Residues in the a! Subunit of Human Choriotropin That Are Important for Interaction with the Lutropin Receptor*

Synthetic peptides were used to probe the structure- function relationships between human choriotropin (hCG) and the lutropin (LH) receptor. Previously, a peptide region of the a subunit of hCG, residues 26-46, had been shown to inhibit binding of ‘‘‘I-hCG to the LH receptor in rat ovarian membranes (Charles- worth, M. C., McCormick, D. J., Madden, B., and Ryan, R. J. (1987) J. Biol. Chern. 262, 13409-13416). To determine which residues are important for this inhibitory activity, peptides were truncated from either the amino or carboxyl terminus, or individual residues were substituted with alanine. The amino-terminal boundary was determined to be Gly-30 and the car-boxyl-terminal boundary, Lys-44. This core peptide contained all the residues needed for full activity of the parent peptide 26-46. Arg-35 and Phe-33 were particularly important residues; when they were substituted with alanine, the peptide inhibitory potencies were decreased. Ser-43, Arg-42, Cys-32, and Cys-31 were also important but to a lesser degree. These results are consistent with predictions based on chemical and enzymatic modification studies and provide insight into which residues are important for interaction between hCG and the LH receptor.

and Ryan et al. (1987Ryan et al. ( , 1989). Although chemically deglycosylated and enzymatically desialylated hCG have been crystallized (Harris et al., 1989;Lustbader et al., 1989), no high resolution structural data is yet available. In the absence of a structure, chemical modification experiments (summarized in Pierce and Parsons (1981)) have demonstrated the importance of particular amino acids for hormone-receptor interactions, as well as subunit interactions.
Synthetic peptides have been used to study the structurefunction relationships between the glycoprotein hormones and their receptors because large amounts of chemically defined and pure material can be generated. Peptides that duplicate defined regions of the a subunit can inhibit binding of lZ51-hCG to the LH receptor in rat ovarian membranes  and can inhibit binding of lZ51-TSH to FRTL-5 cell membranes (Morris et al., 1988). Peptides representing various regions of the @ subunits of hCG , hTSH (Morris et al., 1990), or human follitropin (Santa Coloma and Reichert, 1990) also inhibit binding of radioiodinated hormone to receptor. Additionally, some LY and @ peptides exhibit low but significant biological activity Santa Coloma and Reichert, 1990;Erickson et al., 1990).
Charlesworth et al. (1987) defined a region in the a subunit with high inhibitory activity (IC50 value of 10 pM) in a competitive LH/CG receptor binding assay. This region, a26-46 ( Fig. I), is particularly well conserved across species lines. In several different species, from carp to human, there are 17 of 21 identical residues (Ward and Bousfield, 1990). This region includes Met-29 and Cys-31, which seem to be involved in or near the receptor-binding site, as well as Tyr-37 and Lys-45, which seem to be located in or near the subunit interface region (Pierce and Parsons, 1981). To analyze more precisely which residues are important for receptor interaction, we synthesized a series of peptides truncated from either the amino or carboxyl terminus and a series of peptides in which each residue was individually substituted with alanine. These studies indicate that 1) the core sequence of 30-44 is necessary for full inhibitory activity and 2) Arg-35 and Phe-33 are particularly important for activity, whereas Ser-43, Arg-42, Cys-32, and Cys-31 are necessary but not as crucial for full activity. These results provide insight into which amino acid residues are important for interactions between hCG and the LH receptor and, by analogy, residues important for the interactions between the other glycoprotein hormones and their receptors.

MATERIALS AND METHODS
Peptide Synthesis and Purification-Peptides were synthesized by standard solid phase techniques (Stewart and Young, 1984) either manually or using an automated AB1 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA). Ten truncation peptides were synthesized on p-methylbenzhydrylamine resin with t-butylox- 1. Sequence of the human a subunit. The 01 subunit is the same within a species and highly conserved between species. The peptide 26-46 is underlined.

A P D V Q D C P E~T L Q E N P F F S~~P G A P I L Q C M~C C F S R A Y P T P L R S K K T M L V i K N V T S E S T C E V
ycarbonyl-L-amino acid derivatives added as symmetric anhydrides. Arginine was coupled as the 1-hydroxybenzotriazole active ester. Completed peptides were cleaved from the resin and side chain protecting groups removed by treatment with liquid hydrogen fluoride containing 10% anisole and ethanedithiol, v/v, for 1.5 h a t 0 "C. The remaining truncation peptides were synthesized on DMPP resin with Fmoc-L-amino acid derivatives. Amino acids were coupled onto the growing peptide as the 1-hydroxyhenzotriazole active esters. The peptides substituted with alanine were synthesized on DMPP resin by a modification of the "tea bag" protocol (Houghten, 1985) using activated pentafluorophenyl esters of Fmoc-L-amino acid derivatives. All peptides synthesized with Fmoc derivatives were cleaved from the resin and deprotected by treatment with 91% trifluoroacetic acid, containing 2% anisole, 2% ethanedithiol, and 5% H20, v/v, for 1.5 h at 25 "C. Peptides removed from p-methylbenzhydrylamine and DMPP resins have carboxyl-terminal amides that are used for internal sequences to avoid the carboxyl-terminal charge. Crude peptides were purified by column chromatography (Sephadex G-25, Pharmacia) in 1 M acetic acid and/or reversed phase high performance liquid chromatography (Vydac C-4, a linear gradient of 5-80% acetonitrile in 0.1% trifluoroacetic acid over 40 min). Amino acid composition was verified by analysis of acid hydrolysates (1 h a t 156 "C or 24 h a t 100 "C in 6 M HC1, in uacuo) using precolumn derivatization with phenylisothiocyanate (Heinrikson and Meredith, 1984) and the Waters PicoTag system (Waters Associates, Millipore Corp., Milford, MA). The composition of each peptide was in good agreement with the expected sequence.
hCG/LH Radioligand Receptor Assay-Competition assays were used to measure the inhibitory activity of each peptide. Briefly, hCG (radioiodinated by the method of Greenwood and Hunter (1963) as modified in Lee and Ryan (1972)) was incubated with varying amounts of peptide and 1.25-2-mg equivalents of ovarian membrane preparation (Lee and Ryan, 1973) in assay buffer (40 mM Tris, pH 7.3, 0.025% bovine serum albumin, w/v) for 12-16 h at 25 "C in a shaking water bath. Membrane-bound "'I-hCG was separated from free '""IhCG by centrifugation. The membrane pellets were washed with assay buffer, then counted in a Beckman Gamma 4000 y counter. Total assay volume was 500 pl. Nonspecific binding was determined in parallel tubes that were supplemented with 100-fold excess unlabeled hCG. The percentage of specific binding remaining (%Bo) was calculated for each concentration of standard hCG or peptide. Competition curves (Fig. 2) were generated, and ICs0 values were determined. The IC,,, is that concentration of standard hCG or competitor peptide that inhibits 50% of the '"I-hCG binding to the LH receptor in the membrane preparation.
Hormone Preparations and Chemicals-hCG (-3000 IU/mg) was obtained from Organon Pharmaceuticals and purified as described . Chemicals used for peptide synthesis and purification were supplied by Aldrich, Applied Biosystems, Inc., and Burdick & Jackson Laboratories Inc. (Muskegon, MI). Amino acids were purchased from Advanced Chemtech (Louisville, KY), Applied Biosystems, Inc., and Bachem (Torrence, CA). Resins were obtained from Applied Biosystems, Inc. and Calbiochem. All other reagents were of the highest reagent grade available.
Circular Dichroic Analysis-CD spectra were recorded using a Jasco shown. Increasing concentrations of standard hCG or test peptides were incubated with '"I-hCG and 1.25-2-mg equivalents of ovarian membrane preparation in 40 mM Tris, pH 7.3, 0.025% bovine serum albumin for 12-16 h a t 25 "C. After separation of membrane-bound '*'I-hCG from free "'I-hCG, the percentage of specific binding remaining (%&) was calculated, and competition curves were generated. The IC50 value is that concentration of standard hCG or peptide that inhibits 50% of the "'I-hCG binding to the membrane preparation. Nonspecific binding was less than 10% of total binding. The average ICs,, for hCG was 0.15 2 0.03 nM.
Statistics-Each peptide was tested at multiple concentrations in at least four different experiments. Each assay was done in duplicate.
The mean ICso for each peptide was compared with the mean IC,, for the control peptides using the Student's t test (Brown and Hollander, 1977). Significance was established at p < 0.01.

RESULTS
Amino-terminal Truncations-To define the amino-terminal boundary required for inhibitory activity, a nested set of peptides was synthesized. Each peptide was shortened by 1 residue from the amino terminus. In addition, three peptides were synthesized that were extended beyond Leu-26. Table I summarizes the peptides synthesized and tested for this study as well as their ICbo values. Extending the amino terminus to Ile-25, Pro-24, or Ala-23 did not significantly enhance the inhibitory activity of the peptide 26-46 (Table I) (Fig. 3). Furthermore, peptides 27-46, 28-46, 29-46, and 30-46 had activity similar to that of intact 26-46. When Gly-30 was removed, yielding peptide 31-46, the ICbo increased from 10 to 40 MM, that is, the potency decreased by 4-fold. When Cys-31 was removed, peptide 32-46 exhibited potency comparable with the control peptide 26-46. Peptide 33-46, which lacked Cys-32, had an ICso of 78 MM. The potency of 33-46 decreased almost &fold, compared with 26-46. The shortest peptides in the series, 34-46 through 41-46, had essentially no activity at the highest concentration tested (extrapolated ICEo values >600 PM, data not shown). These data indicate that the amino-terminal boundary required for inhibitory activity was Gly-30. For further experiments, however, Met-29 was used as the amino-terminal boundary, since it had been previously implicated in hormone-receptor interactions (Cheng, 1976a(Cheng, , 1976b. Carboxyl-terminal Truncations-A second set of truncated peptides (Table I) (Fig. 4) was synthesized with the aminoterminal boundary defined as Met-29. These peptides were   35-46, 36-46, 37-46, 38-46, 39-46, 40-46, and 41-46 had less than 5%) of control activity. The inset includes the sequences of three selected peptides, which serve as landmarks. each shortened by 1 residue from the carboxyl terminus. When compared with 29-46, peptides 29-44 and 29-45 had essentially the same inhibitory activity. However, when Lys-44 and Ser-43 were removed, yielding peptides 29-43 and 29-42, the ICsO values increased by 4-7-fold. The shortest peptides of the series, 29-40 and 29-41, lost considerable inhibitory activity. These peptides retained less than 10% of the inhibitory activity exhibited by peptide 29-46. These data indicate that Thr-46 and Lys-45 may be dispensable, but Lys-44 is important for maintaining inhibitory activity of the peptide. Therefore, we conclude that the residues necessary for the inhibitory activity of region 26-46 were contained in the core sequence 30-44, the amino-terminal boundary at Gly-30, and the carboxyl-terminal boundary at Lys-44. Alanine Substitutions-Two groups of peptides were syn- thesized in which each residue was substituted individually with alanine. Alanine was chosen because it is an amino acid abundant in proteins and has a fairly small neutral side group that should not disrupt any structure the peptides may adopt. Glycine, on the other hand, with only hydrogen as the side group, allows too much flexibility in the pept,ide backbone (Schulz and Schirmer, 1979). The first set of peptides was substituted at residues 29-35 in the sequence 29-46. The data in Table I and Fig. 5 show that when Arg-35 was substituted with alanine, the potency of the peptide was decreased to less than 10% of control activity. Substitution of Phe-33 with alanine caused the IC5(, to increase to 55 PM. The potency of the peptide was decreased about 4-fold. Cys-31 and Cys-32 appear to be less important, since the alanine substitutions increased the IC,,, values somewhat hut not significantly. The second set of peptides was substituted at residues 37-44 in the sequence 29-44. The data in Table I indicate that Arg-42 and Ser-43 were important, since substitution with alanine caused the IC5,) values to increase a small but statistically Analysis of circular dichroic data of selected peptides in aqueous and helicogenic solvents Circular dichroic spectra were collected and analyzed as described under "Materials and Methods." TES represents the data from spectra collected from peptide solutions in 5 mM TES, pH 7.0; T F E represents that data from peptide solutions in 90% trifluoroethanol in TES. significant amount. Substitution of the remaining residues with alanine had little or no effect on inhibitory activity. Circular Dichroic Analysis- Table I1 summarizes circular dichroic data obtained for selected peptides. For comparison, the values for the human a subunit are included. Intact glycoprotein hormones exhibit low a helix content (Gamier, 1978) as do the a subunit and the a peptides. Content of a helix is increased somewhat when the peptides are in the helicogenic solvent trifluoroethanol. Most importantly, there is no striking difference in secondary structure in those peptides containing an alanine substitution.

DISCUSSION
Synthetic peptides have proven to be useful probes for the study of structure-function relationships between hCG and the LH receptor. In this study, we have used synthetic peptides to determine the importance of particular amino acid residues for the inhibitory activity of a26-46, a peptide that inhibits binding of lzSII-hCG to rat ovarian membranes . Our data suggest that the core sequence 30-44 contains all the residues necessary for full inhibitory activity. Met-29, which was used as the amino-terminal boundary in subsequent peptides, is involved in the interaction of hormones with receptors, as indicated by chemical modification experiments (Cheng, 1976a(Cheng, , 1976b. Therefore, our data are consistent with the idea that these residues are in or near the receptor interaction region of the a subunit. The carboxyl-terminal boundary appears to be Lys-44; Lys-45 and Thr-46 can be deleted with little effect on inhibitory activity. Lys-49 in the bovine cy subunit can be cross-linked to Asp-113 in the p subunit of bovine LH (Weare and Reichert, 1979), so it appears that respectively, in the bovine a subunit) are in or near the region where the subunit interface and the receptor interaction regions come together. Other data have also suggested that the subunit interface and the receptor interaction regions are, in fact, close together . Interestingly, when Lys-44 is substituted with alanine, the potency of that peptide is the same as peptide 29-44. So it appears that loss of the residue by truncation is more detrimental than substitution with alanine. This might indicate that Lys-44 is required for a critical peptide length and not necessarily for the positively charged side group. Secondary structure predictions for this region indicate that 9 of 21 residues could be involved in turn structures (Erickson et al., 1990). By CD analysis, this region seems to adopt primarily (3 sheet, p turn, and unordered structures (Table 11). Together these data could explain how the subunit interaction region(s) and the receptor interaction region(s) are juxtaposed.
Arg-35 and Phe-33 appear to be particularly important for interaction of a26-46 and hCG with the LH receptor. The positive charge of Arg-35 could be involved in ionic interactions between hCG and the receptor. Phe-33 could be involved in hydrophobic or ring-stacking interactions with hydrophobic or aromatic residues in the receptor. Future experiments using lysine or histidine for Arg-35 and other hydrophobic residues such as tyrosine, tryptophan, or isoleucine for Phe-33 will address these possibilities. and Cys-32 are also important but to a lesser degree. Cys-31 has been shown previously to be involved in receptor interactions by chemical modification experiments (Giudice and Pierce, 1979); however, the other 3 residues have not. Arg-42 could be involved in ionic interactions with the receptor. Ser-43 could potentially serve as a hydrogen bond donor with an appropriate residue(s) on the receptor. It is widely accepted that Cys-31 and Cys-32 are disulfide-bonded with Cys-7 and Cys-10, respectively. Since the Cys-7-Cys-31 bond is the first one reduced (Pierce and Parsons, 1981), it must be relatively surface accessible. Cys-31, Cys-32, and Phe-33 are predicted to be in a turn structure, whereas Arg-35 and Ser-34 are predicted to be in the beginning of a p sheet structure (Erickson et al., 1990). These residues and, in fact, most of the amino acids in this region are absolutely conserved among all species analyzed to date (Ward and Bousfield, 1990). Apparently, in order to maintain functional integrity of the glycoprotein a subunits, these residues have been conserved throughout evolution.
Substitution of Pro-38 or Pro-40 with alanine did not affect the values. This was somewhat unexpected since proline residues can introduce bends in the polypeptide backbone. Substitution with alanine could relieve this bending. These 2 proline residues are also conserved in all species studied to date (Ward and Bousfield, 1990). Pro-38 is predicted to be in a turn structure and Pro-40 at the beginning of a / 3 sheet region (Erickson et al., 1990). Furthermore, the remainder of the region is predicted to be in turn structure or (3 sheet (Erickson et al., 1990). Our findings suggest that substitution of either proline with alanine does not exert major changes in the rest of the peptide structure. In fact, changes in IC5, values appear to be due predominantly to changes in side chains, not gross structural changes (Table 11).
These peptides were also tested for inhibitory activity of TSH binding to porcine thyroid membranes.* Peptides active in the ovarian membrane system with hCG were also active in the TSH competition assays. These data are consistent with the fact that the same a subunit interacts with the two different yet related p subunits. One would expect a similar subunit interaction region(s) on the a subunit, and perhaps some similar and some subtly different receptor interaction region(s). In fact, this is what is seen. Leinung et aL3 demonstrated that Gly-30, Phe-33, Arg-35, Arg-42, and Lys-44 were important for maintaining inhibitory activity of a26-46 in the TSH receptor assay, as was shown in this work for the hCG. LH receptor assay. However, some subtle differences were apparent. Cys-31 and Lys-45 were found to be important in the TSH system but not in the hCG system. In this study, Ser-43 was shown to be somewhat important in the hCG system but not in the TSH system.
The data presented in this report indicate that the core peptide 30-44 has within it two regions of importance. The first region seems to lie between residues 30 and 35, the second between residues 42 and 44. Arg-35 and Phe-33 seem to be particularly important in the amino-terminal region, whereas Arg-42 and Ser-43 appear to be the important residues in the carboxyl-terminal region. Previous studies (Charlesworth et d., 1987) showed that two overlapping peptides from the human a subunit, 21-35 and 31-45, had inhibitory activity. However, the potencies were somewhat less than 26-46 (26-46, 10 pM; 21-35, 12 pM; and 31-45, 18 pM). This is explained by these data. Each of the overlapping peptides contains only one of the important regions in the core peptide 30-44. The peptide 26-46 (Charlesworth et al., 1987) and the core peptide 30-44 contain both sites and have higher inhibitory activity than either of the previously mentioned overlapping peptides.
We have defined a core sequence in the a subunit of hCG, 30-44, that seems to be involved in hormone-receptor interactions. In addition, we have provided insight into which residues are particularly important for those interactions. In this work, we have demonstrated the utility of synthetic peptides as probes into structure-function relationships between hCG and LH receptor.