Properties of the Luteinizing Hormone Receptor of Isolated Bovine Corpus Luteum Plasma Membranes*

SUMMARY Luteinizing hormone binding sites with high affinity and specificity for ovine-luteinizing hormone have been shown to be present in a purified plasma membrane preparation obtained from bovine corpus luteum. The specific binding of W-luteinizing hormone to the membranes is a saturable process with respect to 9-luteinizing hormone. Native luteinizing hormone competes for the binding in a way ex-pected from the biological identity of the 2 molecules. Human ckorionic gonadotropin and pregnant mare serum gonadotropin, two gonadotropins which have luteinizing hormone activity, compete for the binding site of luteinizing hormone, with an affinity which is less than that of native luteinizing hormone. The same holds for the o( and p chains of luteinizing hormone which showed, respectively, 100 and 200 times less affinity than luteinizing hormone for its binding site. hormone, which does not have intrinsic luteinizing hormone does not compete for the binding of luteinizing hormone to an extent greater than its contamination by luteinizing The is and maximum


SUMMARY
Luteinizing hormone binding sites with high affinity and specificity for ovine-luteinizing hormone have been shown to be present in a purified plasma membrane preparation obtained from bovine corpus luteum.
The specific binding of W-luteinizing hormone to the membranes is a saturable process with respect to 9-luteinizing hormone. Native luteinizing hormone competes for the binding in a way expected from the biological identity of the 2 molecules. Human ckorionic gonadotropin and pregnant mare serum gonadotropin, two gonadotropins which have luteinizing hormone activity, compete for the binding site of luteinizing hormone, with an affinity which is less than that of native luteinizing hormone. The same holds for the o( and p chains of luteinizing hormone which showed, respectively, 100 and 200 times less affinity than luteinizing hormone for its binding site. Follicle-stimulating hormone, which does not have intrinsic luteinizing hormone activity, does not compete for the binding site of luteinizing hormone to an extent greater than its contamination by luteinizing hormone allows. The binding of 1251-luteinizing hormone is temperaturedependent and reaches its maximum in 10 min at 37". The rate constant of the luteinizing hormone-membrane association (2.17 X 106, M-' s-l) and dissociation (2.46 X 10m3 SK') have been measured independently at 23 and 10". The dissociation constant (1.13 X 1O-g M) based on these rate constants is similar to that (3 X 10es M) calculated separately from equilibrium data. Measurement of the rate constants at various temperatures gives similar values for the dissociation constant.
This shows that the decrease in dissociation rate is proportionately the same as the decrease in association rate.
Binding is maximal at pH 7.6 and is not affected by Ca2+ concentration in the range of 0.1 to 20 mM. The effects of different enzymatic preparations on the binding site of luteinizing hormone have been investigated. It is not affected by DNase, trypsin, chymotrypsin, pepsin, and collagenase.
Treatment of the membrane preparations by neuraminidase increased the binding capacity for luteinizing hormone by 2-fold.
Phospholipase C, as well as phospholipase A, decreases it by half.
* This work was supported by grants from the Population Council (M.72.138C), the National Science Foundation (GB 341X), and the Rockefeller Foundation.
Study of the specific binding of '*Wuteinizing hormone to subcellular fractions of bovine corpus luteum has shown that the specific binding of luteinizing hormone is localized to the plasma membrane fraction (1). Since the disruption of cellular structures leads to the loss of biological activity, the interaction of LH' with its receptor site can be best defined by looking at the binding of 1251-LH to the membranes.
One should then make certain that this interaction is specific and that 1251-LH does not bind to structures other than its receptor site. The present report presents detailed data on the properties of the binding interaction between bovine corpus luteum plasma membrane and '*"I-LH.
The kinetic data for binding of 'WLH to its receptor site will be further utilized for the isolation of the LH receptor site from plasma membrane of bovine corpus luteum.
EXPERIMENTAL PROCEDURE Materials-Highly purified ovine LH is prepared by the method of Papkoff et al. (2) and is further purified by chromatography on diethylaminoethyl cellulose to remove contaminating thyroid-stimulating hormone as described by Pierce and Carsten (3). The biological activity measured by the ovarian ascorbic acid depletion assay is 2.75 units per mg (957, confidence limits 2.1 to 3.7) referred to the standard NIH-LII-S17.
Thyroid-stimulating hormone activity amounts to 15 to 30 milliunits per mg. Contamination by growth hormone, follicle-stimulating hormone, adrenocorticotropic hormone, or melanocjite-stimulating hormone cannot be detected.
Highly purified PMSG (15,000 i.u. per mg) is prepared by the method of Gospodarowicz and Papkoff (4) and Gospodarowicz (5). The isolation of the two subunits of ovine LH is affected in this laboratory by counter current distribution (12 transfers repeated twice) as described by Papkoff and Samy (6) and as modified by Reichert et al. (7).
Amino acid and carbohydrate analyses show the purity of the LH cy and LH fl subunits (6) which is further confirmed by double diffusion in agar, immunoelectrophoresis, quantitative precipitation curves, steroidogenic activity (8), and physicochemical properties (9). Highly purified FSH is prcparcd by the method of Papkoff et al. (10). Its activity is 30 times the standard NIH-FSH-Sl.
Residual LH contamination is eliminated by chromatography on an anti-LH IgG Sepharosc column, and amounts to 0.005% as determined by radioimmunoassay (11). Iodinalion Procedure--The iodination of LH is performed using lactoperoxidase as the catalytic agent. All reactions arc carried out in small glass tubes at room temperature.
The reactants arc mixed continuously with a small magnetic stirrer.
In order to minimize the introduction of more than 1 atom of iodine per LH molecule, iodination is performed with an equimolar ratio of '*jI:LH.
To minimize the deleterious effect of the oxidizing agent, hydrogen peroxide, it is added in approximately equimolar amounts with respect to the concentration of iodine and LH.
To obtain large quantities of the iodo-LH for chemical and biological analysis, milligrams of purified LH are iodinated with lZ71 to which is added a trace of r*jI. When low specific activity iodinated LH is necdcd, the reagents are added rapidly in t.he following order and amounts: (a) 250 &i of 12"INa in 5 ~1 of 0.1 s NaOH; (b) 588 ng of 12'INa in 100 ~1 of 0.1 N NaOH; (c) 50 11 of 1 M potassium phosphate buffer, pH 7.3; (d) 120 pg of LH in 50 ~1 of 0.25 M potassium phosphate buffer, pH 7.3; (e) 5 pg of lactoperoxidase in 5 ~1 of the same buffer. The reaction is initiated by adding 70 ng of hydrogen peroxide in 10 ~1 of water.
To sustain the reaction, 70 ng of hydrogen peroxide are added four times at intervals of 2 min. At l-mm intervals startiiig at zero time, 5 ~1 of the iodinated solution are withdrawn, mixed with 0.1 ml of .O.l $& bovine serum albumin (w/v), and precilritated with 10% trichloroacetic acid. Precipitates are washed twice with cold lOolo trichloroacetic acid, dissolved in 0.2 ml of 0.2 N KOH, and their radioactivity determined in a liquid scintillation counter (xuclcar Chicago model Unilux II). Usually in 10 min, 90 to 95% of the I*51 is trichloroacetic acidprecipitable.
When iodinated LH of high specific activity is needed, no 12'1 was added, and 10 mCi of rz51Na (588 ng) are added.
The ratios of I251 to LH, lactoperoxidase, and hydrogen peroxide are kept equimolar.
After the reaction is completed, final lmrification is achieved by gel filtration of the iodination solution on columns of Scphadex G-200 equilibrated with 0.9% NaCl, 0.01 M potassium phosphate, pH 7.3.
Integrity of the LH is analyzed by polyacrylamidc gel electrol)horesis (Fig. ld).
One peak of radioactivity is observed, coinciding with the o( subunit of the hormone. Countercurrent dist.ribution of the subunits of LH confirms this observation. All the radioactivity is confined to the o( subunit. This suggests that the tyrosyl residue present in the /3 subunit is not exposed when the LH has its native configuration.
The lack of izsI in the fi subunit also shows that the reaction is highly specific for tyrosyl residues since no other amino acids are labeled.
The integrity of the antigenic site of '*jI-LH is confirmed by a quantitative prccipitin test (Fig. 1B) The equivalence zone for LH is the same as that of '*:I-LH, and over 95% of the r2%LH is precipitated.
When '?"I-LH is applied on an anti-LH IgG Sepharose column (II), 93% is retained on the column, thus proving that the antigenic sites of LH are intact (Fig. 1C). Ninety per cent of the adsorbed iz51-LH is eluted with 6 M guanidine HCl, pH 1.5. The unadsorbed fraction may represent either iodinated peroxidase or denatured LH. When the biological activities of LH and '*"I-LH are compared by the ovarian ascorbic acid depletion assay, their activities are indistinguishable (Table I). Their ability to stimulate progesterone biosynthesis ( Fig. 2A), as well as release of progestins in vitro from bovine luteal cell suspension (Fig. 2B), is also identical over a lOOO-fold range of concentration.
These different criteria indicate that iodination of bovine LH using the lactoperoxidase as a catalytic agent produces no noticeable denaturation of the hormone. The final activity of ovine LH is 1800 Ci per mM, corresponding to an average incorporation of 1 atom of I251 per molecule of LH. With 90% incorporation of ina into LH, no further purification of LH by ion exchange chromatography is required to separate LH from i2"I-LH.
By increasing the concentration of iZ51, an average of more than 1 atom of 125I can be incorporated by a molecule of LH. Since a decrease in biological activity of ovine LH is observed when more than 2 atoms of iodine are incorporated into each molecule B FIG. 1. A, fractionation of a 12% acrylamide gel ~1-1 4.5 (12). lZ51-LH (170.000 corn) mixed with 100 11~ of LH is submitted to electrophoresis for-1 hour 45 min at 8 ma-per tube. At the end of the electrophoresis, the gels are either stained with 10/O Amido schwarz in 7y0 acetic acid solution, or frozen at -20" on Dry Ice, cut at l-mm thickness, dissolved as described by Ward el al. (13) and counted in a liquid scintillation counter. A tracing of the stained gel is shown below the fractionation.
Identical distribution of radioactivity is obtained with and without staining. B, quantitative precipitin curve comparing the antigenicity of LH and ls51-LH (840,000 cpm per tube). The protein precipitate is assayed by the method of Lowry et al. (14). The molecular weight of LH is assumed to be 30,000. C, binding and elution of 12jI-LH (2.5 X lo6 cpm) dissolved in 0.9 M NaCI, 0.01 M potassium phosphate, pH 7.3, applied on a column of anti-LH of LH, the iodination procedure has been designed to limit the incorporation of iodine to 1 atom per molecule. The conditions of iodination described by Miyachi et al. (18) for the iodination of human LH or HCG using lactoperoxidase as a catalytic agent are not satisfactory for ovine LH.
With their conditions 90% of the ovine LH is denatured and aggregated. This is mostly secondary to the pH of 5.6 at which the iodination is performed.
In contrast to human LII or HCG, ovine LH dissociates easily at acidic pII (17) and iodination of tyrosine in the o( chain, as well as in the /3 chain, occurs resulting in denaturation of ovine LH. Furthermore, high concentration of hydrogen peroxide has a deleterious effect on the biological activity of ovine LH (19)  Binding Assuy-The assay for spkfic binding of 'WLH to membranes is a slight modification of that used to measure spccific binding of insulin to intact fat cells (20,Zl) and fat cell mcmbranes (22).
The filters are washrd under vacuum with 3 times 3 ml of ice-cold Krebs-Ringer bicarbonate buffer containing 1 7. albumin (w/v).
Every dctermination of binding is performed in triplicate, and for ever)-sue+ determination parallel, triplicate samples are performed in the prcscnce of native LH (100 pg per ml) to d&ermine the correction for nonspecific binding of LII.
As it has been stressed by others (22), it is imperative that such corrections be performed in ordrr to dcterminc "specific" LH binding accurately. Nonspecific binding to EGWP filters in the absence of membranes is 0.3 5; of the input. I'lasma membranes which hare been heated to 90" for 5 min do not bind more than 0.5% of the input.
The filters are dissolved in 15 ml of 13ray's scintillation fluid containing 4n Cab-0-Sil and counted in a liquid scintillation counter as described.
Counting efficiency is 6Oc/c. Enzyme Efect-Specific procedures used for digestion of plasma membranes with enzymes are descaribed in the appropriate tables.
Generally, the membranes arc incubated with the enzyme in Krebs-Ringer bicarbonate buffer containing 1 to 0.17* albumin (w/v) for 10 to 20 min at 37", then washed twice ba centrifugation using the same buffer (18,000 X g for 10 min). The pellet is resuspended in Krebs-Ringer bicarbonate buffer containing 1 y0 albumin (w/v), and binding of 'WLH is then pcrf'ormed.

Binding
as Function of '2"I-LZI Concenkation-The specific binding of 12"I-LH to purified plasma membranes obtained from bovine corpus luteum is a saturable process with respect to 1261-LH concentrat,ion (Fig. 3). Specific binding of "51.LH can be detected at a, concentration as low as 3 ng per ml (1 x lo-lo &I), and saturation is obtained at 300 ng per ml (1 X 10e8 M).
Displacement of Bound lz51-LH by Native LH and Other Gonadotropins--'WLH specifically bound to plasma membrane is displaced by increasing concentrations of native LH in a fashion predicted by the near-identity of these 2 molecules (Fig. 4). Other polypeptidic hormones which possess LH activity such as HCG and PMSG are able to compete for the receptor site of LH. However, their affinity is about 10 times less than that of LH. Of the two gonadotropins, HCG is the most potent (Fig. 5). Hormones which are free of LH act,ivity such as FSH (Fig. 6) compete very poorly for the LH binding site. Its a&&y allpears to be lO,OOO-fold less than that of LH and can be explained by LH contamination, which is 0.0057; on the basis of radioimmunoassay.
LH 01 and LH p, the two subunits of LH, have, respectively, 50. and loo-fold less activity on a weight basis than LH, thus confirming the fact that to get full steroidogcnic effect the two subunits of LH must be combined (8).
Binding as Function of Plasma Membrane Concentration-The binding of 1251-LH to plasma membrane is directly proportional to the plasma membrane concentration over the range of concentrations that can be used in these procedures (Fig. 7A). This linear relationship in the absence of sat,urating concentration reflects the high affinity of the interacting species and can be Binding a-s Function of Time-The specific binding of i251-LI-I to plasma membrane of corpus luteum at 23" reaches a maximum at 12 min and then stays constant. In contrast, nonspecific binding reaches a maximum at 4 min (Fig. 7B) teraction-The rate of binding of r2"I-LH to its receptor increases with increasing temperature.
It reaches a maximum at 37" (Fig.  8). The rate constants of association can be calculated from these data since they obey second order kinetics.
At 23" the rate of association kr is 2.17 x lo6 RI+ s-r while at 10" it is 4.2 X 10" M-1 s-1. The rate of dissociation decreases with decreasing temperature (Fig. 8). The dissociation data obey first order kinetics, and the half-life of the complex is 5 min at 23" and 16 min at 10". At 23" the dissociation constant is 2.4 X 10e3 s-r, while at 10" it is 7.18 x 10e4 s-r. Values of the equilibrium constant indicate similar binding of LH to its receptor at low temperatures as compared to high temperatures (Table II) Table III. Ca2+ has no effect on the binding of '2"I-LH to its receptor between 1 and 10 mM, but concentrations higher than 20 MM reduce the binding by 20%. When NaCl is present in a concentration higher than 0.5 M, the binding capacity is considerably reduced, and at 2 M no binding of 12%LH is observed. KC1 has the same effect. The specific binding of 12AI-LH to plasma membrane occurs over a relatively narrow range of p1-I (Fig. 10). Maximum binding is observed at pH 7.4 and 7.6. At pH 6 or 8 the binding capacity is only 307, of the binding capacity observed at pH 7.6. However, the low binding capacity of plasma membrane at low pH values such as pH 5 or high pH values such as pH 9 is fully reversible since incubation of plasma FIG. 9. A, double reciprocal plot of data obtained in an esperiment performed as described in Fig. 3. K1.n from the slope of this line is 3.4 X low9 liter ~-1.
H, Scatchard plot of data obtained in an experiment performed as described in Fig. 3. The apparent constant kn is 3 X 1O-9 liter M-l. membrane at those pH values followed by washing and rcsuspension in pH 7.6 fully rcstorcs their capacity to bind '251-LH. Eflect of Enzymes on LH Binding-The effects of several enzyme preparations on the binding of '*"I-LH to plasma membranes of corpus luteum are shown in Table IV. Purified llreparations of trypsin, chymotrypsin, and pepsin do not affwt it. Digestion of plasma membrane preparations with neuraminidase increases the binding capacity of the plasma membrane, while digestion with phospholipase lowers it to half the control value. Collagcnase, one of the enzymes wc used to dissociate lutcJn1 cells EJ Our earlier attempts to study the interaction of '2"I-LH with its receptor site failed because we had labeled the hormone using the chloramine T method.
Even though we have attempted to minimize the damage to the proteins caused by chloraminc T by working with an cquimolar ratio of chloramine 1' to LH, the specific activity of the hormone (300 Ci per mM) is low, as is the incorporation of 1251 (11). To raise t,he specific activity of ' is not observed. So denaturation, which is manifested in only small changes in immunological or biological activity, results in large amounts of spurious binding.
Also, in the case of human LH or HCG, when chloramine T is used as an iodinating agent, the ratio of chloramine T to hormone, the length of exposure, and the temperature are critical factors for the iodination of human LH or HCG if binding studies arc to be conducted with it after-ward (23). Since ovine LH is a much more fragile molecule than human LH or HCG in that its two submits arc easily dissociated by mildly acidic conditions, it is not astonishing that the damage done by exposing ovine LH to chloramine T is more extensive than for HCG or human LH.
Using lactoperoxidase as a catalytic agent WC have, in contrast to the chloramine T method, been able to incorporate 90 to 95% of the 125I into LH. Since we are working in an equimolar ratio of iodine to LH, this means that on the average 1 molecule of LH contains 1 molecule of 1261, The immunological and biological properties of iz51-LH are similar to those of the native hormone and when binding studies of '*"I-LH with its receptor, present in plasma membrane of corpus luteum, are investigated, saturability of the receptor site is obtained.
Evidence for the s1)ecificit.y of the LH-cell interaction is further strengthened by the failure of several polypeptide hormones to compete with LH for binding to cells, We observe no displacement of lz51-LII (2 X lo-i0 M) by growth hormone, prolactin, adrenocorticotropin, or glucagon at concentrations as high as 3 x 10-e M. However, native LH is able to compctc with '2%LH for binding.
Two other gonadotropins which possess LH activity, EICG and PMSG, are able to compete for the receptor site with an activity 10 times less than that of ovine LH, hardly an astonishing fact since we are working with a homologous system. Ovine LH and bovine LII have been shown to be similar, and the receptor site of plasma membrane of bovine corpus luteum should then have a higher affinity for ovine LI-I than for gonadotropin of mlrelated species such as human or horse. The binding competition by LH o( and LH /3 has also been analyzed. It is known that LH (Y and LH /3 have a low steroidogenic activity (9) and ascorbic acid depletion activity (6,8,24,25) when compared to LH. This is reflected in their low binding affinity for the LH receptor site present in plasma membrane fraction of bovine corpus luteum.
Their ability to compete with LH is 2% for LH N and 1% for LH /3. These values represent, respectively, a lOOand 200-fold molar excess over LH and suggest that neither subunit alone is sufficient to convey significant receptor activity. The binding activity of LH a and LII p is comparable to the values reported by us for their steroidogenic activity in bovine corpus luteum (8) and similar to their activity in the radioligand rcccptor assay2 (23). The competition of ovine FSH is negligible. Displacement is observed only at a lO,OOO-fold excess of FSH over LH and most probably reflects the residual contamination of this hormone by LH. Kammerman et al. (26), looking at the competitive binding of HCG and FSH in porcine granulosa cells, have reported similar data.
Of special interest to us is the effect of different enzymes on the binding of i251-LH to its receptor in plasma membrane fraction of bovine corpus luteum.
We have recently reported the isolation and the maintenance in tissue culture (26) of metabolically active bovine luteal cells, obtained from corpus hlteum by an enzymatic treatment (16). Of the three enzymes that we are using for the cellular dissociation, two, collagenase, and try@, do not affect the binding site of LH.
The third, hpaluronidase, reduces the binding affinity for LH by a third.
While it is not yet clear whether this effect is due to proteolytic activity present in this crude enzyme preparation or to hyaluronidase itself, it is evident that its use for cell dissociation presents some dangers if one wants to study the binding of LH to lut,eal cells. However, the kinetics of binding iZ51-LH to cell suspensions obtained from corpus luteum shows the same characteristics3 as those described plasma membrane.
Despite the loss of biological activity xhen the cell is disrupted, the specific binding of LH to its receptor site should permit its identification during the purificat'ion of the receptor sites which will eventually lead to its isolation. ATofe Ad&cl ilz Proof-Since t.his manuscript was submitted for publication Charming and Kammerman (33) have described the chsractcristics of gonadotropin receptors of porcine granulosa cells and Lee and Ryan (34) have reported the binding of human LI-I to homogenatesof lutcinized rat ovaries. KD of 7.9 X lo-r0 M \\'a~ found.
I thank t'he Talone Meat Co. for providing t'he bovine corpora lutea. Wit,hout their cooperation this work would not have been possible.
I also thank Dr. K. Jones for his invaluable assistance and constructive criticism of the manuscript drafts.