bind to the First Loop of the alpha-Subunit Near the beta-Subunit Interface

The presence of bovine alpha-subunit residues in the first alpha-subunit loop blocked binding of the Type Ia antibodies to hCG ( Table 2and Table 3). All these antibodies bound to heterodimers containing human alpha-subunit residues 11-26 much better than to those containing bovine alpha-subunit residues in this region. Within this group of antibodies there were subtle differences in the locations of some antibody binding sites. For example, antibodies A102 and A113 bound well to analogs of bovine alpha-subunit that contained only human residues 11-17, whereas the remainder (i.e. A101, A110, and A111) appeared to also require human residues 18-26. All these antibodies bound hCG as well as or better than the free alpha-subunit suggesting that the conformation of this region of the protein was not greatly influenced by subunit combination. None of these antibodies bound to hCG-receptor complexes (not shown). However, they all inhibited hCG binding to LH receptors (Fig. 3). The inhibition seen in response to A101 was less than that expected based on its affinity for radiolabeled hCG (Fig. 3, inset).



Type Ib Antibodies Bind to a Surface on the First and Third Loops of the alpha-Subunit That Faces the beta-Subunit

All of the type Ib antibodies bound free alpha-subunit better than hCG (16) and recognized an epitope that was distributed across loops one and three (Tables II and III). For example they bound chimeras that contained human residues 11-81 well. However, they bound poorly to analogs in which the only human residues were amino acids 11-26. They bound even worse to analogs in which the only human residues were amino acids 41-81. This suggested that the binding sites for these antibodies included parts of loops one and three, an idea that was confirmed by the finding that they bound well to analogs in which human residues were derived from amino acids 11-17 and 41-81 or 11-26 and 73-75. Thus, the binding sites of these antibodies appeared to include residues in the regions between 11-17 and 73-75. This suggested that these residues were close to one another in the protein, a prediction (33) that was confirmed by the crystal structure(13) . We observed that A109 was an effective inhibitor of hCG receptor binding (Fig. 3). A109 did not bind to hCG-receptor complexes (not shown). The other type Ib antibodies have even higher ability to distinguish the free alpha-subunit and hCG. Due to their low affinities for hCG, we did not test their abilities to inhibit hCG-receptor complex formation or to bind to hCG-receptor complexes.

Type II Antibodies Bind to a Part of the Third alpha-Subunit Loop

In contrast to the binding sites of the antibodies just discussed, type II antibodies recognized an epitope that included residues 64-68 of the third alpha-subunit loop ( Table 2and Table 3). This explained the abilities of these antibodies to bind to hCG at the same time as those in types Ia and b (Fig. 4). This also suggested that residues 64-68 were located on a different face of the alpha-subunit than residues 73-75, an observation that is consistent with the crystal structure(13) . Antibody A201 did not inhibit hCG binding to receptors at the concentrations tested. This was surprising since antibodies to this region did not bind to hCG-receptor complexes. These were the only antibodies we found that did not inhibit hCG binding or bind to hCG-receptor complexes.

The Type III Antibody Binds to a Part of the Third Loop Near the Sites for the Type I and Type II Antibodies

Antibody A112, the only antibody in this class, recognized the region of the alpha-subunit that contained residues 73-75. For example it bound to chimeras containing human residues 11-26/73-75 better than chimeras containing human residues 11-26 or 11-26/64-68 ( Table 2and Table 3). Binding to this region is consistent with its ability to compete with antibodies in types Ia, Ib, and II as well as with the locations of these residues in the crystal structure. A112 was an effective inhibitor of hCG-receptor binding (Fig. 3) and did not bind to hCG-receptor complexes (not shown).

The Type IV Antibody Binds to a Conformational Epitope Derived from the N Terminus and Regions of the alpha-Subunit Derived from Loops One and Three Furthest from the beta-Subunit Interface

A407, the only example of a type IV antibody, had very low affinity for the free alpha-subunit (Table 3) or for hFSH (not shown) indicating that it recognized a conformation unique to hCG. A407 bound to alphabeta heterodimers composed of human alpha-subunit and bovine LH beta-subunit (not shown) but not to those composed of bovine alpha-subunit and hCG beta-subunit ( Table 2and Table 3), suggesting that its epitope was limited to the alpha-subunit. The epitope for A407 was complex. A407 was the only antibody whose binding was influenced by human residues 1-6, 11-17, and 81 ( Table 2and Table 3). It was not influenced by human residues 18-75. A407 bound to hCG at the same time as all type Ia antibodies that recognized residues 11-17 (i.e. A102 and A113) and those that recognized residues 18-26 better than 11-17 (i.e. A101, A110, and A111). Therefore, we concluded that the type Ia binding site included residues in the C-terminal half of the 11-17 sequence (i.e. 14-l7) and the A407 site included residues in the N-terminal half of the same sequence (i.e. 11-13). A407 bound to hCG-receptor complexes (Table 1) and had little influence on hCG binding to rat LH receptors (Fig. 3). This showed that the N terminus and portions of loops one and three furthest from the subunit interface were not near the receptor binding site. Because A407 recognized a conformation dependent epitope, its ability to bind to hCG-receptor complexes suggested that the shape of this portion of the hormone is not altered upon hormone binding. The crystal structure indicates that the N terminus of the alpha-subunit is located near the N terminus of the beta-subunit. The low affinity of A407 for free alpha-subunit and for hFSH suggests that the conformation of this portion of the alpha-subunit is altered on subunit combination and differs in hCG and hFSH.

The Type V Antibody Binds to a Highly Conserved Portion of the Third alpha-Subunit Loop Near the Cysteine Knot

A105, the only type V antibody available had high affinity for heterodimers containing human beta-subunit and bovine alpha-subunit (Table 3). This indicated that it recognized a conserved epitope in both human and bovine alpha-subunits and we were unable to identify the A105 binding site using the chimera strategy. Only two regions of the bovine and human alpha-subunits are highly conserved and located in areas of the molecule not recognized by the other anti-alpha-subunit antibodies. These include most of the second loop and a small portion of the third loop near the cysteine knot. To distinguish these, we monitored the ability of A105 to bind to a fragment of the human alpha-subunit that had been produced by tryptic digestion(14) . This fragment is missing residues 36-51, most of the second alpha-subunit loop. Since A105 recognized this fragment (Table 4), the A105 epitope does not involve the second loop. In support of this conclusion, we also observed that A105 bound to heterodimers containing alpha-subunit analogs in which Phe and Arg were replaced with alanines (not shown).



Deletion of alpha-Subunit Residues 88

N92 Altered the Interaction between the alpha- and beta-Subunits

Heterodimers lacking the C terminus of the alpha-subunit have low affinities for their receptors and this has been interpreted as support for the idea that C terminus of the alpha-subunit has a role in hormone binding(1) . To learn if the C terminus of the alpha-subunit was involved in the binding sites of any of the alpha-subunit antibodies, we tested the abilities of each antibody to bind an analog lacking alpha-subunit residues 88-92. All of these antibodies bound to this analog indicating that residues 88-92 were not part of their binding sites (Table 5). Unexpectedly, antibodies that had higher affinity for the heterodimer recognized the analog lacking the C terminus better than hCG (Table 5). This suggested that this mutation distorted the interaction between the subunits without causing them to dissociate. We also found that deletion of the C terminus led to an increased ability of hCG to be recognized by A105, an antibody that bound to hormone-receptor complexes (Table 4). The binding site for A105 appears to be near the C terminus, and we anticipate that the mobility of the C terminus that prevents it from being seen in the crystal structure (13) also interferes with binding of A105. These observations indicate that it may be premature to conclude that the C terminus of the alpha-subunit contacts the LH receptor based on the reduced abilities of analogs with mutations in this region to bind to LH receptors. In addition, these observations suggested that the ability of mutant subunits to combine into a heterodimer is not sufficient to detect the influences of mutations on hormone conformation.




DISCUSSION

The Identity of alpha-Subunit Residues in Antibody Binding Sites Is Consistent with the Epitope Map and with the Crystal Structure of Deglycosylated hCG

To devise a plausible model of hormone receptor interaction, we needed to correctly identify portions of the hormone that contacted the receptor and/or that were exposed in the receptor complex. Our strategy to identify residues in antibody binding sites depends on the assumptions that (i) residues essential for antibody binding can be detected by comparing the abilities of antibodies to recognize a series of antigen analogs and (ii) mutagenesis in one part of the molecule does not disrupt the overall structure of the protein. The validity of both these assumptions is supported by the crystal structures of lysozyme-antibody complexes (34) and of other proteins(35, 36) . A rule of thumb has been proposed that mutations that reduce the affinity of an antibody for the antigen by more than 10-fold are likely to participate in the binding site(35) . Based on this criterion, the analogs we used should have been sufficient for us to identify residues that are located in the binding sites for all antibodies except A113 and A105. The binding site for A113 determined using the chimeras is consistent with the crystal structure. However, that for A105 could not be determined using the chimeras.

The locations of all the antibody binding sites are consistent with the crystal structure of deglycosylated hCG(13) . This is most convincingly illustrated by predictions about the locations of residues in the twin loops of the alpha-subunit made on the basis of the A109 and A407 binding sites. For example, as predicted from the ability of A109 to bind to chimeras(33) , residues 14-17 and 73-75 were found to be adjacent in the crystal structure. Also, the proximity of residues near the N terminus of the protein and residue 81 had been predicted from the A407 binding site prior to the crystal structure(33) . This suggested that the locations of the antibody binding sites have been correctly identified.

The Abilities of Hormones to Form Heterodimers Is Not Always a Good Indication That the Subunits Have Combined Correctly

Mutations that disrupt hormone binding can alter hormone conformation in an unexpected manner. One reason we chose this indirect approach to study hCG-receptor interactions is that it is not always easy to identify an influence of a mutation on the structure of the hormone. Site directed approaches to studying hormone-receptor contacts make the implicit assumption that residues can be substituted without altering the conformation of the hormone. While this assumption is likely to be true when the mutation does not influence receptor binding as is the case of the analogs used in this study, it needs to be tested thoroughly when analogs are found that have reduced receptor binding. Large changes in hormone structure can usually be detected by monitoring the abilities of the subunits to combine into a heterodimer. However, we have found that this can be misleading. Truncation of the C terminus of the alpha-subunit led to an analog that combines well with the beta-subunit but the heterodimer appears to have a different conformation than hCG (Table 5). Deletion of the N terminus of the beta-subunit led to an analog in which the subunits combined poorly (3) yet this analog and hCG have identical affinities for LH receptors. (^2)We were concerned that we might face the same problem in the studies described to identify antibody binding sites and took several precautions to avoid being mislead. These included (i) using only those analogs that we found would bind well to LH receptors and (ii) examining the binding sites of several antibodies that competed for binding to hCG. This latter control enabled us to test the internal consistency of the data we obtained. The consistency of the epitope maps and the crystal structure of hCG supports the conclusion that the key residues in the antibody binding sites have been identified correctly.

Antibodies to the alpha-Subunit Recognize Most of the Exposed Surfaces of Loops One and Three

Due to their sizes only four Fab fragments can be simultaneously superimposed on the surface of alpha-subunit loops one and three while keeping key residues found to control antibody binding within the region typical of the contact zone between an antibody and an antigen such as lysozyme(29) . Thus, the antibodies we studied recognize most of the surface of the alpha-subunit present in this portion of the hCG (Fig. 5). This also confirms the conclusions we have drawn about the antibody binding sites from mutagenesis studies and epitope mapping, a critical requirement for the model of hCG-receptor interaction.


Figure 5: Composite figure illustrating the relative binding sites of several anti-alpha-subunit antibodies. The alpha-subunit is illustrated in white and the beta-subunit is shown in yellow. The oligosaccharides are illustrated in red. The relative locations of the antibody binding sites are shown by the positions of the space filled coordinates of the crystal structure of anti-lysozyme Fab fragments that have been placed visually. This was done by docking a high resolution structure for a lysozyme-anti-lysozyme Fab fragment complex perpendicular to hCG over residues that were identified as being in the antibody binding sites (Table 2). The view shown in the upper panel explains the relative abilities of the antibodies to bind to hCG at the same time (cf. epitope map, Fig. 4). Top panel, view from above the alpha-subunit looking down on the tips of the first and third loops. Lower panel, view of molecule turned 90° and illustrating the second loop of the alpha-subunit and the beta-subunit. Color code: alpha-subunit, white; beta-subunit, yellow; oligosaccharides, red; type IV site, orange; type V site, blue; type II site, green; and type I site, purple.



Aside from monoclonal antibodies made against synthetic peptides, the binding sites for most anti-alpha-subunit antibodies have not been determined. The identification of key residues in the binding sites of antibodies we report here should have several practical applications. Many of the antibodies we have used in these studies are widely used for research and clinical diagnoses. Knowledge of the portions of hCG recognized by these antibodies will facilitate identifying unknown analytes present in serum or that are produced by tissues. Epitope maps prepared with these antibodies as reference compounds can also be used to predict the binding sites of other antibodies. Finally, since their binding sites are now known, these antibodies can be used to study the influence of mutations on specific regions of the protein and to detect changes in hormone conformation that may influence receptor binding (cf. Table 2and Table 5).

Model to Explain Hormone-Receptor Interaction and Gonadotropin-induced Signal Transduction

The major goal of these studies was to devise a model that explains gonadotropin binding and signal transduction. Based on the data presented here and in a companion study (53) we devised a model (Fig. 6) that accounts for all the properties of the hCG-receptor complex that are well known. This is significantly different from the tether model described previously because it suggests that the N-terminal extracellular domain interacts with the extracellular surface of the transmembrane domain at multiple points. We envision the extracellular domain to have a U- or J-shape and to lie flat on the transmembrane domain thereby maximizing the contacts between the two proteins. High affinity hormone binding results from the insertion of the extracellular portion of the receptor near the curved portion of the U or J into the groove in hCG created by the interface of the second alpha-subunit loop and the first and third beta-subunit loops. Much of the remainder of the hormone fits loosely into the open space in the extracellular domain between its arms and makes few, if any, additional high affinity contacts with either arm (Fig. 6). Binding of hormone alters the conformation of the extracellular domain, an effect that is transmitted to the intracellular domain through the contacts between the two domains. This initiates signal transduction. The oligosaccharides are required for this process because they potentiate the effect of the hormone on the separation of the arms of the receptor extracellular domain. The oligosaccharides on the second loop of the alpha-subunit and the first loop of the beta-subunit influence this by the bulk that they add to the hormone. Consequently, the change in conformation of the receptor extracellular domain is much less for hormone analogs lacking oligosaccharides. This accounts for the inabilities of deglycosylated analogs to stimulate signal transduction and for the restoration of efficacy by antisera to the beta-subunit.


Figure 6: Model of hCG binding to the extracellular domain of the LH receptor that illustrates a mechanism of signal transduction. Note, panels in the left column illustrate a side view of the receptor and those in the right column illustrate the corresponding top view obtained by rotating the side view forward 90°. The free LH receptor (top left and right panels) ribbon model is based on the structure of RNase inhibitor (2) and was prepared by replacing residues in the leucine-rich repeats of RNase inhibitor with those of the corresponding repeats found in the LH receptor. Following extensive energy minimization and molecular dynamics, the receptor model was ``docked'' onto the structure of bacteriorhodopsin (52) to obtain a view illustrating the approximate sizes and orientations of the extracellular and transmembrane domains. The extracellular and cytoplasmic loops of the transmembrane domain are not shown. During docking, the C-terminal residue of the extracellular domain (Arg) was placed adjacent to the N-terminal end of the first alpha-helix of rhodopsin (white). The remaining six helices of rhodopsin (shown in the order green, red, yellow, purple, orange, and magenta) were rotated to make maximal contact with the extracellular domain. The extracellular domain of the receptor is illustrated in blue (residues 1-93 and 170-341) and orange (residues 94-169). The orange-colored section of the extracellular domain illustrates a portion of the LH receptor that appears to control its ligand binding specificity. When this section of the LH receptor was substituted for the homologous region of the FSH receptor, the resulting chimera bound both hCG and hFSH with high affinity(31) . Five of the six receptor oligosaccharides are illustrated in yellow. In the hormone-receptor complex (middle left and right panels) note that, to improve the clarity of these panels, we have omitted the oligosaccharides and the transmembrane domain of the receptor. To dock the hormone to the receptor we moved the groove in hCG formed by alpha-subunit loop two, beta-subunit loop three, and, to a lesser extent, beta-subunit loop one (13) over the orange-colored portion of the receptor (alpha-subunit, red ribbon; beta-subunit, green ribbon; hormone oligosaccharides, red sticks). We then rotated the hormone about this intersection with the receptor to expose hormone residues found to be exposed and hidden based on the antibody binding data described in this and a companion study(53) . Finally, all the atoms in the hormone and extracellular domain of the receptor including those in their oligosaccharides were subjected to energy minimization and molecular dynamics at 300 K to eliminate bad contacts and optimize side chain interactions. This occurred without major structural changes in either the hormone or the receptor and led to the stable complex shown here. hCG beta-subunit loops one and three are immediately above the portion of the receptor known to convey lutropin binding specificity (i.e. colored orange). The second alpha-subunit loop contacts the concave portion of this region of the extracellular domain on its inner surface. hCG alpha-subunit loops one and three and beta-subunit loop two project into the large cavity formed by the U shape of the receptor extracellular domain and are nearest its C-terminal arm. The N-terminal ends of both hormone subunits (free ends of the alpha- and beta-subunit ribbons shown in middle left panel) and the oligosaccharides of the beta-subunit (red oligosaccharide chains that point upward in middle left panel) are on the surface of the hormone furthest from the plasma membrane. Only those oligosaccharides on the alpha-subunit are shown in the top view (middle right panel). Note the proximity of the oligosaccharide needed for signal transduction (i.e. that at Asn) and the N terminus of the extracellular domain of the receptor (middle right panel). Signal transduction results from the steric effect of the oligosacharide at Asn on the N-terminal end of the extracellular domain to widen the distance between its N- and C-terminal arms. This may be accentuated by steric interaction of alpha-subunit loops one and three with the C-terminal end of the extracellular domain. The model does not require a direct interaction between the hormone and the transmembrane domain for signal transduction. Given the proximity of the hormone and the transmembrane domain in this model, these cannot be excluded. In antibody-hCG receptor complexes (lower left and right panels) the crystal structure of an Fab fragment-lysozyme complex was ``docked'' to residues of epitopes known to be exposed in hCG-receptor complexes. To minimize complexity, we illustrate only four of the five known antibody binding sites and none of the oligosaccharides on the hormone or the receptor. The orange and yellow ribbons correspond to Fab fragments docked over exposed residues of the alpha-subunit (i.e. A105, yellow; A407, orange; cf. Table 2). The red and blue ribbons correspond to Fab fragments docked over exposed residues of the beta-subunit (i.e. B111, red; B112, blue). Key residues in the binding sites of these antibodies include 108-114 and 77, respectively. Not shown is the binding site of B105, an antibody that recognizes a portion of hCG containing residue 74 (cf. (53) ). The binding site for this antibody is most easily visualized as lying between that of the red and blue ribbons on the lower left panel.



There are few other configurations of the receptor that would account for the portions of the hormone in the receptor complex that can be detected by antibodies and that would also permit the hormone to be near the transmembrane domain. As discussed later, the conformation of another leucine-rich repeat protein, RNase inhibitor is horseshoe-shaped(30) . This provides considerable precedent for the model we have proposed. In addition, the model is consistent with the known interactions between hCG and the LH receptor discussed as follows.

First, the model of hCG-receptor interaction accounts for portions of the hormone that can be recognized by antibodies that bind to hormone-receptor complexes described here and in a companion study (53) (Fig. 6). In the alpha-subunit this includes residues near the N terminus and most of the portions of the first and third alpha-subunit loops furthest from the beta-subunit interface. In the beta-subunit this includes residues on the surfaces of the first and third loops that are furthest from the alpha-subunit interface. Surfaces recognized by antibodies face away from all parts of the receptor and are not likely to be near other proteins in the cell membrane.

Second, the model explains why many residues in both alpha- and beta-subunits usually proposed to be near the receptor interface can be replaced without altering receptor binding or hormone activity(24, 31) . In the alpha-subunit these include most of the residues found in loops one and three near the beta-subunit interface, a few residues in the second loop including Arg-Lys(38) , and a residue that can be cross-linked to the beta-subunit (i.e. that corresponding to human alpha-subunit Lys in pig and bovine LH)(39, 40) . In the beta-subunit these residues include nearly all the amino acids found in the second loop (24) and in the seat belt, the region that has the major influence on receptor binding specificity (24, 31) . The model suggests that the side chains of these residues do not make specific contacts with the receptor even though many of these residues are in the large groove of the extracellular domain. Their functions in signal transduction, if any, occurs primarily by steric effects. Mutations of residues that are found in this groove have relatively little influence on hormone function unless they disrupt the structure of the hormone.

Portions of the alpha- and beta-subunits that become located between the arms of the receptor extracellular domain lose their abilities to be recognized by antibodies after they bind to LH receptors. These epitopes appear to be obscured by the receptor. Antibodies that bind to these regions are very effective in blocking hormone binding, a phenomenon that appears due in part to their abilities to prevent this region of the hormone from entering the space between the arms of the receptor. As noted earlier, Pantel et al.(41) have found that these antibodies can recognize hCG that is bound to truncated LH receptors, an observation that is consistent with this view.

Third, the structure of a U- or J-shaped extracellular domain could readily create a narrow projection that fits into the hormone groove created by the apposition of the second alpha-subunit loop and portions of the first and third beta-subunit loops. Entry of a portion of the receptor into this groove could also account for the change in conformation of the beta-subunit that occurs on binding to receptors (28, 53) .

Fourth, the model of hCG-receptor interaction explains why the oligosaccharides are essential for signal transduction. In the model, binding of hormone to the receptor is distinct from signal transduction. Signal transduction occurs by an influence of the hormone on the distance between the arms of the extracellular domain. While it is possible that the hormone reduces this distance by binding to both arms of the extracellular domain, we think this is very unlikely since the residues in the portion of the hormone near the arms of the extracellular domain (24) and the leucine-rich receptor repeats in these arms (31) can be changed without disrupting signal transduction. More likely, we anticipate that the hormone will increase the distance between the two arms and we propose that this is the role played by the oligosaccharides in signal transduction. The sugars appear to function primarily by their bulk rather than by a specific interaction with the receptor, an idea that is consistent with the following observations. (a) It explains why both hLH and hCG are potent lutropins even though their sugars are quite different(42) . It also explains the full efficacy of hormones having high mannose sugars that have been expressed in Baculovirus(43) . (b) The bulk of the sugars resolves the differences in the influence of the oligosaccharides on efficacy(44) . That on the alpha-subunit at Asn has the least effect on hormone efficacy because it extends beyond the arms of the receptor. The alpha-subunit oligosaccharide at Asn has the greatest influence on efficacy because it is closest to the receptor. The oligosaccharides on the beta-subunit have an intermediate influence on efficacy. These project away from the hormone-receptor complex but are sufficiently near the arms of the extracellular domain that they could interact with them. (c) The influence of bulk accounts for the observations that sequential removal of the oligosaccharides from hCG using exoglycosidases is accompanied by a graded loss in efficacy(45) . (d) Finally, the observation that antibodies to hCG can restore efficacy to deglycosylated hCG (46, 47) is also consistent with the idea that the oligosaccharides function primarily by steric effects. Binding of antibodies to the exposed region of the beta-subunit would increase the bulk of the portion of the hormone that is found between the arms of the receptor extracellular domain.

Fifth, the model explains the observation that hCG will bind and activate FSH receptor analogs that have relatively few residues derived from the LH receptor(31) . Because the arms of the extracellular domain make few specific high affinity contacts with the side chains of the hormone, hCG analogs bind with high affinity to LH/FSH receptor chimeras that are derived mostly from the FSH receptor(31) . Further, because the ``contacts'' needed for signal transduction are ``steric'' in nature, both hCG and hFSH can elicit signal transduction from LH/FSH receptor chimeras(31) . Only a small part of the extracellular domain of the receptor appears essential for hormone binding. Thus, the model also explains why LH receptors that have been truncated at amino acid 206 are able to bind hCG with high affinity (4) .

Sixth, the model does not require the hormone to interact with the transmembrane domain of the receptor. However, since much of the hormone is present in the space between the arms of the extracellular domain, these interactions are not precluded. This would account for the reports that hCG can bind and activate the transmembrane domain of the LH receptor (7, 8) and that a synthetic peptide corresponding to the C terminus of the alpha-subunit can stimulate cyclic AMP accumulation (48) . The interaction of a portion of the hormone with the transmembrane domain might also serve to attract the hormone into the groove in the extracellular domain and potentiate its effect on the conformation of the receptor. This would explain the observation that binding of hCG to a membrane bound receptor occurs with higher affinity than to soluble receptors.

Leucine-rich Domains Have Horseshoe Shapes Similar to the Shape Proposed for the Extracellular Domain of the LH Receptor

The structure of the extracellular domain of the receptor in the model is similar to that of one other leucine-rich repeat protein, ribonuclease inhibitor(30, 49) . The horseshoe shape of the RNase inhibitor may be characteristic of all leucine-rich repeat proteins suggesting that there will be considerable structural similarity between the RNase inhibitor and the extracellular domain of the glycoprotein hormone receptors. However, binding of ligand to the receptor is likely to be quite different than binding of RNase to the inhibitor. The interaction between the RNase inhibitor and RNase resulted in the formation of several hydrogen bond and van der Waals contacts that stabilize the high affinity of the inhibitor for the enzyme. These included residues on much of the enzyme and portions of the inhibitor throughout its concave surface, albeit primarily at its C-terminal end. Binding of the two proteins resulted in a minor separation of the N- and C-terminal ends of the horseshoe(49) . The affinity of lutropins for their receptors is much weaker than binding of RNase to its inhibitor. Thus, fewer hormone and receptor residues appear to make key high affinity contacts. Unlike the RNase-inhibitor interaction, the apex of the receptor extracellular domain appears to have the dominant role in gonadotropin binding(31) . Indeed, the C-terminal end does not appear to form high affinity contacts with hCG and can be deleted without much effect on binding affinity(50) . Also, as noted earlier, substantial portions of both the hormones and the receptors can be changed without disrupting their functions(24, 31) . The difference in RNase-inhibitor and gonadotropin-receptor interactions is probably related to their functions. Effective inhibition of RNase appears to require a large number of specific high affinity contacts with only a small change in the shape of the inhibitor(49) . In contrast, a substantial change in the glycoprotein hormone receptors may be needed for signal transduction. As noted earlier this appears to be facilitated by steric effects caused by the oligosaccharides rather than by specific protein-protein contacts.

Signal Transduction by the Glycoprotein Hormones Is Fundamentally Similar to That Thought to Occur in Other G-protein-coupled Receptors

The discovery of the large extracellular domain of the LH receptor was viewed as surprising and suggested that signal transduction in these receptors might be different from that in other G-protein-coupled receptors(2, 3) . This was reinforced by the observation that the extracellular domain of the receptor was responsible for virtually all its high affinity hormone binding. The model we have described suggests that the gonadotropin receptors function more like the other G-protein-coupled receptors than initially expected. We consider the N-terminal domain of the glycoprotein hormone receptor to be an extension of the extracellular side of the transmembrane domain needed to accommodate the hormone. Because the extracellular domain and the transmembrane domain appear to be joined by a specific interaction(5) , a change in the extracellular domain would be rapidly transmitted to the transmembrane domain. The interaction between the extracellular and transmembrane domains appears specific for the gonadotropin receptors and cannot be replaced by any G-protein coupled transmembrane domain(5) . This explains why it is difficult to express the extracellular domains of these receptors separately from their transmembrane domains and why co-expression of the two domains is required to restore efficient signal transduction (51) . Disruption of the interaction between the extracellular and transmembrane domains would be expected to lead to desensitization.


FOOTNOTES

*
These studies were supported in part by National Institutes of Health Grants HD14907, HD24650, and HD15454. A preliminary account describing how the antibody binding site data illustrated here can be used to devise protein models using distance geometry algorithms has been published elsewhere (33). 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.

§
To whom correspondence should be addressed: Dept. of Obstetrics/Gynecology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 908-235-4224; Fax: 908-235-4225.

Present address: Ares Advanced Technology, Inc., 280 Pond Street, Randolph, MA 02368.

(^1)
The abbreviations are: hCG, human chorionic gonadotropin; LH, lutropin; hLH, human LH; FSH, follitropin; hFSH, human follitropin.

(^2)
Slaughter, S., Wang, Y., Myers, R. V., and Moyle, W. R. (1995) Mol. Cell. Endocrinol., in press.


ACKNOWLEDGEMENTS

We thank Drs. R. Canfield, S. Birken, A. Krichevsky, R. Ryan, G. Armstrong, R. Wolfert, S. Berube, and R. Krogsrud for the antibodies and hormone standards used in these studies. We thank Dr. Neil Isaacs for the crystal coordinates of deglycosylated hCG. Thanks also go to Drs. Irv Boime and Om Bahl for reading the manuscript.


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