©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Groove between the - and -Subunits of Hormones with Lutropin (LH) Activity Appears to Contact the LH Receptor, and Its Conformation Is Changed during Hormone Binding (*)

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

Laurey Cosowsky (1) S. N. Venkateswara Rao (1) Gordon J. Macdonald (2)(§) Harold Papkoff (3) Robert K. Campbell (1)(¶) William R. Moyle (1)(**)

From the  (1)Departments of Obstetrics/Gynecolgy and (2)Neuroscience and Cell Biology, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854 and (3)Department of Animal Science, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Gonadotropins are heterodimeric glycoprotein hormones that control vertebrate fertility through their actions on gonadal lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) receptors. The beta-subunits of these hormones control receptor binding specificity; however, the region of the beta-subunit that contacts the receptor has not been identified. By a process of elimination we show this contact to be the portions of beta-subunit loops one and three found in a hormone groove created by the juxtaposition of the alpha- and beta-subunits. Most other regions of the beta-subunit can be recognized by antibodies that bind to human chorionic hormone (hCG)-receptor complexes or replaced without disrupting hormone function. Using a series of bovine LH/hCG and human FSH/hCG beta-subunit chimeras we identified key hCG beta-subunit residues in the epitopes of two antibodies that bind to hCG-receptor complexes. These epitopes include the surfaces of beta-subunit loops one and three near residue 74 on the outside of the hormone groove and parts of the C-terminal end of the ``seat belt'' that holds the two subunits together. The antibody that recognized residue 74 bound to receptor complexes containing most mammalian lutropins better than to the free hormones, an indication that the outside surface of the beta-subunit groove is altered during hormone binding. This region of the beta-subunit is furthest from the alpha-subunit and is recognized equally well in the free beta-subunit and in the heterodimer. Thus, the receptor associated increase in antibody binding appears due to an interaction of this portion of the beta-subunit with the receptor and not to an effect of the receptor on the relative positions of the alpha- and beta-subunits. Unlike most previous studies designed to identify portions of the beta-subunit likely to contact the LH receptor, this indirect approach provides data that are more easily interpreted because it does not rely on the use of mutations that disrupt hormone function. The approach described here should be valuable for studying the receptor interactions of other complex ligands.


INTRODUCTION

The placental hormone hCG^1 and the pituitary gonadotropins hLH and hFSH are essential for vertebrate reproduction. These hormones are heterodimers composed of a conserved alpha-subunit and a hormone-specific beta-subunit(1) . The crystal structure of deglycosylated hCG (2) is likely to be representative of each glycoprotein hormone. Both subunits are composed of three large loops held together by a cysteine knot. Loops one and three of the beta-subunit form a pair held together by a disulfide at one end of the molecule; loop two is at the opposite end (Fig. 1). The subunits of hCG are aligned such that paired loops from one subunit are adjacent to the single loop of the other subunit. beta-Subunit residues 91-110 form an additional loop termed the ``seat belt'' that wraps around the second loop of the alpha-subunit to stabilize the heterodimer. The seat belt loop is primarily responsible for the ability of the beta-subunit to control receptor binding specificity(3, 4) . While the amino acid sequences of several gonadotropins(5, 6, 7, 8, 9, 10, 11, 12, 13) and their receptors (14, 15, 16) have been known for some time, little is known about the portions of the hormone that are responsible for their abilities to bind tightly to their receptors.


Figure 1: Ribbon diagram of hCG beta-subunit folding pattern. The loops described in the text are noted (dark blue). The oligosaccharides were added by modeling (red sticks). They are expected to be highly mobile at least until the hormone binds to the receptor. A few key residues in three major epitopes are illustrated here. Those in epitopes for dimer-specific antibodies are illustrated in red. Antibodies to these regions bind to hCG at the same time as antibodies to other residues noted on the diagram. Arg (orange label) is located at the tip of the third loop and is a key residue in the binding site of B105. Asn (cyan blue) is located nearby and is a key residue in the binding sites of antibodies B108 and B112. Residues 108-114 (green label) are found near the C terminus (C) and form the binding sites for B111. Note that residues 112-114 are not seen in the crystal structure and are not shown here. The upper and lower arrows noted for the seat belt point to its N and C termini.



Many efforts to identify gonadotropin residues that contact the receptor have employed hormone analogs that have lost all or part of their receptor binding abilities. Gonadotropin analogs made by chemical, enzymatic, and genetic modifications(1, 3, 4, 17, 18, 19, 20, 21, 22, 23) have shown that modification of several residues reduce receptor binding. These could be involved in essential receptor contacts. Unfortunately it is not always easy to interpret the results of this type of study since mutagenesis can alter the structure of the hormone in an unknown fashion. Since the free subunits do not bind to receptors(1) , any mutation that alters the interaction of the subunits has the potential to disrupt binding.

Other investigators have made extensive use of synthetic peptide pieces of the hormone to identify receptor binding sites(24, 25, 26, 27, 28, 29, 30) . A peptide corresponding to the second loop of the hCG beta-subunit has the greatest activity albeit only at concentrations exceeding 0.01 mM(24) . However, this region of hCG can be changed to the sequence found in the hFSH beta-subunit without reducing its ability to bind to LH receptors or to induce testosterone synthesis (3) . This observation suggests that the second beta-subunit loop does not participate in essential high affinity receptor contacts. At the high concentrations needed for activity in studies employing synthetic peptides (i.e. 0.01-10 mM), it is often difficult to distinguish specific and nonspecific effects.

Interpretation of data obtained using synthetic peptides is usually based on the assumption that the peptide binds to the receptor to block its interaction with the labeled hormone. However, the peptide might function by binding to the hormone and disrupting the interaction between the alpha- and beta-subunits. In the absence of data indicating that the peptide has no effect on the hormone, conclusions as to the binding site of the peptide may be misleading. Efforts to make more active compounds by combining several peptides that have low activity have been very disappointing(31, 32) .

The only methods to directly identify hCG residues that contact LH receptors involve use of x-ray crystallography and/or NMR spectroscopy. However, is it not yet clear that crystals of the hormone-receptor complex can be obtained that include the transmembrane domain. The complex is likely to be too big for current NMR techniques. To surmount these difficulties we have used a process of elimination to distinguish portions of the beta-subunit involved in key receptor contacts. This involves (i) finding mutations that do not alter the ability of the hormone to bind to receptors or elicit signal transduction and (ii) identifying the binding sites of monoclonal antibodies that bind receptor-bound hCG. Because the data obtained do not depend on analogs that have altered receptor binding or signal transduction activity, there is no risk that the results will be confounded by the influence of a mutation on the conformation of the hormone or the interaction between the subunits. Although this approach will not identify specific receptor contacts, it can be used to orient the hormone in the receptor and to delineate portions that are available to make high affinity receptor contacts.

A few anti-hCG beta-subunit antibodies to an overlapping epitope have been reported to bind to hCG-receptor complexes(33) . Using a series of hCG/hLH beta-subunit chimeras, we identified beta-subunit Asn as a key residue in the binding sites for two of these antibodies(33) . This approach was successful for identifying key residues in the binding sites of antibodies that recognize hCG much better than hLH. However, it could not be used with antibodies that had high affinities for both human hormones. Using hCG/bLH and hCG/hFSH beta-subunit chimeras we have now identified additional beta-subunit residues that form parts of epitopes that are exposed in hCG-receptor complexes. When these observations are combined with information on residues that can be changed without disrupting hormone function and with the locations of residues in the crystal structure of deglycosylated hCG(2) , they suggest that beta-subunit residues in the first and third loops adjacent to the second loop of the alpha-subunit are the parts of the beta-subunit most likely to form key contacts with the LH receptor. The conformation of this portion of the hormone is changed when lutropins bind to LH receptors.


MATERIALS AND METHODS

Hormones

Highly purified hCG (CR119 (11600 IU/mg) and CR121 (13450 IU/mg)) were obtained from Dr. Robert Canfield, Columbia University, New York, NY. The other species of vertebrate LH and CG were all prepared (H.P.) essentially by methods previously described (34, 35, 36) . These included several mammalian lutropins (ovine, equine, bovine, porcine, canine, leporine, rodent, and kangaroo), equine CG, three species of non-mammalian lutropins (ostrich, turtle, and bullfrog), and two non-mammalian follitropins (ostrich and bullfrog).

Antibodies

B410 was obtained from Dr. Janet Roser, University of California, Davis, Davis, CA, and corresponds to antibody 518B7 that has been described previously(37) . The sources of A113, B105, B111, and B112 (33) and the IODO-GEN procedure used to radioiodinate these proteins have also been described previously(38) . Radiolabeled antibodies had a specific activity of approximately 50 µCi/µg (i.e. 3-4 molecules of iodine per molecule of antibody).

Construction of Hormone Analogs

The structures of the bLH/hCG beta-subunit chimeras and the other analogs used in this study are illustrated in Table 1. Preparation of hCG/hFSH chimeras has been described elsewhere(3, 4) . Preparation of the bLH/hCG beta-subunit chimeras began with vectors pSVL-hCGalpha and pSVL-hCGbeta` that express hCG alpha- and beta-subunits, respectively, and that have been previously described(3) . The bLH beta-subunit cDNA was obtained from Dr. Scott Chappel (Ares Advanced Technology, Randolph MA). We prepared pSVL-bLHbeta, a vector that expressed bLH beta-subunit in COS-7 cells, by removing the cDNA from the source vector by HindIII digestion, filling in the ends using the Klenow fragment of DNA polymerase, and ligating the blunt-end fragment into the SmaI site of pSVL (Pharmacia Biotech Inc.). Vectors which expressed hCG/bLH beta-subunit chimeras CbLbeta36-145, CbLbeta1-35, CbLbeta88-145, CbLbeta1-87, CbLbeta103-145, and CbLbeta1-102 were constructed using three conserved restriction sites (i.e. NaeI, PvuII, and PpuMI) present in the hCG and bLH beta-subunit cDNAs (Fig. 2). These enabled us to scan the beta-subunit to identify regions likely to contain the B105 epitope. Analogs containing fewer hCG or bLH beta-subunit residues were prepared by polymerase chain reaction-directed mutagenesis using a technique termed ``splicing by overlap extension'' (37) and the primers shown in Table 2. In this procedure the N-terminal (i.e. ``upstream'') and C-terminal (i.e. ``downstream'') coding regions of the desired analog were made in separate polymerase chain reactions and then these polymerase chain reaction products were combined to give the full-length construct.




Figure 2: Schematic illustration of the restriction site swapping that was used to prepare bLH and hCG chimeras. Note the chimeras are named by their content of hCG beta-subunit residues. Thus, CbLbeta1-35 represents a chimera in which residues 1-35 are derived from hCG beta-subunit and the remainder are from bLH beta-subunit.





DNA Isolation, Cell Culture, and Transfections

Plasmid DNA was isolated by alkaline lysis and purified by polyethylene glycol precipitation(39) . COS-7 cells maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM glutamine were co-transfected with pSVL-hCGalpha and the appropriate beta-subunit coding vector using a calcium phosphate precipitation procedure(40) . After a 24-h incubation at 37 °C the cell monolayers were washed, and the media were replaced with fresh serum-free Dulbecco's modified Eagle's medium. Three days later, the media were collected, centrifuged to remove cellular debris, and concentrated approximately 50-fold. This was accomplished by placing the media into a dialysis bag and laying the bag in Aquacide II (Calbiochem). Residual salts were removed by dialysis against 0.02 M phosphate buffer (pH 7.4).

Antibody Assays

To learn which vertebrate gonadotropins bind B105, we employed a solid phase assay that measured the ability of radiolabeled antibodies to bind hormone adsorbed to the wells of a polystyrene microtiter plate. Wells were incubated with 1 µg of hormone diluted in 50 µl of 0.02 M sodium phosphate, 0.9% NaCl solution (pH 7.2). This permitted the hormone to adsorb to the surface. After 1 h at 37 °C, the solution was replaced with 200 µl of the buffer containing 200 µg of bovine serum albumin for an additional hour at 37 °C to block the adsorption sites remaining on the plastic surface. Next, the albumin solution was replaced with 50 µl of albumin solution containing 2-3 ng of radiolabeled B105 (approximately 50,000-150,000 cpm) to permit the B105 to bind to the adsorbed hormone. After a final hour at 37 °C, the radiolabeled solution was aspirated, the wells were rinsed with albumin containing buffer, the plates were cut apart with scissors, and each well was analyzed in a gamma counter. For controls we measured the binding of labeled antibody to wells that had not been exposed to hormone and, in some cases, to wells that contained an excess (1 µg) free hormone or unlabeled antibody added together with the radiolabeled antibody. All three controls had the same level of background.

To quantify the hCG/bLH beta-subunit chimeras made in cell culture we used a monoclonal sandwich immunoassay procedure that has been described previously (41) except that A113, an antibody to the alpha-subunit of hCG, was used to capture the analogs, and either radiolabeled B105 or B410 were used as detection reagents. B410 is an antibody that binds hCG and bLH with high affinity (42) and was used to estimate the amount of dimer in the media. The amounts of culture media that gave similar activity for the analogs in the B410 sandwich assay were used to assess B105 binding.

Receptor Binding Assays

We employed a bio-immunoradiometric assay (Bio-IRMA) to determine the position of the antibody binding site relative to that of the receptor(43) . This assay depends on the ability of a radiolabeled monoclonal antibody to bind to hormone-receptor complexes and has been described in detail elsewhere (43) . To detect differences in the affinity of antibodies for free and receptor bound hormones we performed the assay in a simultaneous fashion (i.e. the membrane bound receptors, hormone, and radiolabeled B105 were mixed together at the start of the experiment). After 1 h at 37° the reaction mixture was diluted to 2 ml with a 0.9% NaCl solution containing 1 mg of bovine serum albumin/ml, and the membranes were pelleted by centrifugation at 1000 g. The supernatant was aspirated, and the radiolabel remaining in the pellet was analyzed in a gamma counter. Bio-IRMA designed to characterize the abilities of B105, B108, B111, and B112 to bind to hCG-receptor complexes were also performed in a sequential fashion. Briefly, we incubated homogenates of rat ovaries with amounts of hCG that permit them to bind to LH receptors in the membranes. The membranes were collected by sedimentation at 1000 g and resuspended in hormone-free buffer. Radiolabeled antibody was added and permitted to bind to hormone-receptor complexes in the membranes. Bound and free labels were separated by sedimenting the membranes at 1000 g and the label bound to membrane hormone-receptor complexes was analyzed in a gamma counter.

Procedures for measuring the abilities of hormone analogs to bind to LH receptors by competition with I-hCG have been described elsewhere(41) . 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(41, 43, 44) . Briefly, the antibody was incubated with hCG for 30-60 min prior to addition of cells. After further incubation at 37 °C for 60 min, the cells or membranes were collected by centrifugation and radiolabeled hCG that had been bound was measured in a gamma counter. As a control, we monitored the ability of unlabeled hCG to block the binding of the radiolabeled hCG.

Model of hCG

A crystal structure of deglycosylated hCG has recently been published (2) and the coordinates were kindly given to us by Dr. Neil Isaacs. We added oligosaccharides to this structure using the Sybyl modeling package (Tripos Associates, Inc., St. Louis, MO). Following energy minimization and molecular dynamics calculations in which the positions of the Calpha coordinates were held constant, we arrived at a structure that had no bad contacts and that should be reflective of glycosylated hCG.


RESULTS

The Most Conserved beta-Subunit Epitope Does Not Contact the Receptor

When these studies were started, we expected the most conserved part of the lutropin beta-subunit to be nearest the LH receptor interface. This is because a large number of lutropins are able to bind to rat LH receptors and stimulate steroidogenesis. Surprisingly, antibodies that bound to several different mammalian lutropins (i.e. B105, B110, and B410) also bound to these hormones when they had bound to rat LH receptors, indicating that a conserved epitope was exposed in the hormone-receptor complex (see Fig. 3for data on B105, remainder of data not shown). Studies were then designed to identify amino acid residues that were part of this epitope. To accomplish this we focused on identifying the binding site for antibody B105, an antibody that had high affinity for hLH and hCG and very low affinity for most other mammalian lutropins including those from cattle, rabbits, pigs, dogs, horses, rats, and kangaroos (data not shown). Except for sea turtle LH, B105 did not bind to LH from most non-mammalian species including those from ostriches or bullfrogs. B105 did not bind to hFSH, equine FSH, ovine FSH, ostrich FSH, or bullfrog FSH. This indicated that the binding we observed was specific to a conserved mammalian lutropin epitope that remains exposed when these hormones bind to rat LH-receptor complexes.


Figure 3: Simultaneous Bio-IRMA of hCG and several mammalian LH. hCG and various mammalian LH were incubated with rat luteal LH receptors and radiolabeled B105 in a simultaneous Bio-IRMA as previously described(43) . Values shown are the amount (count/min ± S.E.) of radiolabeled B105 that was bound to the rat receptors in the presence of different amounts of hormone. The bars above the arrows indicate values obtained in the presence of 0.01 ng of hormone. The bars illustrated sequentially to the right of that marked by the arrow refer to 0.1 ng (&cjs2090;), 1 ng (&cjs2089;), 10 ng ( ), 100 ng ( ), and 1000 ng (black square) of hormone, respectively. Neither FSH nor any of the non-mammalian hormones tested gave measurable activity in this assay (not shown).



To identify residues likely to be in the B105 epitope, we used a homology scanning mutagenesis procedure that depended on measuring antibody binding to chimeras of hCG and bLH beta-subunits. The beta-subunits of hCG and bLH were used to make the chimeras because these hormones had high and low affinities for B105, respectively. Thus, chimeras that bound B105 well would contain hCG residues in the B105 epitope, respectively; whereas those that bound B105 poorly would have bLH residues in the B105 epitope. As a control to determine the amount of chimera made by the transfected COS-7 cells, we measured the binding of the chimeras to B410, an antibody that had high affinity for both hCG and bLH. B105 bound well only to those LH analogs that contained Arg (Table 3). This included hCG/bLH beta-subunit chimeras that had hCG residues 36-145, 1-87, and 1-102 (i.e. CbLbeta36-145, CbLbeta1-87, and CbLbeta1-102). B105 did not bind to hCG/bLH chimeras that contained bLH residues in these regions (i.e. CbLbeta1-35, CbLbeta88-145, and CbLbeta103-145). An analog of hCG (hCGbeta:R74P) containing a single substitution (i.e. Arg was replaced by Pro, the corresponding residue from bLH beta-subunit) did not bind B105 any better than bLH. Conversely, an analog of bLH (bLHbeta:P74R) in which Pro was converted to Arg bound B105 with the same high affinity as hCG. This indicated that the conserved epitope was formed by a region of hCG near Arg, namely the third beta-subunit loop(2) .



Other Surfaces of the beta-Subunit Are also Exposed in the hCG-Receptor Complex

We observed that several antibodies bound to hCG-receptor complexes (see Table 4for a partial list). Since antibodies are large proteins, this suggested that a substantial percentage of the beta-subunit surface was not obscured by the LH receptor or other nearby proteins. To test this idea and to identify those antibodies that bound to a different region of the beta-subunit, we mapped the binding sites of all the hCG antibodies relative to one another (see Fig. 4for a partial map). These maps showed that the exposed surface was large enough to be recognized by antibodies that could bind to hCG at the same time. For example, antibodies B105, B111, and B112 were all found to bind to hCG-receptor complexes (Table 4). B112 and B111 bound to hCG at the same time, yet each competed for binding to hCG with B105. The B112 binding site has been shown to involve Asn(33) and we focused on identifying residues that were involved in the B111 site.




Figure 4: Epitope map of selected beta-subunit antibodies. Sandwich assays were performed in which hCG was captured using each of the antibodies listed and detected using radiolabeled B105, B108, B111, or B112. Epitopes for antibodies that bind to hCG at the same time are shown in different or non-overlapping circles. Epitopes for antibodies that do not bind hCG at the same time are shown in the same or overlapping circle.



To identify key residues in the B111 binding site, we used a series of hCG/hFSH chimeras that contained hCG beta-subunit residues 1-93 and varying numbers of hFSH residues between amino acids 94 and 145. While all of these were capable of binding B105, only a few were able to bind to B111 (Table 5). Comparisons of the sequences of the analogs (Table 1) with their abilities to bind B111 (Table 5) showed that the B111 binding site involved residues 108-114. For example, B111 did not bind CFC94-114 and had only minimal ability to bind to CFC101-114, suggesting that substitution of FSH residues in place of hCG beta-subunit residues 94-114 or 101-114 destroyed the B111 binding site. B111 bound CFC101-106 like hCG and much better than CF101-109114 and hCGbetaG103V114. Because residue 107 is conserved in hCG and the hCG/hFSH beta-subunit chimeras, these observations suggest that residues 108-114 account for most of the B111 binding site. Residues C-terminal of 114 also appear to have a small influence on B111 binding since B111 had reduced affinity for analogs that were truncated at residue 114 (i.e. CF101-109114 and hCGbetaG102Vbeta114). Because B111 binds to hCG-receptor complexes and recognizes residues 108-114, we conclude that the region of hCG that includes the C-terminal portion of the seat-belt loop does not contact the LH receptor.



Antibodies That Bind to hCG-Receptor Complexes Inhibit hCG Binding to Receptors

We observed that most antibodies that bind to hCG-receptor complexes can also inhibit binding of hCG to rat LH receptors (Fig. 5). This suggests that antibodies can inhibit receptor binding even though they recognize epitopes that are not part of the high affinity hormone binding site. Most of these block binding only partially, yet those that appear to bind closest to the part of the hormone that interacts with the receptor (e.g. B108 and B112) block receptor binding completely. Not all antibodies bind to hCG-receptor complexes. Key residues in the binding sites of many antibodies that do not bind to hCG-receptor complexes are known (33) , and we had hoped that these data would provide additional information on portions of the beta-subunit likely to contact LH receptors. Most antibodies that do not bind to hCG-receptor complexes recognize part of the second loop and residues 91-94 of the seat belt. They bind to hCG at the same time as B105 (Fig. 4) and are good inhibitors of hCG receptor binding. While these observations do not prove that these portions of the beta-subunit contact the receptor, they are consistent with the idea that the second loop and the N-terminal part of the seat belt are near the hCG receptor interface.


Figure 5: Comparison of the abilities of antibodies B105, B108, B111, and B112 to inhibit binding of hCG to rat LH receptors. The antibodies were added to I-hCG prior to adding the radiolabel to CHO cells expressing rat LH receptors or to rat luteal membranes. Values are means of triplicates and the bars extend to the limit of the S.E.



The Conformation of the beta-Subunit Is Altered when hCG Binds to LH Receptors

Using a Bio-IRMA(43) , we found that the affinity of B105 for most non-human mammalian lutropins was increased when they were complexed with rat LH receptors (Fig. 3). While we did not measure this increase in affinity directly, it must have been substantial because the assay was performed by incubating the hormone, membrane-bound rat LH receptors, and radiolabeled antibody B105 in the same tube. In this format the antibody can bind to free hormone as well as to hormone-receptor complex; the receptor can bind to free hormone or antibody-hormone complex. When less than 4 ng of hCG (i.e. 0.1 pmol) were used in the assay, the amount of radiolabeled B105 bound to the hormone-receptor complex was proportional to the amount of hCG bound to the receptor. When a higher amount of hCG was used in the assay, the amount of hCG bound to both the antibody and the receptor was also increased, but binding of labeled B105 to hormone-receptor complexes was reduced. This is because B105-hCG-complexes are unable to bind to LH receptors that are already occupied by hCG. We estimated the magnitude of the increase in affinity of B105 for the receptor-bound hormone by comparing the binding of B105 to hCG- and lutropin-receptor complexes when large amounts of the hormones were used in the assays. In the assays illustrated in Fig. 3there was a massive excess of hormone relative to receptor when 1 µg of hormone (i.e. 25 pmol of hCG or 30 pmol of lutropin) was mixed with 0.1 pmol of receptors and 0.01-0.02 pmol of radioiodinated B105. When 1 µg of hCG was used in this assay, the B105 and the receptors were almost fully occupied by hCG, and less than 0.3% of the radiolabeled B105 became bound to the hormone-receptor complex. In contrast, when 1 µg of any non-human mammalian LH was mixed with receptors and B105, we observed that 7-10% of the B105 became bound to hormone-receptor complexes. By dividing the percentage of B105 bound when 1 µg (30 pmol) of non-human mammalian LH was used in the assay by that percentage observed when 1 µg (25 pmol) of hCG was used in the assay, it can be seen that the affinity of B105 for receptor-complexes containing any non-human lutropin was at least 20-fold greater than that for the free hormone, and probably much more. This strongly suggests that binding of bLH and other non-human lutropins to receptor complexes is accompanied by a change in the molecule that reduces the inhibitory influence of Pro on B105 binding to mammalian lutropins.

Unexpectedly, eCG was much less active than eLH in the Bio-IRMA (Fig. 6). This was surprising since (i) both eLH and eCG have the same primary amino acid sequences(45) , (ii) both can be recognized by B105 (not shown), and (iii) both bind to LH receptors (Fig. 5). The only difference between eLH and eCG is the extent and type of their glycosylation. This observation suggests that the oligosaccharides of eCG block binding of B105 to eCG, but only when the hormone has bound to LH receptors.


Figure 6: Panel A, inhibition by hCG, eLH and eCG of radiolabeled hCG binding to the rat luteal receptor. All the other mammalian LH (not illustrated) used in these studies had activities equal to or lower than that of eLH. Panel B, activities of hCG, eLH, and eCG in the sequential Bio-IRMA. See the text and Moyle et al.(43) for the details of this assay. Values are means of duplicates and the bars extend to the individual measurements.




DISCUSSION

The Binding Sites Reported Here Are Consistent with the Crystal Structure of Deglycosylated hCG and with the Epitope Map

A summary of the antibody binding site data described here is shown in Table 6. Use of this approach to identify portions of hCG that are exposed in hormone-receptor complexes depends on the correct identification of antibody binding sites. The consistency of the data with the crystal structure of hCG provides strong support for the conclusions that we have drawn about the identities of beta-subunit regions that are recognized by antibodies and the proposed orientation of the antibody binding sites. The idea that Arg is an important component of the B105 epitope agrees with the observations that all bLH/hCG chimeras containing Arg at residue 74 bound B105 well and that all chimeras containing Pro at this site bound B105 poorly. However, given that antibody combining sites are usually much larger(46) , other residues including those common to both bLH and hCG beta-subunits are likely to be involved in the B105 epitope. While we did not attempt to identify other residues in the B105 site, we observed that B105 competed for binding to hCG with B111 and B112, two antibodies that can bind to hCG at the same time. Thus, the surface recognized by B105 would be expected to lie between those recognized by B111 and B112. This is consistent with the crystal structure of chemically deglycosylated hCG and the location of the key residues in the B105, B111, and B112 binding sites(2) .



Substantial Portions of the hCG beta-Subunit Face Away from the Receptor Interface after hCG Binds to LH Receptors

Epitope maps suggest that most anti-beta-subunit antibodies bind to one of three regions in hCG. The first includes the second loop and the N-terminal part of the seat belt(33) . Most antibodies to this region (i.e. sites for B101, B107, and B109) bind to hCG much better than to the free beta-subunit. The second epitope region includes the binding sites for antibodies that recognize epitopes in loops one and three (i.e. the binding sites for antibodies B105, B108, B110, B111, B112, and B410). The third region includes the binding site for antibodies that recognize the C-terminal part of the seat belt loop (i.e. antibody B111). Surprisingly, two of these three epitope regions can be detected in hCG-receptor complexes. Antibodies are large proteins and the observation that at least two of the three major beta-subunit epitope regions can be detected after hCG binds to LH receptors implies that only a small fraction of the beta-subunit surface is available to participate in essential high affinity contacts with the LH receptor.

All of the beta-subunit antibodies that bound to hCG-receptor complexes inhibited hCG binding, albeit to different degrees. For example, B108 and B112 inhibited hCG binding considerably better than antibodies B105 and B111. This was related to the surface of the beta-subunit to which they bind. The surface recognized by B108 and B112 is closer to the alpha-subunit interface than that recognized by either B105 or B111(2) . Thus, binding of B108 and B112 may be near the hormone-receptor interface or in the pathway taken by the hormone on its way to contacting the receptor.

Parts of the beta-Subunit That Contact LH Receptors

Our goal was to distinguish portions of hCG beta-subunit that do not contact the LH receptor and to combine this information with the crystal structure to identify regions that would be available to interact with the receptor. Portions of the beta-subunit noted earlier that can be recognized by antibodies that bind to hCG-receptor complexes clearly do not contact LH receptors. However, this strategy can detect only those portions of the hormone for which we have antibodies and which are likely to be oriented away from the receptor. Other parts of the beta-subunit near the receptor interface that do not form contacts needed for high affinity binding can be deduced by comparing the abilities of mammalian lutropins or mutant analogs to bind rat LH receptors.

Second beta-Subunit Loop (Residues 38-57)

Initially, we thought that the second loop would contact the receptor and control high affinity binding. The idea that residues 38-57 contact the receptor was supported by the observations that peptides from this area can inhibit hCG binding to LH receptors (24) and by our observation that antibodies B101(41) , B107, or B109 (^2)do not bind to hCG-receptor complexes. These antibodies recognize the second beta-subunit loop and/or residues 91-94 in the seat-belt(33, 47) . Thus, we were astonished to find that analogs of hCG in which beta-subunit residues were replaced with the corresponding residues from hFSH beta-subunit bound to LH receptors and elicited signal transduction indistinguishably from hCG(3) . Only Thr, Val, and Gln are common to this region of hCG and hFSH. While it is conceivable that these beta-subunit sequences are responsible for high affinity binding of both hCG and FSH, this seems unlikely. The abilities of the synthetic peptides to bind LH receptors may be explained by the observation that they were active only at superpharmacological concentrations (i.e. >0.01 mM). Our failure to detect B101, B107, or B109 binding to receptor bound hCG is consistent with the idea that portions of the second loop and the seat belt are obscured by the LH receptor even though this region does not appear to make key receptor contacts.

Seat Belt

Because it is responsible for gonadotropin receptor binding specificity(4) , the seat belt (i.e. residues 91-110) would be expected to contact the LH receptor. Residues 91 and 92 are unlikely to participate in LH receptor binding since they differ in hCG, hLH, and in many naturally occurring analogs that have high affinity for the LH receptor (Table 1). Residues between Cys and Cys are also unlikely to contact LH receptors since they can be changed to the sequence found in FSH beta-subunit without disrupting LH receptor binding or efficacy(4) . The remaining seat belt region between Cys and Cys, termed the ``determinant'' loop, has been proposed to have a role in LH receptor binding(48) . However, with the exception of residue Cys, Asp, and Cys, this region is not well conserved among most lutropins that bind to the rat LH receptor. Indeed, we have found that most hCG analogs that have at least one positive charge between residue 94-96, either Ser or Thr for residues 97 and 98, and Asp have high affinity for LH receptors(4) .^2 The role of Asp in receptor contact cannot be determined since mutations of this residue reduced hCG function(21) . Loss of function mutations have proven valuable for identifying receptor contacts in growth hormone(49, 50) . However, these are of limited use in interpreting the interaction of gonadotropins with their receptors. Unlike growth hormone, gonadotropins are heterodimers whose activity depends on the correct interaction of both subunits with one another and/or with the receptor. Thus, loss of function caused by a mutation could be due to an effect on a receptor contact or to an influence on the interaction between the subunits needed for hormone activity. Therefore, in the absence of data showing that a mutation of Asp does not alter the interaction between the subunits, conclusions about its role in receptor contacts are premature.

N Terminus of the beta-Subunit

Deletion of the N terminus of the beta-subunit strongly inhibits its ability to combine with the alpha-subunit(51) . However, the small amount of dimer that does form is biologically active, suggesting that this region does not contact the receptor. This observation also suggests that the ability of a beta-subunit mutant to combine with the alpha-subunit conveys little about the conformation of the subunits in the heterodimer and, by itself, is not sufficient to prove that the conformation of the heterodimer has not been altered by a specific mutation.

Portions of the Twin Loops Containing the N-Linked Oligosaccharides

Another portion of the beta-subunit unlikely to form specific contacts with the LH receptor is the region that contains N-linked oligosaccharides at residues Asn and Asn. These can be removed without reducing the affinity of the resulting analog for the LH receptor(52) . Indeed, removal of the oligosaccharides is usually associated with an increase in affinity. The influence of the oligosaccharides is limited to hormone efficacy, an observation that is explained by the model of hCG-receptor interaction in a companion study(53) .

The Remaining Region of hCG beta-Subunit Is the Portion of the Protein in Loops One and Three That Face alpha-Subunit Loop Two

Models describing the interaction of the gonadotropin beta-subunit and the receptor need to account for the observations that a substantial portion of the beta-subunit of receptor-bound hCG can be recognized by monoclonal antibodies and that most residues in the remainder of the molecule can be changed without disrupting high affinity LH receptor binding or signal transduction. The regions that remain include residues of loops one and three that face alpha-subunit loop two, beta-subunit residues that are moderately conserved in most mammalian glycoprotein hormones.

The Conformation of the Third beta-Subunit Loop Is Changed after hCG Binds to LH Receptors

The observation that B105 binds better to most mammalian lutropins when they are bound to LH receptors suggests that the conformations of the hormones are altered after they bind to receptors. This finding is consistent with the idea that portions of the beta-subunit loops one and three nearest the alpha-subunit interface participate in receptor binding. To accommodate the receptor, loops one and three may need to be moved slightly. Given the internal hydrogen bonds and the disulfide between Cys and Cys, a slight movement of residues in the surface of these loops nearest the alpha-subunit could be transmitted to the antibody binding sites found on the ``backside'' of these same loops.

Other features of the lutropins also appear to be changed when they bind to LH receptors. Unlike the binding of B105 to most mammalian lutropins, the affinity of B105 for hCG-receptor complexes was lower than that for hCG(44) . Since hCG contains a long C-terminal extension, the reduction in affinity might be caused by a change in the position of the C-terminal extension of hCG after receptor binding. However, other observations argue against this idea. As shown here (Fig. 3) B105 bound to eLH-receptor complexes better than to eLH, yet B105 bound to eCG much better than to eCG-receptor complexes (Fig. 6). This implies that the N-linked or O-linked oligosaccharides are more important than the C-terminal extension. Both eLH and eCG have the same amino acid sequence and contain a C-terminal extension like hCG. The hormones differ primarily in their oligosaccharides. The N-linked oligosaccharides of eCG are di- and tri-branched and contain large amounts of sialic acid whereas those on eLH are mono- or di-branched and more often sulfated than sialylated(54) . Thus, the N-linked sugars on eCG are considerably bulkier than those on eLH and might interfere with the binding of B105 to eCG LH-receptor complexes. We have proposed a model of hCG receptor interaction in which the oligosaccharides influence signal transduction by their bulk (53) . In this model the positions of the N-linked oligosaccharides would be expected to be different in the free hormone and in the receptor bound hormone. If the position of oligosaccharides were constrained such that they were directed more toward the B105 binding site when the hormone bound to the receptor, the increased sizes of the oligosaccharides in eCG might interfere with B105 binding. This would account for the ability of B105 to bind to receptor complexes that contain eLH but not the bulkier eCG.

In conclusion, these observations suggest that the portions of the beta-subunit most likely to contact the LH receptor are nearest its interface with the second alpha-subunit loop. This is in a region of the beta-subunit that appears to have a different conformation in receptor-bound hormone. Most other regions of the beta-subunit do not appear to make specific high affinity hormone contacts, an important consideration in the model of hormone-induced signal transduction proposed in a companion study(53) .


FOOTNOTES

*
The preparation of the LH from different species was supported in part by National Institutes of Health Grant HD05722 (to H. P.) and National Science Foundation Grant DB-8710021 (to H. P.). The remaining studies were supported by National Institutes of Health Grants HD14907, HD24650, and HD15414. 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.

§
Supported by a grant from the UMDNJ Foundation.

Current address: Ares Advanced Technology, Inc., 280 Pond St., Randolph, MA 02368.

**
To whom all 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.

(^1)
The abbreviations used are: hCG, human chorionic gonadotropin; LH, luteinizing hormone; eLH, equine LH; eCG, equine CG; oLH, ovine LH; bLH, bovine LH; pLH, porcine LH; lLH, leporine (rabbit) LH; pLH, porcine LH; kLH, kangaroo LH; fLH, bullfrog LH; cLH, chicken LH; hFSH, human follicle-stimulating hormone; hTSH, human thyroid-stimulating hormone; Bio-IRMA, biological receptor based immunoradiometric assay. Chimeras of hCG and hLH beta-subunits are named on the basis of their hCG content. For example, CbLbeta1-35 contains hCG residues 1-35 and bLH residues 36-121.

(^2)
W. R. Moyle, unpublished observations.


ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Donna M. Anderson and Diana Dean-Emig at various stages of this research. We thank Dr. Neil Isaacs for the crystal coordinates of deglycosylated hCG. We thank Drs. Robert Canfield, Glenn Armstrong, Robert Wolfert, and Steve Birken for hormones and antibodies used in these studies. We also thank Drs. Irving Boime and Om Bahl for reading the manuscript.


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