Physical Characteristics of the Gonadotropin Receptor-Hormone Complexes Formed in Vivo and in Vitro (Gonadotropin Receptors/Ovary/Solubilization)
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1272-1275, April 1975 Physical Characteristics of the Gonadotropin Receptor-Hormone Complexes Formed In Vivo and In Vitro (gonadotropin receptors/ovary/solubilization) M. L. DUFAU, E. J. PODESTA, AND K. J. CATT Section on Hormonal Regulation, Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Elwood V. Jensen, January 1>, 1976 ABSTRACT The physical properties of detergent- Lubrol PX and Triton X-100 have shown sedimentation con- solubiliked gonadotropin receptor-hormnone complexes, of 7.0 and 8.8 respectively. The partition coefficients determined by density gradient centrifugation and gel stants 5, filtration, were compared after in vivo and in vitro labeling (K..) of such complexes determined by gel filtration on 6% ofspecific ovarian binding sites with radioiodinated human agarose columns were 0.32 and 0.29, respectively (12). chorionic gonadotropin (hCG). Following intravenous ad- To examine the relevance of such "in vitro" labeled recep- ministration of biologically active "2'I-labeled hCG, up to tor-hormone complexes to the complex formed in vito, we 50% of the gonadotropin tracer was bound to the lutein- ized ovaries of immature female rats treated with pregnant have administered 12I-labeled hCG to immature female rats mare serum/human chorionic gonadotropin. Comparable treated with pregnant mare serum/human chorionic gonado- binding of "i6I-labeled hCG was observed after equilibra- tropin and examined the gel filtration and' sedimentation tion of ovarian particles with the labeled hormone in vitro. properties of the complexes subsequently extracted from the The sedimentation properties of the solubilized receptor- with non-ionic detergents. Such studies have hormone complexeg formed in vivo were identical with luteinized ovaries those derived for the corresponding complexes formed shown that the physical properties of the receptor-hormone in vitro and extracted with Triton X-100 and Lubrol PX, complex formed in vivo are identical in several respects with with sedimentation constants of 8.8 S' for the Triton- those of the corresponding complex formed in vitro. These solubilized complex and 7.0,S for the complex extracted results indicate that the receptor-hormone complex extracted with Lubrol PX. During analytical gel filtration of the Triton-solubilized receptor-hormone complex on Sepha- with detergent from particulate receptors labeled in vitro rose 6B in 0.1% Triton X-100, the partition coefficient provides a valid experimental counterpart of the complex (K.y) of the "in vivo" complex (0.32) was not significantly formed in vivo after interaction of circulating gonadotropin different from that of the complex formed in vitro (0.29). with the specific receptor sites of the luteinized rat ovary. Gel filtration of the Lubrol-solubilized ovarian particles on Sepharose 6B in 0.5% Lubrol PX gave K., values for the MATERIALS AND METHODS "in vivo" and "in vitro" labeled complexes of 0.36 and 0.32, respectively. These findings demonstrate that the Preparation of Radioiodinated Gonadotropin. Purified hCG physical properties of size and shape which determine the (10,000 IU/mg, donated by Dr. R. Canfield, Department of partition coefficient and sedimentation characteristics Medicine, Columbia University, N.Y.) was labeled with 125J of detergent-solubilized gonadotropin receptor-hormone by the following modification of the lactoperoxidase method complexes formed in vitro are not distinguishable from mm those of the complexes extracted after specific interaction (13). The reaction was performed in a 10 X 75 poly- of the ovarian gonadotropin receptors with radioiodinated propylene tube to which was added: 0.5 M sodium phosphate hCG in vivo. buffer (pH 7.0), 25 ,ul; hCG 1 'mg/ml in 0.05 M phosphate buffer (pH 7.5), 25 Ml; Nal"I obtained from Amersham- Specific gonadotropin receptors for luteinizing hormone (LH) Searle, 1 mCi; lactoperoxidase (Sigma) 100 ,gg/ml in 0.05 M [and human chorionic gonadotropin (hCG) I have been re- phosphate buffer, 10 jl; and freshly prepared 1:30,000 solu- cehtly identified in homogenates of the testis and ovary from tion of H202, 10 Al- After mixing, the reaction was allowed to rat, pig, and cow. Such receptors show high specificity and proceed for 2 min at room temperature, followed by addition affinity for LH/hCG, and have been applied to radioligand- of 100 ,l of a solution of bovine serum albumin, 1 mg/ml in receptor assay and structure-function studies of LH and hCG phosphate-buffered saline. The mixture was then transferred (1-7). In addition, the specific gonadotropin receptors have to a column (0.5 X 14 cm) of Sepharose-concanavalin A (Phar- been solubilized from particulate binding fractions of gonadal macia) for purification of 12"I-labeled hCG by group specific homogenates by treatment with non-ionic and ionic deter- affinity chromatography for glycoprotein (14). After elution gents (7-11). Recently, the physical properties of gonado- of the iodide peak and labeled non-glycoprotein with the buff- tropin receptors extracted with detergents from gonadal ered saline containing bovine serum albumin, 1 mg/rl, the homogenates, before or after labeling with [124I]iodo-hCG in radioiodinated glycoprotein hormone was eluted with 0.2 M vitro, have been described in detail (7-12). The gonadotropin- a-methyl-D-mannoside in buffered saline/serum albumin solu- receptor complexes extracted from ovarian particles with tion. The central 3/4 of the "'I-labeled hCG peak was pooled and stored as frozen aliquots at -70'°The specific activity of Abbreviations: hCG, human chorionic gonadotropin; LH, the radioactive chorionic gonadotropin was approximately 20 luteinizing hormone. UCi/Mg, or 40,000 dpm/ng. 1272 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Gonadotropin Receptors Labeled In Vivo and In Vitro 1273 In Vivo Labeling of Ovarian Receptor Sites. Twenty-one-day- old immature female Sprague-Dawley rats were injected sub- 5000 IgG BSA cutaneously with 50 IU of pregnant mare serum gonadotropin (Equinex), followed after 65 hr with a single dose of 25 IU 0. 5-20% Sucrose human chorionic gonadotropin (Pregnyl, Organon). Forty- 4000 0.1% Triton X-100 eight hours after hCG treatment, the rats were injected with O In Vitro 1O0uCi of 125I-labeled human chorionic gonadotropin as a single o 8.8S In Vivo bolus of 0.2 ml via the jugular vein. The animals were sacri- *r3000 8.8S C ficed 2 hr after injection and the ovaries were excised, weighed, z and analyzed in a gamma spectrometer to determine the up- 0 two ovaries '12000 take of labeled gonadotropin. The total uptake by 0 was always close to 50% of the administered dose of 125IJ 0 labeled hCG. II 1000 Solubilization of Ovarian Receptor-Hormone Complex. The labeled ovaries were minced and homogenized in an all-glass 0I homogenizer with 10 volumes of 50 mM Tris-HCl buffer (pH 10 20 30 40 7.4). After centrifugation at 120 X g for 30 min at 40, the FRACTION NUMBER (TOP) supernatant solution was recentrifuged at 20,000 X g for 30 FIG. 1. Density gradient centrifugation of soluble ovarian min at 4°. The supernatant solution was then discarded, and receptors prelabeled with [1251]iodo-hCG in vivo and in vitro the pellets corresponding to two ovaries were solubilized in before extraction with Triton X-100. Each of the solubilized either (a) 1% Triton (Rohm and Haas) in Tris* HCl buffer, by hormone-receptor complexes gave a single 8.8S peak of radio- extraction with 0.5 ml of detergent solution for 30 min at 40, activity which was precipitable with 12% polyethylene glycol. followed by dilution to a final concentration of 0.1% Triton; Marker proteins were immunoglobulin G and bovine serum or (b) 1% Lubrol PX. (Sigma) in Tris - HCl, by extraction with albumin (BSA). 5 ml of detergent solution for 30 min at 4°. The detergent- solubilized preparations were then centrifuged in a Beckman and boundary spreading on the lower side of the sedimentation L2-75B ultracentrifuge for 2 hr at 360,000 X g in an SW60 profile was not a constant feature of the Triton-extracted rotor, and the clear supernatant solution was used for char- receptor complex. acterization by gel filtration and density gradient centrifuga- When the Lubrol-solubilized complex labeled in vivo was tion. The efficacy of extraction with each of the detergents similarly analyzed by centrifugation in 5-20% sucrose, a single was about 80%. When in vitro labeling was performed, the species was again observed, with sedimentation coefficient of 20,000 X g ovarian homogenate from animals treated with 7.0 S in three separate experiments. This value was in exact pregnant mare serum/human chorionic gonadotropin was agreement with that obtained for the sedimentation coefficient equilibrated with '25I-labeled hCG for 16 hr at 4°, and showed (7.0 S) of the complex obtained by extraction with Lubrol comparable binding (50-60%) of the labeled hormone. PX of ovarian particles labeled in vitro (Fig. 2). Thus, each of the detergent-extracted forms exhibited sedimentation prop- Physical Characterization of Detergent-Solubilized Receptors. The solubilized preparations (200 ,A) were subjected to den- sity gradient analysis in the presence of standard proteins in 5-20% sucrose gradients containing either 0.1% Triton or 0.5% Lubrol PX, by centrifugation for 16 hr at 38,000 rpm in 0.E an SW40 rotor (9). The extracts were also chromatographed 0 *CD with standard proteins on a Sepharose 6B column (1 X 60 o 8000 cm) previously equilibrated with 0.5% Lubrol or 0.1% Triton 06 (11). Polyethylene glycol precipitation was performed upon 0 the fractions obtained from density gradient and gel filtration 1' 6000 analysis to further demonstrate the presence of macromolecu- a lar hormone-receptor complexes (8).