Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein.

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein, probably N. , in light membranes of calf testis. Moreover, another component containing only high affinity binding sites for GTP represents a discrete molecular entity free from FSH receptors and is capable of hydrolysis of GTP. The availability of separate components having either high affinity FSH receptors associated with a guanine nucleotide regulatory protein or high affinity GTP-binding sites free of FSH receptors should facilitate further studies on molecular mechanisms of FSH activation of adenylate cyclase.
High affinity receptors for FSH' present on testicular membranes (1-5) mediate several biochemical actions of FSH on testis (6-8). One of the early steps in FSH action is the stimulation of adenylate cyclase activity with a concomitant increase in cyclic AMP production (9-11). We have shown that rat testicular plasma membrane system responds to GTP and its analogues by increased adenylate cyclase activity in response to FSH (12). Moreover, specific high affinity binding sites for these nucleotides are shown to be present in Sertoli cell membranes (13). It has been speculated that the primary control for the stimulation of adenylate cyclase activity by FSH and guanine nucleotides is through interaction between FSH-occupied receptor and stimulatory guanine regulatory protein (NJ. No information, however, is available on the molecular mechanism of the FSH receptor-guanine nucleotide-binding protein interaction. Such studies require an approach involving the solubilization of FSH receptors, GTPbinding protein, and catalytic unit of adenylate cyclase in a functional state. Previous efforts in this regard have not been successful due to rapid destabilization of components of the adenylate cyclase system, once solubilized by detergents (3, 11,14). In a recent report in this journal (l), we described a new protocol for solubilizing FSH receptors from lighter membranes of bovine calf testes. The detergent-soluble FSH receptor preparation so obtained retained receptor and guanine nucleotide-binding activities and responded to exogenous FSH by activation of adenylate cyclase. In this report we extend these studies to probe the molecular and functional relationship between FSH receptors and guanine nucleotide binding sites. Our present data suggest that FSH receptors The abbreviations used are: FSH, follitropin; '"1-hFSH, radioiodinated human follitropin; Gpp(NH)p, guanosine 5'-imidotriphosphate; GTPase, guaninetriphosphatase; DTT, dithiothreitol; App(NH)p, adenosine 5'-imidotriphosphate; N'-protein, guanine nucleotide-binding regulatory component of adenylate cyclase; N., stimulatory guanine nucleotide-binding protein of adenylate cyclase; DC-2-Fr 11, concentrated light membranes of calf testis; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl-l-piperazineethanesulfonic acid. are physically associated with a putative guanine nucleotide regulatory protein, probably N., in a GTP-sensitive state. Moreover, we have observed a separate component having high affinity nucleotide-binding sites for GTP that is free from FSH receptors and capable of hydrolysis of GTP.

Methods
Preparation of Testicular Light Plasma Membranes-FSH receptor-rich light plasma membranes were isolated and purified from 11.5 kg batches of bovine calf testes, according to a procedure previously described by us (1). These membranes (DC-2-Fr 11) contained approximately 780 fmol of high affinity FSH receptors/mg of membrane protein and were stored at -80 "C until used.
Solubilization of FSH Receptors-A previously described procedure (1) was employed to solubilize FSH receptors from light plasma membranes. In brief, membrane-enriched fraction (DC-2-Fr 11) was suspended to a concentration of 20 mg of protein/ml in 10 mM Tris-HCl buffer, pH 7.2, containing 1 mM MgCl,, 0.001% NaN3, 0.004% 2-mercaptoethanol, and 40 pM p-hydroxymercuribenzoate sodium. Triton X-100 (20%) was added subsequently to a final concentration of 0.2%. The sample was sonicated four times (6 s each time) at 50 watts in the cold (4 "C), and then treated with 0.2 volumes of petroleum ether to extract the free lipids. The aqueous phase was separated by centrifugation at 10,OOO X g and was recentrifuged at 145,000 X g for 1 h in a refrigerated ultracentrifuge. The supernatant was recovered and used in further studies. This detergent-soluble fraction contained both high affinity FSH receptors (approximately 1150 fmol/mg protein), Gpp(NH)p-binding activity and retained significant FSH-induced adenylate cyclase activity (1).
Affinity Chromatography with GTP-Sephurose-GTP-Sepharose derivative (Pharmacia) was washed with 20 mM Tris-HC1 buffer, pH 7.5, containing 1 mM DTT, 1 mM EDTA, 6 mM MgCl,, 2.5% sucrose, and 0.05% Triton X-100 (buffer-A), packed by centrifugation, and resuspended in two times its volume with buffer-A. Aliquots of active fraction eluted from Sepharose-GB column (GB-Fr-1, see "Results") were added to two volumes of suspension (1.5 ml) of either Sepharose-4B (control) or GTP-Sepharose derivative. The mixture was shaken gently for 45 min at room temperature. Sample treatment with GTP-Sepharose was performed in the absence and presence of 250 p~ of Gpp(NH)p. The reaction mixture was separated by centrifugation at 2000 X g for 10 min in the cold into supernatant (unretained material) and the matrix-bound materials. The GTP-Sepharose-bound material was washed twice with buffer-A and eluted by bringing the total volume to 2.25 ml with buffer-A containing 500 p~ Gpp(NH)p. After 45 min of incubation with gentle shaking, the released proteins were separated by centrifugation. Breakthrough and bound materials were assayed for "'I-FSH binding and protein content.
Preparation of FSH-Receptor Complex-Five ml of GB-Fr-1 (FSH receptor-containing fraction) was preincubated with 100 ng of 12' 1-hFSH for 16 h at 4 "C. The receptor-(GB-Fr-1) bound hormone was separated from free hormone by gel filtration on a column (2.5 X 35 cm) of Ultrogel AcA-34 equilibrated with 10 mM Tris-HC1 buffer, pH 7.2, containing 1 mM MgC12, 0.001% NaN3, and 0.0975% Triton X-100. Fractions of 2 ml were collected and assayed for radioactivity in a gamma counter. Fractions collected in void volume were pooled and used immediately in incubation experiments with GTP/other nucleotides. Preincubation of GB-Fr-1 with "'I-hFSH in the presence of nonradioactive hFSH (1000-fold molar excess) eliminated the radioactive peak (receptor-bound "'1-hFSH) in the void volume.
Experiments to examine the effects of GTP on I2'I-hFSH binding to FSH receptors were carried out under standard assay conditions (1). Aliquots of GB-Fr-1 (60-150 pg of protein) were incubated with approximately 2.5 ng of "'I-hFSH in the absence or presence of various concentrations of GTP or FSH or both (see Figs. 4 and 5) for 16 h, at 4 "C. Receptor-bound 'Y-hFSH was separated from free hormone by PEG precipitation as described earlier (1). The radioactivity in the pellet, representing '=I-hFSH-receptor complex was counted in an autogamma counter with an efficiency of 75% for "' I.
Treatment of Membranes with Cholera Toxin or Pertussis Toxin-Treatment of testis membranes with activated pertussis toxin or cholera toxin was performed according to the procedures of Lin et al.
(15) and Katada and Ui (16). Highly pure testis membranes (5 mg of protein/ml) were incubated with 25 pg of activated pertussis toxin at 30 "C for 15 min in 1 ml of 25 m~ Tris-HC1 buffer, pH 7.5, containing 2.5 mM MgC12, 1 mM ATP, 1 mM NAD, 10 mM thymidine, and 0.5 mM DTT. For cholera toxin treatment, membranes were incubated with 250 pg of activated cholera toxin in 1 ml of 40 mM Tris-HC1 buffer, pH 7.5, containing 2.5 mM MgC12, 1% BSA, 1 mM NAD, 10 mM thymidine, and 1 mM DTT. Controls were performed without treatment with toxin or without NAD. Treated membranes were washed twice with 25 mM Tris-HC1, pH 7.5, containing 2.5 mM MgC12, and then with 10 mM Tris-HC1, pH 7.2, containing 1 mM MgC12 and 0.001% NaN3. lZ5I-hFSH binding to receptors in these membranes, and subsequent dissociation of bound 'T-hFSH from membranes, in the presence and absence of GTP were performed as described above.
Analytical Methods-Protein determinations were carried out according to the method of Lowry et al. (18) with minor modifications (1). Bovine serum albumin was used as a standard.
Hormone-binding Assays-Highly purified human FSH (LER-1781-2, 4000 IU/mg) was radioiodinated using the lactoperoxidase method (19) with some modifications (20). We used either the method of gel filtration through a column of Sephadex G-100 or disc gel electrophoresis (21) to separate radioiodinated hormone from free iodide. The specific activity of the '=I-hFSH, as determined by self displacement (22), was 22-31 pCi/pg. The percent bindability of radioiodinated preparations to excess receptor was 25-32%.
Detergent-extracted receptor preparations and column effluent fractions were assayed for lZ6I-hFSH-binding activity, as described earlier (1). The quantitative determination of FSH-binding capacity of detergent-extracted preparations/column effluent active fractions was carried out by equilibration with approximately 2.5 ng of FSH tracer, in the presence of increasing concentrations of unlabeled hFSH (LER-2030-3A, 1 to 10, OOO ng). The assay procedure was the same as described earlier (1). The affinity constant and FSH receptor concentration were estimated from competitive data using the LI-GAND program of Munson and Rodbard (23).
To determine the 5'-nucleotidase activity, the method of Windall and Unkeless (24) was employed with minor modifications (25). The results were expressed as micromoles of inorganic phosphate generated per hour per milligram of protein.
Binding of [3H]Gpp(NH)p to detergent-extractedpreparations and column effluent fractions was determined according to the method of Pfeuffer and Helmreich (26), with some modifications as described earlier (1).
GTPase Assay-The GTPase assay was performed as described by Cassel and Selinger (27), but modified by Sunyer et aE. (28). The fmal concentrations in the reaction medium consisted of 25 mM Tris-HC1 buffer, pH 7.5,5 mM MgClz, 1 mM EDTA, 20 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase (200 units/mg), and 1 mM CAMP. Labeled GTP substrate was repurified according to the method of Iyengar and Birnbaumer (29), aliquoted, and stored frozen at -20 "C. Ten to 20 pg of sample protein were incubated with 25 nM GTP[?-32P] (100,000-400,000 cpm) in 100 pl of above reaction medium for 4 min at 30 "C. Parallel set of tubes also received App(NH)p and ATP, according to the assay procedure. App(NH)p, an inhibitor of a number of ATPases (30, 31), when added to the assay, was at 0.4 p~ to decrease the rate of GTP hydrolysis by nonspecific NTPases (27). Moreover, the suppression of the transfer reaction caused by a nucleotide triphosphate regeneration system was further enhanced by the addition of 1 mM ATP providing a greater specificity to the assay. The assay reaction was stopped by immediately placing the tubes on ice and adding 0.75 ml of ice-cold stopping solution (20 mM phosphoric acid, pH 2.1, 5% (w/v) activated charcoal). The tubes were allowed to stand for 30 min at 4 "C, centrifuged at 3,600 X g for 20 min, and 0.5 ml of the supernatant was added to 4.5 ml of scintillation mixture (3a70B, Research Products International). Background radioactivity ranged from 2.5 to 7.5% of the assay.

RESULTS
Gel Filtration on a Sephurose-GB Column-Fractionation of Triton X-100-soluble light plasma membrane protein in aliquots of 185 mg, on a column of Sepharose-GB (71 x 2.8 cm) resulted in two major fractions (Fig. L4). Sepharose-GB-Fraction-1 (GB-Fr-1) representing column effluent fractions 72-100 contained FSH-binding activity. Some Gpp(NH)p-binding activity was also coeluted in this fraction.
Most Gpp(NH)p-binding activity was, however, eluted in more retarded Sepharose-GB-Fraction-2 (GB-Fr-2) (column effluent fractions 105-140) and was clearly separated from the hormone-binding activity. The peak 5'-nucleotidase activity was eluted in the void volume (Fig. lB), emerging prior to hormone-binding activities.
To ensure that fractions GB-Fr-1 and 6B-Fr-2 were free of contamination from one another, they were concentrated 2.5fold by ultrafiltration, and then each fraction was rechromatographed through the same column of Sepharose 6B. Only tubes about the apex region of elution profile for each fraction were pooled and used for further study.
Further characterization of these fractions by chromatog- Fr. Number
Affinity Chromatography on GTP-Sephurose-When 6B-Fr-1 was incubated with GTP-Sepharose approximately 85% of FSH-binding activity was retained by the affinity matrix. About 82% of the bound receptor was eluted by 0.5 mM Gpp(NH)p. When affinity chromatography was carried out in the presence of 250 ~L M Gpp(NH)p (which effectively competes with Sepharose-bound GTP for the N-protein), the affinity matrix failed to retain significant FSH receptor activity. In control experiments, underivatized Sepharose-4B failed to retain significant receptor activity. These results suggest that the FSH receptors and Gpp(NH)p-binding component coeluted in GB-Fr-1 were physically associated.
It was of interest, therefore, to know if FSH receptors and Gpp(NH)p-binding component present in GB-Fr-1 were functionally related. To this end, we studied the FSH receptor sensitivity to guanine nucleotides, a heterotropic property which previously has been well-characterized in several other receptor systems, as an evidence of functional association between receptor and guanine nucleotide-binding protein (32-35).
Effect of GTP on lZ51-FSH Binding to GB-Fr-1-When 1251-FSH and increasing concentrations of GTP were simultaneously incubated with GB-Fr-1, GTP effectively inhibited the in Relation to GTP-binding Sites binding of FSH tracer to GB-Fr-1 in a dose-dependent manner ( Fig. 2A). The slopes of the dose response lines for GTP (-3.28) and unlabeled FSH (-1.85) were different (Fig. 2B).
Here the slope represents the fall in the percent bound (expressed as probits) per log dose of FSH or GTP. The concentration of GTP, on a mass basis, required to cause 50% inhibition of lZ51-FSH binding was 92-fold higher than that of unlabeled FSH. Moreover, the interactions of GTP and FSH appear to be noncompetitive at FSH-binding site, since a higher occupancy of receptors with lZ51-FSH (close to saturation) did not decrease the GTP effect on hormone binding. Kinetics of lZ5I-FSH Binding as Influenced by GTP-To ascertain the mechanism of the GTP effect on lZ51-FSH binding, we examined the kinetics of '251-FSH binding to 6B-Fr-1 (Fig. 3). In the absence of GTP, specific lZ5I-FSH binding was saturable. Scatchard analysis indicated a single class of high affinity FSH-binding sites with a dissociation constant (Kd) of 0.31 nM and a maximal number of binding sites (Bmax) of 4530 fmol/mg. On the other hand, the presence of GTP at 0.5 pmol/assay tube reduced receptor affinity from 0.31 to 1.49 nM without affecting the Bmax value. Thus, it appears that GTP reduces lZ51-FSH binding predominantly by decreasing the apparent affinity of binding sites for FSH.
Nucleotide Specificity for Dissociation of Bound "'1-FSH from GB-Fr-I-FSH-Receptor complex was prepared by incubating lZ5I-FSH with GB-Fr-1, in the cold (4 "C) for 16 h, and by separating the unbound lZ51-FSH by filtration through Ultrogel AcA-34 column. Aliquots of the FSH-receptor complex were then incubated with increasing concentrations of various nucleotides for 15 min at 30 "C to study dissociation of specifically bound lZ51-FSH. In a parallel experiment, a nucleotide triphosphate regeneration system (20 mM creatine phosphate and 10 units of creatine phosphokinase) were added to assay tubes to prevent possible interconversion of nucleotides due to transfer reaction. In summary, GTP was most effective in causing the dissociation of bound lZ51-hFSH. Halfmaximal dissociation of lZ51-FSH was evident at approxi-I \.  mately 0.45 pmol of GTP/assay tube (final concentration 7 to 9 X M; n = 3). GDP required 9.2-fold higher concentration than GTP. The relative potencies of ATP, ADP, and AMP were less than 5% which can be attributed to the known contamination of guanine nucleotide in these commercial preparations. UTP, ITP, App(NH)p, CAMP, cGMP, and GMP were ineffective. Gpp(NH)p, a nonhydrolyzable analogue of GTP mimicked the GTP effect, but required 15 times higher concentration than that of GTP. It has also been shown in previous studies that concentrations similar to, or greater than, this were required by Gpp(NH)p to influence the binding of other hormones that stimulate adenylate cyclase (36-37).
Effect of GTP on Time Course of Dissociation of Bound lZ51-FSH-The effect of GTP (1 pmol/assay tube) on dissociation of FSH bound to receptor in GB-Fr-1 was rapid in onset. A significant dissociation was observed within 1 min of the addition of GTP to the incubation medium (Fig. 4A). In the absence of GTP, no significant dissociation of bound '251-FSH was observed after 60 min of incubation. The addition of excess unlabeled FSH (4 pg/tube) to the incubation medium did not influence the rate of dissociation of lZ51-FSH in either the absence or presence of GTP.
Effect of GTP on Gel Filtration Profile of lZ5I-FSH-Receptor Complex-In previous experiments, the modulation of FSH binding to receptor by GTP was shown indirectly based on the failure of lZ5I-hFSH to coprecipitate with receptor during PEG separation. The present experiment was carried out to demonstrate directly that lZ51-hFSH was released as free hormone from receptor-hormone complex in response to GTP effect. 1251-FSH-Re~eptor (GB-Fr-1) complex was prepared and separated from unbound '251-FSH by gel filtration as described under "Methods." Equal aliquots of FSH-receptor complex were then incubated in the absence or presence of GTP (1 pmol/assay tube) for 10 min, at 30 "C. Following incubation, they were fractionated on a column of Ultrogel AcA-34. Gel filtration profile of the hormone-receptor complex incubated in the absence of GTP revealed a single major peak emerging in the void volume. On the other hand, the profile of hormone-receptor complex after incubation with '251-FSH.GB-Fr-1 complex was prepared as indicated under "Methods." Aliquots of the complex (10,000 cpm) were incubated in the presence or absence of 1 pmol of GTP/ assay tube or in the presence of 4 pg of nonradioactive hFSH/assay tube for indicated time intervals, at 30 "C. Data are expressed as percent of radioactivity present in the complex prior to the addition of GTP. Each data point represents the mean of triplicate determinations and these results are representative of two separate experiments. Panel B, dissociation of bound '*'I-hFSH from GB-Fr-1 exposed or unexposed to higher concentration of Triton X-100, in the presence of GTP. GB-Fr-1 was exposed to 0.4% Triton X-100 (final concentration), while maintaining an optimum ratio between sample protein and Triton X-100 (l), and chromatographed on Sepharose-4B column. The first retarded peak containing all receptor activity with negligible Gpp(NH)p-binding activity (A) was incubated with "'I-hFSH under standard assay conditions (see Methods). Untreated GB-Fr-1 (0) was similarly incubated with lZ5I-hFSH. In either case, hormone-receptor complexes were separated by gel permeation chromatography on Ultrogel AcA-34 column. Aliquots of complex (approximately 10,000 cpm) were incubated at 30 "C for 15 min in the presence or absence of indicated concentrations of GTP. Values are expressed as a percent of the radioactivity present in the complex prior to the addition of GTP. Each data point represents the mean of triplicate determinations. G T P showed a marked decrease in radioactivity in this peak with a concomitant increase in free lZ5I-FSH (Fig. 5).
Additional evidence to indicate that FSH receptor-associated GTP-binding activity was involved in GTP regulation of hormone binding to FSH receptor in GB-Fr-1 was obtained as follows. We exposed Sepharose GB-Fr-1 t o a higher concentration of Triton X-100 (0.4%), while maintaining an appropriate ratio between sample protein and Triton X-100 previously shown to preserve receptor activity (1). Detergents at sufficiently elevated concentrations are known to uncouple the functional units of adenylate cyclase (38, 39). Exposure of Sepharose GB-Fr-1 to 0.4% Triton X-100 resulted in a significant dissociation of GTP-binding activity from GB-Fr-1, as evidenced by the elution profiles of activity from the Sepharose-4B column. Ninety % of associated GTP-binding activity now eluted in a retarded peak after GB-Fr-1. lZ5I- Aliquots of complex (approximately 10,000 cpm) were incubated, either at 30 "C for 15 min or at 4 "C for 16 h, in the presence (0) or absence (A) of GTP (1 pmol/assay tube) and fractionated on a column of Ultrogel AcA-34 presaturated with 2% BSA and washed extensively. Total lZ5I radioactivity in column effluents was assessed, and recovery of lZ5I radioactivity exceeded 80% of that applied. hFSH bound to receptor in GB-Fr-1 (after treatment with Triton X-100) was now insensitive to GTP effect (Fig. 4B). Association of FSH-binding Activity with a Cholera Toxinreactive Guanine Nucleotide Regulatory Protein-Our observation that FSH receptor sensitivity to GTP is demonstrable in fraction GB-Fr-1 containing both receptor and GTP-binding activities, but not in the Triton X-100-treated fraction containing receptor activity lacking in GTP-binding activity, suggests that the FSH receptor may be physically associated with a putative guanine nucleotide regulatory protein. We made two additional observations which are consistent with this concept. Pretreatment of the pure testis membranes with cholera toxin plus NAD reduced the FSH binding to receptor (Table I). This is due to a decrease in affinity of receptor (not shown), and a similar effect on hormone binding following modification of guanine nucleotide regulatory protein by toxin has been shown previously for other receptor systems (15,40,41). Importantly, pretreatment of membranes with cholera toxin plus NAD, completely eliminated G T P effect on FSH binding to its receptors (Table I). In contrast, treatment of membranes with pertussis toxin and NAD had no effect on GTP action. These results suggest that the GTP-sensitive FSH receptor activity is associated with N,, but not Ni.
T h e likelihood that the nucleotide regulatory protein N, was in association with the FSH receptor was further investigated as follows. Purified bovine testis membranes were treated with cholera toxin in the presence of [~u-~'P]NAD under conditions in which the N, protein is specifically labeled by ADP-ribosylation of its a-subunit (42-45). Membrane proteins extracted with Triton X-100 were then chromatographed on a Sepharose-GB column (Fig.  6). A significant peak of 32P radioactivity was eluted after void volume. In the presence of cholera toxin pretreatment, this peak was very small (Fig. 6 ) . Comparison of the elution profiles of membrane protein labeled either by ["PIADP-ribosylation or prebound

TABLE I Effects of cholera toxin and pertussis toxin treatments on GTP effect on '"I-hFSH binding to its receptors in pure membranes of bovine testis
Bovine testis membranes were subjected to the indicated treatments and then incubated with lz5I-hFSH to prepare hormone-receptor complexes, as described under Methods. The complexes were incubated in the presence or absence of GTP (2.5 pmol/ml), as described earlier (legend for Fig. 5) for 15 min at 30 "C. This experiment was performed twice with similar results.
Percent specific binding of '%I-hFSH" with lZ5I-hFSH indicates that ADP-ribosylated protein and the '251-hFSH-labeled receptor coelute from the column. These results are consistent with the present concept that FSH receptors are associated with a guanine nucleotide regulatory protein, probably N.. Gpp(NH)p-binding Properties of FSH Receptor-associated and Receptor-free Fractions-Specific binding of [3H] Gpp(NH)p to fractions GB-Fr-1 (FSH receptor-associated) and GB-Fr-2 (receptor-free) were determined by Scatchard analysis. Differences exist in their [3H]Gpp(NH)p-binding properties. The Scatchard plot for the nucleotide-binding component in GB-Fr-1 indicated the presence of low affinity (9.2 X 10' M-') and high capacity binding sites, while GB-Fr-2 showed the presence of only high affinity (1.37 X lo7 M-') and low capacity binding sites (Fig. 7). Importantly, the higher nucleotide concentration required by GTP for its effect on FSH binding to GB-Fr-1 is compatible with the observed low affinity and high capacity binding sites present in this fraction.

Treatment
The binding of [3H]Gpp(NH)p in both GB-Fr-1 (receptorassociated) and GB-Fr-2 (receptor-free) displayed a high degree of specificity. GTP at concentrations of 2.5 X M and 0.8 x M were required to inhibit 50% of total [3H] Gpp(NH)p-bound to GB-Fr-1 and GB-Fr-2, respectively. ATP, App(NH)p, ADP, and AMP failed to inhibit the binding of tracer to both fractions (Fig. 8). Thus, the presence of highly specific Gpp(NH)p/GTP-binding sites in these fractions indicate a higher degree of specificity in GTP interactions with these fractions, particularly GB-Fr-1, while modulating FSH binding to its receptors.
A recent report (46) on @-adrenergic receptor system has suggested that enhanced GTPase activity in N. following its interaction with receptor provides a sensitive monitor for receptor-N. coupling. It was, therefore, of interest to compare GTPase-specific activities of GB-Fr-1 and GB-Fr-2. Each fraction contained GTPase activity. GB-Fr-2 was 2-3 times less active than GB-Fr-1 (Table 11). The GTPase activity could be  the presence or absence of indicated concentrations of various nucleotides for 120 min at 30 "C. Separation of bound and free-labeled Gpp(NH)p was accomplished as described earlier (1). Results are expressed as a percent of [3H]Gpp(NH)p bound in the absence of nucleotides. Each point is the mean of triplicates that agree within 5% of the mean value.

TABLE I1
Comparison of G T P hydrolytic (GTPase) specific activity of GB-Fr-1 and 6B-Fr-2 Aliquots of GB-Fr-1 and 6B-Fr-2 (10-20 pg of protein) were incubated in assay medium containing 5 mM MgC12 and 25 nM GTP(7-?'P) (100,000-400,000 cpm) as described under Methods. The assay reaction was stopped by placing the tubes on ice and adding 0.75 ml of 20 mM phosphoric acid, pH 2.1, containing 5% (w/v) activated charcoal. ["'PIPi released was separated and counted thereafter. Specific activity (pmol of Pi/mg of protein/min). Three assays with triplicate determinations for each point.
* One assay with triplicate determinations for each point.
shown to be specific in both the fractions for guanosine-5'triphosphate by the simultaneous addition of 1.0 mM ATP or 0.4 p M App(NH)p.

DISCUSSION
The present report provides the first evidence for a physical and functional association between unoccupied FSH receptors and a guanine nucleotide regulatory protein, probably N.. We have also observed a separate pool of guanine nucleotidebinding sites with a high affinity for Gpp(NH)p representing a discrete molecular entity free from FSH receptors and in a GTP sensitive state. These findings are consistent with, and extend, our previous observations that GTP and its analogues can act either alone or with FSH to stimulate testicular adenylate cyclase activity (12).
Our conclusion that FSH receptors and a guanine nucleotide regulatory protein, probably N., are physically associated, is based on several lines of evidence. 1) Detergent-soluble FSH receptors and Gpp(NH)p-binding sites coeluted from Sepharose-GB column, and most FSH receptors and GTPbinding activity were retained by a GTP-Sepharose affinity matrix. Their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p, indicating that associated guanine nucleotide-binding protein was involved in FSH receptor retention by the affinity matrix. 2) The physically associated soluble FSH receptors and guanine nucleotide-binding protein are functionally related. This is shown by the negatively cooperative binding interactions of guanine nucleotide and FSH. In the absence of GTP, FSH bound with high affinity; whereas, in the presence of GTP, the binding of FSH to receptors decreased in a dose-dependent manner. Analysis of equilibrium-binding studies suggested that the decreased binding could not be accounted for by, a decrease in the number of receptorsper se. Rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Also, an increase in dissociation rate prompted by G T P was reflected in significant decrease in FSH receptor affinity. Thus, in the absence of exogenous GTP, no significant dissociation of bound I2'1-FSH was observed even after 1 h, whereas in the presence of this agent, the dissociation was rapid (less than 1 min). 3) Detergents at sufficiently elevated concentrations are known to uncouple the functional units of adenylate cyclase (38, 39). Exposure of GB-Fraction-1 to higher concentration of Triton X-100 in the present study reduced significantly the receptor-associated GTP-binding activity. Importantly, this also rendered FSH-binding to its receptor insensitive to GTP effect. 4) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a high peak of 32P radioactivity coeluted with FSH receptor activity from Sepharose-GB column.
Recent studies have shown that the &-subunit of N, regulatory protein is the site of cholera toxin-induced ADP-rybosylation (42-45). We infer, therefore, that the Triton-solubilized FSH receptors are associated with a guanine nucleotide regulatory protein, probably N, which is consistent with the ability of FSH to stimulate testicular adenylate cyclase (9-11). 5 ) Examination of differential effects of cholera toxin (plus NAD) and pertussis toxin (plus NAD) on GTP regulation of ligand binding also provided some insight about the putative guanine nucleotide regulatory protein associated with the FSH receptor. Pretreatment of the pure testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, completely eliminated GTP effect on FSH binding to its receptors. These results are consistent with the concept that GTP-sensitive, FSH receptor activity is associated probably with N,, but not Ni.
In a previous study, it has been suggested that the detergent deoxycholate mimics nonspecifically the function which is normally fulfilled by the guanine nucleotide-binding protein in inducing the high affinity state of the P-adrenergic receptor (52). In the present study, the high affinity state of soluble FSH receptors in the absence of GTP cannot be attributed to similar effect, if any, of Triton X-100. This is because the ability of detergent to induce receptor affinity is unique to deoxycholate and is not common to all detergents (52). Control experiments employing Triton X-100 ruled out such a possibility in the present study. Moreover, we noticed that FSH receptor activity is more labile in the presence of in-creased concentrations of Triton X-100 (I), and an appropriate ratio of protein to detergent is critical in maintaining receptor activity after chromatography on Sepharose-GB. Through a series of chromatographic profiles of receptor activity, we established an optimum level of detergent relative to sample protein, which we used for column fractionation (as indicated under "Methods").
We have recently shown the presence of specific binding sites for Gpp(NH)p/GTP in large excess to FSH receptors in rat Sertoli cells (13). The specific binding of Gpp(NH)p/GTP to bovine testicular membranes represent two classes of binding sites, i.e. high affinity and low capacity, and low affinity and high capacity.' Interestingly, the present studies indicate that the high affinity sites for G T P could be separated from FSH receptors without affecting the ligand-binding properties of either. At this point, however, it is not clear whether the high affinity GTP-binding sites represent a separately existing component in bovine testicular light membranes. Alternatively, the component may have separated during solubilization by detergent, due to dissociation from other associating membrane receptors/proteins. Nevertheless, our results suggest that FSH receptors exist in a high affinity state, independent of their association with high affinity GTP-binding sites.
Another noteworthy result of the present studies is the observation of significant GTPase activity in both FSH receptor-associated and receptor-free guanine nucleotide-binding components in the absence of hormone. Recently Cerione et al. (46) suggested that an induction of GTPase activity in guanine nucleotide-binding protein (N,) following its interaction with P-adrenergic receptor provides a sensitive moniter for P-adrenergic receptor-N. coupling. This was based on their observation that when large amounts of /3-adrenergic receptor and N. were inserted into phospholipid vesicles, a significant induction of GTPase activity was noticed even in the absence of hormone. Our observation that the FSH receptor-associated guanine nucleotide component has higher GTPase activity than that of receptor-free component is consistent with their findings.
The present studies provide evidence for a physical association between FSH receptors and a guanine nucleotide regulatory protein, probably N,. Our results (a significant decrease in FSH receptor affinity in response to GTP effect and loss of sensitivity to GTP in FSH binding after cholera toxin treatment of bovine testis membranes) suggest that GTP interaction with a guanine nucleotide regulatory protein may lead to the conformational change of FSH receptor protein, through a functional association between these two proteins. These results, together with the finding that FSH receptor interactions induce adenylate cyclase with concomitant increase in CAMP in bovine testis membranes (9-ll), raise the possibility for a bidirectional coupling of N, to adenylate cyclase and FSH receptor. The availability of two separate components having either high affinity FSH receptors or high affinity GTP-binding sites should facilitate further studies on the molecular mechanisms of FSH activation of adenylate cyclase.