Molecular and Cellular Endocrinology, 87 (1992) 9-11 0 1992 Elsevier Scientific Publishers Ireland. Ltd. 0303-7207/92/~05.0~

MOLCEL

02799

A synthetic peptide corresponding to amino acids 9-30 of the extracellular domain of the follitropin (FSH) receptor specifically binds FSH Bosukonda Dattatreyamurty Department

of Biochemistv and Molecular Biology, A-10, Albany Medical Colle.ger,Albuny, NY 12208, LISA (Received

Key words: Follitropin

and Leo E. Reichert, Jr.

receptor;

Hormone

binding:

2 March

1992; accepted

Synthetic

peptide;

24 April 1992)

Anti-peptide

antibody

Summary As deduced on the basis of cloning experiments, the putative extracellular domain of pituitary glycoprotein hormone (Iutropin (LH), thyrotropin (TSH), and FSH) receptors (ret) is sufficiently large to suggest involvement in hormone binding. Comparison of the amino acid sequences of the extracellular domains of the glycoprotein hormone receptors indicates that the FSH receptor has a peptide sequence in the external domain close to the amino terminus (residues 9-30) which has no sequence homology to receptors for LH or TSH. To examine whether this region is involved in FSI-I-receptor interaction, we studied the hormone-binding properties of a corresponding synthetic peptide in several systems. (11 Binding of ““I-hFSH to receptor-containing bovine testis membranes was inhibited by preincubation with FSH ret-(9-30) peptide amide in a concentration-dependent manner. (2) ‘2sI-labeled ret-(9-30) peptide amide bound to ovine, bovine, or human FSH preparations, and the binding was inhibited by solubilized bovine FSH receptor. ‘251-labeled ret-(9-30) peptide amide, however, did not bind to LH or TSH. (3) ““I-hFSH bound to unlabeIed ret-(9-30) peptide amide, and the binding was inhibited by excess unlabeIed FSH, but not by LH or TSH. (4) Scatchard analysis indicated that the FSH ret-(9-30) peptide amide contained a single class of FSH binding sites with a K, = 1.1 X 10’ M-‘. (5) The binding of “‘I-labeled ret-(9-30) peptide amide to hFSH, bFSH or oFSH was effectively inhibited by rabbit polyclonal antibodies raised against recd9-30) peptide amide but not by preimmune rabbit serum. In conclusion, our results represent the first demonstration that the region corresponding to residues 9-30 in the extracellular domain of the follitropin receptor specifically binds to FSI-I.

Introduction Follitropin IFSHI is a pituita~ glycoprotein hormone which acts on specific cells in the ovaries ‘and testes thereby regulating gonadal functions.

Correspondence to: L.E. Reichert, Jr., Department of Biochemistry and Molecular Biology, A-10, Albany Medical College, Albany, NY 12208, USA. Tel. (518) 445-5365; Fax 618) 445-5365. Supported by NIH Grant HD-13938.

Like other pituitary glycoprotein hormones, lutropin (LH) and thyrotropin (TSH), follitropin is a heterodimer. Each of these hormones has a common (Y subunit and a hormone-specific p subunit which determines the specific biological activity of the hormone (Pierce and Parsons, 1981). The actions of follitropin are mediated by plasma membrane bound specific high affinity receptors that are coupled to high affinity GTPbinding sites of G, protein (Dattatreyamurty et al., 1987; Zhang et al., 19SS), resulting in the

activation of adenylate cyclase in their target ceils (Zhang et al., 1991). Follitropin receptor has been purified from bovine calf testis and its biochemical properties have been described (Dattatreyamurty et al., 1990). Recent cloning studies using molecular probes derived from selected coding regions of the LH receptor cDNA have provided information about the primary structure of rat FSH receptor (Sprengel et al., 1990). The deduced primary structures of the glycoprotein hormone receptors have several important features in common. Each has a relativeIy large putative extracellular hydrophilic N-terminus domain thought to be involved in hormone-receptor interaction (Salesse et al., 199.1). Comparison of the primary amino acid sequences of the glycoprotein hormone receptors, however, has revealed that the extracellular domain of the FSH receptor (ret) has two unique regions (FSH ret 9-30 and 279-315) which have no sequence homology with LH or TSH receptors (Salesse et al., 1991). This raises the possibility that regions specific to the FSH receptor might involve in specific interaction of the receptor with FSH. We therefore synthesized a peptide corresponding to the first unique region near the amino terminus (residues 9-30) of FSH receptor extracellular domain and examined its specific binding activity for FSH. In this report, we describe several lines of evidence indicating that the FSH ret-(9-30) peptide represents a specific hormone-binding region of the follitropin receptor. Materials

and methods

Peptide synthesis A 23-mer peptide amide corresponding to residues 9-30 in the N-terminus of rat FSH receptor sequence (Sprengel et al., 1990) was synthesized and kindly provided by Dr. Jean Rivier, Clayton Foundation Laboratories for Peptide Biology, the Salk Institute, San Diego, CA, USA, under contract No. l-HD-7-2907 from the Contraceptive Development Branch, Center for Population Research, NICHHD. The C-terminal tyrosinc amide residue is not part of the selected sequence in rat FSH receptor, but was included to allow radioiodination of the peptide. The peptide amide was purified by preparative reverse

phase liquid chromatography on octadecyl-sihca (Waters Delta-Pak C ,$, Milford, MA, USA: 100 A pore diameter) using a linear acetonitrile gradient (5-100%) in 0.1% trifluoroacetic acid at 30°C. Homogeneity of the peptide was checked by analytical high-performance liquid chromatography (HPLC). Amino acid composition (BidIingmeyer et al., 1984) and peptide sequence were determined to verify the synthesis. Reduction and S-acetamidomethylation of FSH receptor peptide decide The receptor-(9-30) peptide amide contains a cysteine residue at position 15. To prevent formation of a peptide dimer, the receptor peptide amide was reduced and alkylated. The peptide was dissolved in cysteine alkylation buffer (6 M guanidine HCI, 0.25 M Tris base, and 1 mM EDTA, pH 8.2) at a concentration of 2.7 mg/ml, and reduced in the presence of a 100-fold molar excess of 2-mercaptoethanol. The solution was flushed with N2 and incubated for 1 h at room temperature in the dark. The reduced peptide was then incubated with a 2-fold molar excess (relative to 2-mercaptoethanol) of iodoacetamide, for 2 h at room temperature as described above. Finally, the alkylated peptide amide was purified by gel filtration through Sephadex G-25. Preparation of radioiodinnted receptor peptide amide The alkylated receptor peptide amide (100 pg) was radioiodinated with carrier-free 2.5 mCi of Na’z’I, by using the lactoperoxidase method (Miyachi et al., 1972) with some modifications (Dattatreyamurty et al., 1986). We used the method of gel filtration through a column of Sephadex G-25 to separate radioiodinated peptide from free iodide. The column was eluted with 50 mM phosphate buffer, pH 7.5, and 1 ml fractions were collected into tubes containing 1 mi of 0.1% ovalbumin. The specific activity of the alkylated and radioiodinated peptide amide was 25-27 pCi/pg. Receptor peptide-Affi-Gel 10 coup~i~?g Briefly, Affi-Gel 10 (packed volume of 5 ml) was washed and resuspended in coupling buffer (0.1 M NaHCO,, pH 8.01, and coupled with 3 mg

11

of receptor-(9-30) peptide amide in a final volume of 7.5 ml, according to the manufacturer’s instructions. After coupling, the Affi-Gel was suspended in a blocking solution (0.2 M glycine, pH 8.0) and incubated for 2 h at room temperature. The affinite matrix was washed with 0.1 M NaHCO,, pH 7.5, followed by 0.05 M Hepes buffer, pH 7.4, containing 1 M KCl, and lastly by 0.05 M Hepes buffer, pH 7.4. Finally, the peptide-AffiGel conjugate was suspended in 0.02 M Hepes buffer, pH 7.4, containing 10 mM MgCl, (affinity matrix to buffer ratio of 1: 1.7) to give an approximate peptide concentration of 100 pg/ml. Preparation of radioiodinated hFSH Highly purified human FSH (LER-1781-2, 4000 IU/mg) was radioiodinated using the lactoperoxidase method (Miyachi et al., 1972) as described previously (Dattatreyamurty et al., 1986). A method of polyacrylamide gel electrophoresis (PAGE) (Schneyer et al., 1986) was used to separate radioiodinated hormone from free iodide. The radioiodinated FSH had a specific activity of 20-25 pCi/pg, and a minimum of 30% bindability to excess receptor. Binding studies The isolation of an FSH receptor-enriched membrane fraction from bovine calf testis homogenate and preparation of detergent-solubilized FSH receptor were as previously described (Dattatreyamurty et al., 1986). Five different experimental approaches were used to study the interaction between receptor-(9-30) peptide amide and FSH. (1) Experiments to examine the effect of peptide on 12’I-hFSH binding to membrane-bound receptors were carried out under standard assay conditions, as previously described (Schneyer et al., 1986) with the following modifications. A fixed amount of ““I-hFSH (N 200,000 cpm or - 2 ng) was preincubated in the absence or presence of increasing amounts of unlabeled ret-(930) peptide amide and buffer (50 mM Hepes, pH 7.5, containing 0.1 M sucrose, 5 mM MgCl,, 0.1% ovalbumin) to a total volume of 275 ~1 for 45 min at room temperature. 3 mg of wet weight bovine testis membrane preparation in 100 ~1 buffer were then added, vertexed and further incubated

for 18 h at 20°C. After incubation, receptor bound 12’I-hFSH was separated from free hormone by centrifugation (30,000 x g, 15 min). Radioactivity in the pellet was counted in an autogamma counter with an efficiency of 85% for 12’I. (2) In another set of experiments, 5 pg of oFSH, bFSH, or hFSH, or l-5 kg of heterologous hormones bLH or bTSH, were dissolved in 10 ~1 of 25 mM Tris-HCl buffer, pH 7.0, and immobilized on PVDF membranes (Immobilon-P) by using a dot-blot apparatus WacuSlot-VS). The sample-containing membranes were incubated in 20 mM Hepes buffer, pH 7.4 containing 3% ovalbumin for 14 h, in cold (4”C), to block excess protein binding sites on the membrane. The blocking buffer was then replaced with 20 mM Hepes buffer, pH 7.4 containing 0.5% ovalbumin and 10 mM MgCl,, and the sample-containing membranes were further incubated with 12’1labeled receptor peptide amide (730,000 cpm/ml) in the presence or absence of solubilized FSH receptor (4 pmol/ml) for 18 h, with slow shaking, at room temperature. Finally, the membranes were washed 3 times with 25 mM Hepes buffer, pH 7.4 containing 5 mM MgCl,, air dried and kept for autoradiography overnight. (3) Alternatively, the binding of hormone to ret-(9-30) peptide amide was also examined, by immobilizing 5 Fg of unlabeled receptor peptide amide on PVDF membranes and preincubating with buffer alone (20 mM Hepes buffer, pH 7.4 containing 10 mM MgCl, and 0.5% ovalbumin) or buffer containing excess unlabeled oFSH, bTSH or bLH for 5 h at room temperature. 12’I-hFSH (600 000 cpm/ml) was then added and the blots were further incubated for 18 h at room temperature. The experimental conditions were the same as described in the second experiment. The membranes were washed with 25 mM Hepes buffer, pH 7.4 containing 5 mM MgCl,, and kept for autoradiography for 3 days. (4) The quantitative determination of FSHbinding property of ret-(9-30) peptide amide was carried out by incubation of a fixed amount (+- 5 kg/50 ~1) of peptide-Affi-Gel 10 conjugate suspension in 20 mM Hepes buffer, pH 7.4 containing 10 mM MgCl,, with approximately 5 ng of ‘*‘I-hFSH, in the presence of increasing concentrations of unlabeled hFSH (5 ng-8 pgg>, in a

17

total volume of 220 ~1. The binding reaction between radioiodinated FSH and receptor peptide was carried out for 18 h at 20°C to reach equilibrium. Other assay conditions were the same as described earlier (Schneyer et al., 1986). To separate bound “‘I-hFSH from free ligand, a rapid filtration method was employed using the Millititer vacuum fiItration system (MiIlipore) according to the manufacturer’s instructions. The radioactivity of “‘I-hFSH bound to receptor peptide was counted in a gamma counter. The affinity constant (K;,) was estimated from competitive data using the LIGAND program of Munson and Rodbard (1980). Criteria used for determining whether a Scatchard plot is.a single-site model or a two-site model are as indicated in the program. (5) The specificity of the binding of ret-(9-30) peptide amide to FSH was confirmed by inhibition studies using a rabbit polyclonal antibody raised against ret-(9-30) peptide amide. “‘Ilabeled ret-(Y-30) peptide amide f 13.7 x 10” cpm in 170 pi of 0.05 M phosphate-buffered saline (PBS)/O.l% ovalbumin) was preincubated with 375 ~1 of rabbit anti-receptor peptide antiserum for 2 h at room temperature and then overnight in cold (4°C). The incubation mixture was subjected to a column of Ultrogcl AcA 34 previously equilibrated and washed with PBS/ovalbumin, to separate the labeled receptor peptide-antibody complex from free “51-labeled receptor peptide. hFSH, bFSH and oFSH preparations, each 5 pg, were immobilized on to PVDF membrane, and the sample-containing membranes were incubated with 3% ovalbumin to block excess protein binding sites on the membranes as described above. The sample-containing membranes were then incubated with ‘2’I-Iabeled receptor peptide-antibody complexes (complex containing 600,000 cpm/ml; final dilution of antibody was 1 : 50) for 18 h, with slow shaking, after which the membranes were washed, dried and kept for autoradiography overnight as described above. Control experiments were done using equivaient amounts and finai dilutions of preimmune rabbit serum. Results and discussion It has been well established that an initial event in foliitropin action is the specific binding

of hormone to follitropin receptors present on target cell plasma membranes. Recent studies from this laboratory (Santa Coloma et al, 1990; Santa Coloma and Reichert, 1990) and others (Erickson et al., 1990) have identified receptorbinding regions on hFSH, but the hormone-binding regions on the follitropin receptor are not yet known. To this end, we began our investigation using a synthetic peptide approach which has proven to be useful in previous studies of membrane receptors including receptors for acetylcholine (Mulac-Jericevic et al., 19SS>, TSH (Attassi et al., 1991; Mori et al., 1991) and thrombin (Ngaiza and Jaffe, 1991). In common with other gIycoprotein hormone receptors, the follitropin receptor has a large extracellular domain (Sprengel et al., 1990) which is likely to contain hormone-binding regions. Comparison of the amino acid sequences of the extracellular domains of the glycoprotein hormone receptors (Salesse et al., 19911, however, revealed two unique regions (residues 9-30 (Fig. 1A), residues 279-315) in the extracellular domain of follitropin receptor which have no sequence homology to the lutropin and thyrotropin receptors (Salesse et al., 1991). Moreover, of these two regions, the first region (residues 9-30) is well conserved between rat and human follitropin receptors (Sprengel et al., 1990; Minegish et al., 1991) (Fig. 1B) and, therefore, has appeared to have a high probability of representing a specific hormone-binding region on the receptor. A synthetic receptor-(9-30) peptide amide corresponding to the first unique region of rat follitropin receptor was chosen for further study. Five different experimental approaches were used to study the interaction between FSH and ret-W30) peptide amide. In the first set of experiments, we examined the effect of synthetic receptor peptide on the uptake of ‘*‘I-hFSH by membranebound FSH receptors. In this experimental system, a fixed amount of particulate receptor ~membrane-bound) and variable concentrations of receptor peptide (in solution) are interacting with a fixed amount of ligand 12’I-hFSH. As shown in Fig. 2, the ret-(9-30) peptide amide was capable of inhibiting the uptake of lz51-hFSH by membrane-bound receptors, in a dose-dependent manner.

13

As this is an indirect evidence, we carried out additional experiments wherein we examined the direct binding of FSH ret-(9-30) peptide amide to FSH. For this, we first radioiodinated the receptor peptide amide. Since the sequence region (residues 9-30) of the rat follitropin receptor does not contain tyrosine residues, we added a single residue of tyrosine amide at the Cterminus to facilitate radioiodination of the peptide. The reduced and alkylated receptor peptide amide was radioiodinated using the lactoperoxidase method to a specific activity of 25-27 pCi/Fg. The radioiodinated intact receptor peptide was separated from damaged peptide and free iodide by gel filtration through a Sephadex pepG-25 column. When I25I-labeled ret-(9-30) tide amide was incubated with oFSH immobilized on PVDF membrane (Immobilon-P), it effectively bound to the hormone, and the labeled peptide

A 8 rFSHR: rLHR

20

Cys Ser Asn Arg Val Phe La

I

:

Cys

I

Cys Gin Asp Ser LysVal

/

Pro Glu Pro Cys Asp Cys Ala Pro Asp Gly Ala Leu Arg

II

Cys His Gin Glu Asp Asp Phe Arg Val Thr Cys Lys Glu LKU

rFSHR:

Glu Ik Pro Thr Asp Leu Pro Arg Asn Ala Ile Glu La

30

I

:

Cys

rTSHR:

Pro Gly Pro Arg Ala Gly Leu Ala Arg Leu Ser La

I

His Gin Ile

I

Pro Ser

Leu Pro Pro Ser Thr

Gin Thr Leu

B 10 rFSHIk

Ser

Asn

Arg

Val

Phe

L.eu Cys

Gin

Asp

Ser

Lys

Glu

hFSHR:

20 rFSHR: hFsHR:

Val

Thr

Glu

Ile

Pro

Thr Asp ser.

Leu

Pro

Arg

3 ‘D

5 2 E f ?!-

-\

60--

-T \

7\

\ 60--

‘\

\

‘p

40--

\

A

4 0’

1 “g/ml

(a-4)

1000

100

10 hF&l

or

pg/ml

Rec.

paptide

(A-

-A)

Fig. 2. Effect of increasing concentrations of FSH ret-(9-30) FSH peptide on the uptake of ‘2’I-hFSH by membrane-bound receptors. 12’I-hFSH (approximately 200,000 cpm) was preincubated in the absence or presence of indicated concentrations of receptor peptide or unlabeled hFSH for 45 min at room temperature. 3 mg of wet weight bovine calf testis membrane preparation were then added and further incubated for 18 h at 20°C. Specific binding for ‘2’1-hFSH in the absence and presence of receptor peptide or hFSH was determined as described in Materials and methods. Values (B/B,,%) are expressed as percent of specific binding determined in the absence of receptor peptide or hFSH (B,,). Each point is the average of triplicate determinations.

Thr

ITSHR:

rLHR

6-v-&

100 -c

Asn

Fig. 1. A: Sequence comparison between the glycoprotein hormone receptors. Only the N-terminus end (residues 8-34) revealing the highly variable region among the receptors is shown here. For complete sequence comparison, see Salesse et al. (1991). rFSHR, rat FSH receptor; rLHR, rat LH receptor; rTSHR, rat TSH receptor. Identical residues (1) are indicated. The highly variable (unique) region corresponding to residues 9-30 in rFSHR (shown in bold letters) is chosen for study. B: Sequence comparison between rat FSH receptor (rFSHR) and human FSH receptor (hFSHR). Only the Nterminus region corresponding to Y-30 residues is shown here. Amino acid residues identical to those of rFSHR are indicated as dots.

binding was inhibited by the presence of solubilized bovine follitropin receptor (Fig. 3, panels A, B and 0. The labeled receptor peptide specifically bound hFSH as well as bFSH (Fig. 3, panels A, B and C), suggesting that these hormones may share comma binding region(s) for the receptor peptide amide. Interestingly, the sequence corresponding to the ret-(9-30) peptide appears to be conserved in both rat and human follitropin receptors (Sprengel et al., 1990; Minegish et al., 1991). In a third set of experiments, we used an alternative approach where we examined the direct interaction of FSH with ret-(9-30) peptide amide. When 12’1-hFSH was incubated with unlabeled receptor peptide immobilized on PVDF membrane, the labeled hormone bound to the receptor peptide amide, and the binding of labeled hormone was inhibited by excess unlabeled oFSH (Fig. 4). In the fourth set of experiments, we determined the affinity of the ret-(9-30) peptide amide for hFSH. When 12”1-hFSH and increasing concentrations of unlabeled hFSH were incubated

with ret-(9-30) peptide amide in~mobiIized on Affi-Gel 10, hFSH effectively inhibited the binding of “‘I-hFSH to receptor peptide amide in a

concentration-dependent manner. Scatchard analysis indicated that the ret-(9-30) peptide amide contained a single class of FSH binding

B

I

7_

2

a

3

910

M-H

140

C 120

3

loo,-

: 6 E

80--

5

40-e

1

T

2

3

2

3

4

5

6

E

5

60--

1

20 --

hFSH

oFSH

bFSH

URP

bLH

bTSH

4

5

6

Fig. 3. Direct binding of “‘I-labeled FSH receptor peptide amide to immobilized FSH, LH or TSH preparations. Unlabeled hFSH, oFSH or bFSH, each in triplicates (5 pg/slot) or unlabeled bTSH (1 or 5 pg/slot) in duplicates or bLH Cl.25 or 5 pg/slot) or unrelated proteins (URP) such as bovine serum albumin, and ovine prolactin (oPrl), each 5 pg/slot were immobilized on PVDF membranes, and the membranes after blocking with buffer containing 3% ovalbumin, were incubated with “‘I-labeled receptor peptide amide (730.000 cpm/ml) in the absence (panel A) or presence (panel B) of solubilized calf testis FSH receptor (4 pmol/ml). The procedures for the binding experiments and subsequent autoradiography arc as described in Materials and methods. Representative autorad~o~rams of three separate experiments are shown here. Panel .4: Autoradiogram showing the binding of “‘I-labeled receptor peptide amide to oFSH (slots I,2 and 3), hFSH (slots 4, 5 and hf, hFSH (slots 7,8 and 91, bLH (slot IO, 5 /Lg) or URP (unrelated proteins): BSA (slot II), oPrl (slot 12). Panel B: Autoradiogram showing inhibition of the binding of radiolabeled receptor peptide to hFSH, oFSH or bFSH in the presence of excess unlabeled solubilized FSH receptor. Panel C’: Summary of the data (mean i SD) from densitometric scanning of autoradiograms (panel A and panel D). Results are expressed in arbitrary densitometric units (ADU) with 100% assigned for hFSH (panel A, slots 7, 8 and 9). * Non-detectable. Panel D: Aut~~~diog~ms showing the absence of binding of radiolabeled receptor peptide to bLH (slot I (5 pg) and slot 4 (1.25 pg)) or bTSH (slots 2 and 5 (1 pg/slotf: slots 3 and h (S pg/slot)). Panel E: PVDF membranes containing immobilized hLH (panel D. slots 1 and 4) and bTSH (panel D, slots 2, 3, 5 and 6) were incubated with the appropriate dilution (I :400) of respective anti-LH (panel E‘, slots 1 and 4) or anti-TSH (panel E. slots 2, 3, 5 and 6) antibodies. The presence of hormones on these blots was then detected by the Problot Western blot AP system (Promega, Madison, USA) according to the manufacturer’s inslructions.

15

7 Z 8 B &

A

3

lOO-80-s 60.-

$ 40-20-s

Control

FSH

LH

TSH

peptide amide. Unlabeled receptor peptide amide (5 pg/slot) Fig. 4. Direct binding of ‘251-hFSH to immobilized FSH ret-(9-30) in duplicates was immobilized on PVDF membranes, and the membranes after blocking with buffer containing 3% ovalbumin were preincubated with buffer alone (lane 1) or buffer containing excess unlabeled oFSH (lane 2), bLH (lane 3), or bTSH (lane 4) for 5 h at room temperature. ‘*‘I-hFSH (600,000 cpm/ml) was then added and the blots were further incubated for 18 h at room temperature. The procedures for the binding experiments and subsequent autoradiography are as described in Materials and methods. A representative autoradiogram of three separate experiments is shown here. Panel A: An autoradiogram showing ‘2’I-hFSH binding. Panel B: Summary of the data (meanf SD) from densitometric scanning of autoradiograms. Results are expressed in arbitrary densitometric units (ADU) with 100% assigned for controls (panel A, lane 1). * Non-detectable.

sites with an association constant of 1.1 X 10h M-’ (Fig. 5). This value is lower than that of native FSH receptor (1 x 10’” M-‘) for the hormone (Dattatreyamurty et al., 1986, 19901, and probably reflects the presence of additional hormone interaction regions on FSH receptor, as previously postulated (Santa Coloma et al., 1990; Santa Coloma and Reichert, 1990).

F

i3

1

K 0. * 1 . 1 x lo6

M-’

i

4 nMFSH Bound [B]

8

Fig. 5. Scatchard plot for the binding of 12’1-hFSH to FSH ret-(9-30) peptide amide. A constant amount of ‘*‘I-hFSH was incubated with receptor peptide amide immobilized on Affi-Gel 10 ( - 5 pg), in the absence or presence of increasing concentrations of unlabeled hFSH as described under Materials and methods. The affinity constant K, was determined from competitive data by Scatchard analysis using the LIGAND program of Munson and Rodbard (1980).

The specificity of FSH binding to ret-(9-30) peptide amide was confirmed by two independent approaches. When ‘2”I-labeled receptor peptide amide was incubated with bLH or bTSH immobilized on PVDF membrane, the radiolabeled receptor peptide failed to bind these heterologous hormones (Fig. 3, panel D). This, however, was not due to the absence of hormones on the membranes since after Western blotting with the respective hormone antibodies, these membranes showed the presence of heterologous hormones on them (Fig. 3, panel E). Also, the ‘*“I-labeled receptor peptide amide did not bind unrelated proteins (URP) such as bovine serum albumin and ovine prolactin (Fig. 3, panel A). Secondly, when lz51-hFSH was incubated with unlabeled ret-(9-30) peptide amide immobilized on PVDF membrane, in the presence of excess FSH, LH or TSH, only FSH, but not LH and TSH, effectively inhibited the binding of ‘251-hFSH to the receptor peptide amide (Fig. 4). These results are consistent with the predicted hormone specificity of the follitropin receptor sequence region (residues 9-301, based on comparison between amino acid sequences of glycoprotein hormone receptors. In the fifth set of experiments, the specificity of ret-(9-30) peptide amide binding to FSH was further confirmed by inhibition studies with rab-

16

bit anti-receptor (9-30) peptide antibody. When rabbit antibody was preincubated with ‘251-labeled ret-(9-30) peptide amide, it effectively inhibited the binding of radioiodinated receptor peptide to hFSH, bFSH or oFSH immobilized on PVDF membrane (Fig. 6, panel B). Preimmune rabbit serum which served as control in this experiment did not inhibit the binding of ‘2”1-labeled ret-(930) peptide amide to the FSH preparations (Fig. 6, panel A).

6 7 8 9

12 v) 1

10

9

In conclusion, several lines of evidence indicate that the first unique region corresponding to residues 9-30 in the extracellular domain of the follitropin receptor specifically binds FSH. Recent evidence suggests that the extracellular domain of the FSH receptor has the ability and specificity to bind the hormone (Braun et al., 1991). Our results are consistent with and extend this observation. It is, however, not yet clear whether the hormone-binding region of FSH re-

I

O

[

I

c”

1l2

[

1

6

2

7

3

8 9

4 10

5

$

[

12 1cl3 I

O I

::

1n $

loo--

E %

00-e

E

60--

$

40-20-s 0’ hFSH

oFSH

bFSH

peptide amide to FSH by antibodies against receptor peptide. Unlabeled Fig. 6. Inhibition of the binding of ‘251-labeled ret-(9-30) oFSH (slots 1, 2, 6 and 7), hFSH (slots 3, 4, 8 and 9) or bFSH (slots 5 and lo), each 5 pg/slot, was immobilized on PVDF membranes. After blocking with buffer containing 3% ovalbumin, the membranes were incubated with preformed “51-labeled final dilution of antibody was I : 50; see ret-(9-30) peptide amide + anti-receptor peptide antibody complex ( _ 600,000 cpm/ml, Materials and methods for details). The procedures for the binding experiment and subsequent autoradiography are as described in Materials and methods. Representative autoradiograms of three separate experiments are shown here. Panel A: An autoradiogram showing the binding of ‘z51-labeled receptor peptide to oFSH, hFSH or bFSH in the presence of preimmune rabbit serum. Panel receptor peptide amide to oFSH, hFSH or bFSH by rabbit B: An autoradiogram showing inhibition of the binding of “sl-labeled anti-receptor (9-30) peptide antibody. Panel C: Summary of the data (mean k SD) from densitometric scanning of the autoradiogram (panel A). Results are expressed in arbitrary densitometric units (ADU) with 100% assigned for hFSH (panel A. slots 3, 4. X and 9).

17

shown here represents a separate or part of a single large binding site involved in hormone recognition. It has been previously shown that the binding of cu-ncurotoxins to acetylcholine receptor involves several contact regions on the receptor ~~u~ac-Jericevic and Atassi, 1987; MulacJericevic et al., 1988). Also the receptors for other glycoprotein hormones, LH and TSH, were shown to have more than one hormone-binding region (Atassi et al., 1991; Nagayama et al., 1991; Ji and Ji, 1991). It is likely that other potential hormone-binding regions of the FSH receptor might come in close proximity to the hormone-binding region shown here in three-dimensional structure to contribute to the architecture of the hormone-binding site. The fact that the FSH receptor peptide (9-30) region interacts with FSH from different species, but not LH or TSH, suggests that this peptide region may have a discriminatory role in the recognition of hormone by the receptor. Recently, we have shown that the j3 subunit of hFSH which confers the hormone specificity to FSH has at least three receptorbinding regions (Santa Coloma et al., I990; Santa Coloma and Reichert, 1990; Grass0 et al., 1991). Studies are currently underway to more precisely determine the regions of FSW /? subunit interacting with FSI-I receptor peptide (9-30). ceptor

We wish to thank technical assistance.

Carol Kowal for expert

References Atassi. M.Z., Mansbo~tj~ T. and Sakata, S. fl99lf Proc. Natl. Acad. Sci. USA 88, 3613-3617. Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1984) J. Chromatogr. 336, 93-104. Braun, T., Schofield, P.R. and Sprengel, R. (1991) EMBG J. IO, 1885-1890.

Dattatreyamurty, B., Schneyer, AL. and Reichert. Jr., L.E. (1986) J. Biol. Chem. 261, 13104-13113. Dattatreyamurty, B., Figgs, L.W. and Reichert, Jr., L.E. (1987) J. Biol. Chem. 262, 11737-11745. Dattatreyamurty, B., Zhang, S.-B. and Reichert, Jr., L.E. (1990) J. Biol. Chem. 265, 5494-5503. Erickson, L.D., R&a, S.A., Bergert, E.R., ~har~es~orth~ M.C., McCormick, D.J. and Ryan, R.J. if9901 Endocrinology 126, 2555-2560. Grasso, P., Santa Coloma, T.A. and Reichert, Jr., L.E. (1991) Endocrinology 128, 2745-275 1. Ji, I. and Ji, T.H. (1991) J. Biol. Chem. 266, 13076-13079. Mine&h, T., Nakamura, K., Takakura, Y., ihuki, Y. and fgarashi, M. (19911 Biochem. Biophys. Res. Commun. 175,

1125-1130. Miyachi, Y., Vaitukaitis, J.L., Nieschlag, E. and Lipsett, M.B. (1972) J. Clin. Endocrinol. Metab. 34, 23-28. Mori, T., Sugawa, H., Piraphatdist, T., Inoue, D., Enomoto, T. and fmura, H. (1991) Biochem. Biophys. Res. Commun. 178, 165-172. Mulac-Jericevic, B. and Atassi, M.Z. 119X7) Biochem. J. 248, 847-X.52. Mulac-Jericevic, B., Manshouri, T., Yokoi, T. and Attassi, M.Z. (1988) J. Protein Chem. 7, 173-177. Munson, P.J. and Rodbard. D. (1980) Anal. Biochem. 107, 220-239. Nagayama, Y.. Wadsworth, H.L., &hazenbalk, G.D., Russo, D., Seto, P. and Rapoport, R.B. il99l) Proc. Natl. Acad. Sci. USA 88, 902-905. Ngaiza, J.R. and Jaffe, EA. (1991) Biochem. Biophys. Res. Cnmmun. 179, 1656-1661. Pierce, J.G. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495. Satesse, R.. Remy? 3.J., L&n, J.M., Jai&& 3. and Gamier, J. (1991) Biochimie 73, 109-120. Santa Coloma, T.A. and Reichert, Jr., L.E. (1990) 3. B&l. Chem, 265, 5037-5042. Santa Caloma, T.A., Dattatreyamurty, B. and Reichert, Jr., LX. (1990) Biochemistry 29, 1194-1200. Schneyer, AL., Sluss, P.M., Dattatreyamurty, B. and Reichert, Jr,* L-E. il9Xhf End~~r~noIogy f 19, 1446-1453. Sprengel, R., Braun. T., Nikolics, K., Segaioff, DIL. and Seehurg, P.H. (1990) Mol. Endocrinol. 4, 52.5-530. Zhang, S.-B., Dattatreyamurty, B. and Reichert, Jr., L.E. (1988) Mol. Endocrinol. 2, 148-158. Zhang, S.-B., Dattatreyamurty, B. and Reichert, Jr., L.E. (1991) Endocrinology 128, 295-302.