Proc. Nati. Acad. Sci. USA
Vol. 75, No. 10, pp. 4769-4773, October 1978 Biochemistry
Gonadal luteinizing hormone receptors and adenylate cyclase: Transfer of functional ovarian luteinizing hormone receptors to adrenal fasciculata cells (gonadotropin receptors/adrenal cells/cyclic AMP/steroidogenesis/receptor transfer)
MARIA L. DUFAU, KEIKO HAYASHI, GRACIELA SALA, ALBERT BAUKAL, AND KEVIN J. CATT Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, Maryland 20014
Communicated by Roy Hertz, July 17, 1978
ovary and testis by treatment with 0.5% Lubrol PX as described (6). In the present study, detergent solubilization was performed in the presence of 10 mM sodium fluoride to preserve enzyme activity throughout the fractionation procedures. The soluble extracts were fractionated by chromatography on 1.5 X 90 cm columns of Sepharose 6B (3, 6), 1 X 15 cm columns of DEAEcellulose, and 1 X 14 cm columns of Sepharose-concanavalin A. All chromatography columns were equilibrated with 0.5% Lubrol PX/5 mM sodium fluoride/5 mM magnesium chloride in the appropriate buffers before use (50 mM Tris-HCl, pH 7.4, for Sepharose 6B and concanavalin A-Sepharose; 5 mM TrisHC1, pH 7.4, for DEAE-cellulose). Aliquots from the original extract and eluate fractions after chromatography'were assayed for LH receptor binding (7) and adenylate cyclase activity
ABSTRACT Luteinized rat ovaries contain a high concentration of particulate luteinizing hormone (lutropin, LH) receptors and a small quantity of lipid-associated receptors that float in the 360,000 X g supernatant fraction of ovarian homogenates. During fractionation of Lubrol-solubilized LH receptors and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from the ovary an testis, LH rece tors and adenylate cyclase were coincident on gel filtration, but could be resolved during ion-exchange chromatography of soluble ovarian preparations and were completely separated by lectin-affinity chromatography on Sepharose-concanavalin A. For further analysis of receptor-adenylate cyclase coupling, the lipid-rich fraction of ovarian luteal cells was used to transfer gonadal LH receptors to isolated adrenal fasciculata cells. The lipid vesicles obtained from ovarian homogenates by flotation at 360,000 X g contained 5-10% of the ovarian LH receptors and were devoid of adenylate cyclase activity. During incubation of lipid-associated receptors with dispersed rat fasciculata cells at 16°C, progressive incorporation of LH binding sites into the adrenal cells was observed. When adrenal cells bearing heterotopic LH receptors were incubated with 1 nM human choriogonadotropin, cyclic AMP production was consistently stimulated, with an accompanying increase in corticosterone production. These results indicate that LH receptors exist as separate entities from adenylate cyclase in the gonadal cell membrane and can become functionally coupled to adenylate cyclase to evoke cyclic AMP production and steroidogenesis in the host adrenal cells to which they are transferred.
(8).
Adrenal Cell Preparation. Cells from the fasciculata-reticularis layer of adrenal glands from male Sprague-Dawley rats were dispersed essentially as described (9), by incubation of minced adrenal tissue at 370C for 15 min with 2 mg of crude collagenase (Worthington type II) per 25 .g of DNase (Sigma H-li) per ml in medium 199 containing 0.2% bovine serum albumin. After two cycles of this treatment, the dispersed adrenal cells were pooled, filtered through gauze into plastic centrifuge bottles, and sedimented at 120 X g for 10 min. The cell pellet, usually containing about 15 million cells from 100 adrenal glands, was suspended in medium 199/bovine serum albumin and incubated batchwise for 2 hr at 37°C (10). After this period of preincubation, the cells were collected by centrifugation and suspended in 6 ml of medium 199/bovine serum albumin containing the ovarian lipid-associated LH receptor sites. Transfer of LH Receptors. LH receptors were incorporated by incubation of the ovarian lipid fraction with adrenal cells at 16°C for up to 4 hr. After incubation, the cells were centrifuged and washed twice with 50 ml of medium 199/bovine serum albumin. The initial supernatant and washes containing the lipid material and nonincorporated receptors were saved for quantitation of receptor binding activity. The adrenal cells bearing LH receptors, or control cells previously incubated with lipid material without receptors (binding activity was irreversibly inactivated by heating the lipid preparation at 37°C for 30 min), were suspended in medium 199/bovine serum albumin to a final concentration of about 250,000 cells per ml. Adrenal Cell Incubation and Sample Preparation. Corticotropin (ACTH, porcine, Sigma, 150 international units/mg) or (hCG, 10,000 international units/mg) was added as 100-,ul
The majority of particulate high-affinity receptors for luteinizing hormone (lutropin, LH) in the testis and ovary are membrane bound, consistent with the initial action of gonadotropins upon the cell membrane (1, 2). These membrane receptors can be solubilized together with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by nonionic detergents such as Triton X-100 and Lubrol PX (3, 4). In addition to the membrane-bound receptors that require detergent for solubilization, the luteinized ovary contains up to 10% of receptors that are spontaneously "soluble" and can be recovered in the floating lipid fraction of the 360,000 X g supernatant fraction (5). In this report, we describe the chromatographic resolution of solubilized LH receptors and adenylate cyclase in detergent extracts of testis and ovary and the results of studies on the transfer of lipid-associated ovarian LH receptor to adrenal fasciculata cells. MATERIALS AND METHODS Solubilization and Chromatography of LH Receptors and Adenylate Cyclase. LH receptors and adenylate cyclase were extracted from particulate membrane-rich fractions of the The publication costs of this article were defrayed in part by page
Abbreviations: LH, luteinizing hormone (lutropin); Pi/NaCl, Dulbecco's phosphate-buffered saline (pH 7.4); hCG, human choriogonadotropin; ACTH, corticotropin.
charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4769
4770
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Dufau et al.
aliquots to sample vials containing 1 ml of cells and 100 Ail of 1.25 mM 1-methyl 3-isobutylxanthine (Aldrich Chemical Co). Incubations were performed at 370C under 95% 02/5% CO2 with shaking at 100 cycles/min, usually for 2 hr, and terminated by transferring the vials to an ice bath. All subsequent steps were at 0-40C. The vials were decanted into 16-ml polyethylene tubes and centrifuged at 250 X g for 10 min. The supernatant solution was assayed for corticosterone and extracellular cyclic AMP released into the incubation medium. The cells were washed twice with ice-cold medium 199 containing 0.1% bovine serum albumin and resuspended in 1 ml of Dulbecco's phosphate-buffered saline (pH 7.4) (Pi/NaCl) containing 1 mM theophylline. After sonication for 15 sec, the preparation was heated at 1000C for 12 min and analyzed for total and intracellular cyclic AMP (11). Assay of Corticosterone and Cyclic AMP. Corticosterone was measured by the radioimmunoassay procedure of Gross et al. (12) and cyclic AMP was measured by a modification (11, 13) of the method of Steiner et al. (14) with addition of the acetylation step as described by Harper and Brooker (15). Preparation of Ovarian Soluble Lipid-Associated Receptors. The lipid-associated soluble receptors were prepared as described (5) from luteinized ovaries of immature female rats killed 9 days after administration of gonadotropins. The ovaries from gonadotropin-primed rats were dissected free of adventitial tissue and frozen in liquid nitrogen prior to storage at -600C. For preparation of lipid-associated LH receptors, ovaries were minced, washed several times in P1/NaCl, then homogenized in 2-3 vol of Pi/NaCl by 20 strokes in a Dounce glass homogenizer. After dilution to the equivalent of one ovary per ml of Pi/NaCl, the homogenate was centrifuged at 1000 X g for 15 min. The pellet was discarded and the supernate was centrifuged again at 20,000 X g for 30 min. The supernatant fraction was then centrifuged in a Beckman model L75 preparative ultracentrifuge at 360,000 X g for 3 hr at 4°C, and the floating lipid layer- was collected and suspended in medium 199/bovine serum albumin. Aliquots of this preparation were assayed for LH/hCG receptor binding capacity, and the remainder of the lipid fraction was used for transfer of the LH receptors to adrenal fasciculata cells. Lipid was analyzed after removal of neutral lipids by silicic acid chromatography; phospholipids were fractionated by two-dimensional thin-layer chromatography on Silica gel H and quantitated by phosphorus analysis (16). Cholesterol (total and free) and fatty acid composition of triglycerides and cholesterol esters were determined by gas-liquid chromatography. Assay of Soluble and Cell-Bound LH Receptors. LH receptors were measured in equilibrium binding assays by incubation with increasing concentrations of unlabeled hormone in the presence of a constant amount of tracer '25I-labeled hCG (50,000 cpm, about 0.01 nM hCG). The labeled hormone was prepared by enzymatic radioiodination as described (17). Bound and free tracer were separated by centrifugation for binding assays in adrenal cells (7), and by double precipitation with polyethylene glycol (3) for the soluble ovarian lipid-associated receptors or detergent-solubilized testicular and ovarian receptors. Binding capacities were calculated by direct analysis of the binding curve and/or by Scatchard analysis (18), as described (5). RESULTS AND DISCUSSION Initial studies on the fractionation of detergent-solubilized receptors from testis and ovary on Sepharose 6B (6) showed three peaks of receptor binding activity, one which eluted at the void volume and two other peaks with Kav of 0.32 and 0.58. Adenylate cyclase activity coeluted only with the two higher mo-
Proc. Nati. Acad. Sci. USA 75 (1978)
lecular weight peaks of receptor binding activity. The recovery of enzymatic activity was relatively low during the initial experiments; considerable losses were observed during the fractionation step, with retention of only about 5% of the initial enzyme activity. In more recent analyses, solubilization of adenylate cyclase in the presence of 5mM sodium fluoride and 5 mM magnesium chloride was found to preserve the activity of the enzyme, and the recovery of adenylate cyclase activity after fractionation was between 75 and 98%. Consequently, the present studies were performed on solubilized ovarian fractions prepared and analyzed in the presence of sodium fluoride and magnesium chloride. Gel filtration analysis of Lubrol-solubilized ovarian receptors and adenylate cyclase on Sepharose 6B showed two peaks of LH receptor binding with coincident peaks of adenylate cyclase activity (Fig. 1). The major peak was eluted with Kav of 0.28, and there was a minor peak at the void volume. Identical results were obtained during fractionation of detergent-solubilized testicular extracts. In these studies, the small molecular weight species (Kav 0.58) of receptor binding activity (6) was not observed. These results suggest that sodium fluoride and magnesium chloride may stabilize the receptor molecule, preventing dissociation of a portion or subunit of the LH receptor containing the binding site. Similar results have been observed upon fractionation of the solubilized hormone-receptor complex in the absence of sodium fluoride and magnesium chloride. In this case, the hormone acts as the stabilizing agent, perhaps in a manner related to the ability of hormone to preserve the LH receptor from the rapid degradation that usually occurs after detergent solubilization (3, 4). The coincident elution of gonadotropin receptors and adenylate cyclase activity during gel filtration in this and previous experiments (6) suggested that the solubilized receptor molecule may exist as a loose complex containing both the hormone binding site and adenylate cyclase. Alternatively, the receptor binding site and adenylate cyclase activity could be physically separate but coincident during gel filtration on Sepharose 6B. However, further studies by group-specific affinity chromatography on Sepharose-concanavalin A clearly showed that testicular LH receptor sites were eluted separately from the corresponding adenylate cyclase activity (Fig. 2). The enzyme Kav
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Biochemistry: Dufau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
4771
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FIG. 4. Ion-exchange chromatography of detergent-soluble ovarian LH/hCG receptors and adenylate cyclase on a 1 X 14 cm column of DEAE-cellulose with elution by a linear gradient (0-0.5 M) of sodium chloride: Salt concentration (M) is shown in parentheses.
was not adsorbed by the lectin affinity column, and eluted with the majority of the protein in the soluble preparation, while all of the LH/hCG receptor binding activity was adsorbed to the column and could be subsequently eluted by 0.2 M a-methylmannoside. During chromatography of soluble ovarian preparations on Sepharose-concanavalin A, two peaks of receptor binding activity were observed (Fig. 3). The unadsorbed protein contained a minor receptor peak and all of the adenylate cyclase activity in the preparation. The major receptor peak was retained by the affinity column and could be subsequently eluted with 0.2 M methylmannoside as a broad peak of binding activity. The appearance of two species of ovarian receptors was also noted during fractionation of detergent-solubilized ovarian preparations on DEAE-cellulose chromatography (Fig. 4). The individual receptor peaks were eluted by 175 and 255 mM NaCl, while adenylate cyclase activity was eluted as a sharp peak at 220 mM NaCl (Fig. 4). In contrast, the receptor activity of the ovarian lipid-associated receptor did not adsorb to concanavalin A-Sepharose and eluted with most of the protein contained in the preparation. Unlike the detergent-solubilized ovarian preparation, the lipid fraction did not contain detectable adenylate cyclase activity when assayed in five separate- experiments. The preceding studies indicated that the gonadotropin receptor and adenylate
cyclase are physically separate entities that can be resolved during appropiate fractionation procedures. The finding that detergent-solubilized receptors and enzyme activities can be resolved by chromatography does not exclude the possibility that the two species are noncovalently associated in the lipid bilayer of the plasma membrane. However, recent studies using cell fusion (19, 20) and reconstitution of detergent-extracted adenylate cyclase components (21) have also indicated that hormone receptors and adenylate cyclase exist as separate entities within the cell membrane. The lipid-associated LH receptors of the ovary provided a further approach to analysis of the functional properties of LH receptors from gonadal tissue after incorporation into heterologous cells, in this case the steroidogenic cells of the adrenal gland. For this purpose it was important to optimize the conditions for cellular responses to hormone stimulation; preincubation of cells was found to be necessary to observe parallel stimulation of cyclic AMP and corticosterone by- ACTH over
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Proc. Natl. Acad. Sci. USA 75 (1978)
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the most sensitive range of steroidogenic responses (10). Also, it was noted that in adrenal fasciculata cells, in contrast to Leydig cells (10, 11), extracellular cyclic AMP, intracellular cyclic AMP, and receptor-bound cyclic AMP rose in parallel with corticosterone. In the Leydig cells, cyclic AMP bound to protein kinase and intracellular cyclic AMP were the parameters that most clearly increased in parallel with steroidogenesis over the more sensitive range of the dose-related responses to hormone action (11). The ovarian lipid-associated receptor preparation used as the source of receptors for transfer in this study contained 78% -J
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triglyceride, 17% esterified cholesterol, 1.1% free cholesterol, and 3.5% phospholipid. Phospholipid analysis showed a major content of phosphatidylcholine (54%) and phosphatidylethanolamine (24%), with lower proportions of sphingomyelin (9.4%), phosphatidylinositol (5.8%), phosphatidylserine (1%), and phosphatidylcholine (1.8%). Morphological analysis of the dispersed lipid receptor preparation, performed after negative staining with phosphotungstic acid on carbonized Formvar-300 mesh copper-covered grids, showed fat globules with a size range of 85-350 nm. When adrenal cells were incubated for 4 hr at 160C with the ovarian lipid-associated receptor preparation, incorporation of vesicles within the cytoplasm could be subsequently observed by electron microscopy. It is likely that the fat globules undergo endocytosis and/or fusion with the plasma membrane and that the lipophilic molecules in the vesicle are incorporated into the cell's plasma membrane. During studies on the uptake of ovarian LH receptors by adrenal cells (Fig. 5), it was found that these receptors were incorporated into the cell membrane and were subsequently available to bind '25I-labeled hCG. The number of LH receptors increased with time; at 4 hr, the cell bound an average of 200 fmol of hCG per 106 cells, or about 120 receptors per cell. The efficiency of uptake of the LH receptors was relatively high, and 80-90% of the added receptors were incorporated by the cells, evidently into the cell membrane since they were accessible for binding of 125I-labeled hCG. No specific binding of '25I-labeled hCG to control adrenal cells was observed, consistent with the absence of LH receptors in the adrenal gland. The absence of a functional response of the rat adrenal to gonadotropin was previously demonstrated by the inability of hCG to increase the weight of the ventral prostate of castrated animals, whereas high doses of ACTH stimulated adrenal androgen production and caused growth of the accessory organs (22). Stimulation of control adrenal cells without LH receptors by a maximum ACTH concentration (1 nM) provoked a 7-fold
Biochemistry:
Dufau et al.
increase of cyclic AMP production over control values (Fig. 6). In contrast, no stimulation of cyclic AMP or steroid production'. was elicited by 10-100 nM hCG in control cells. In adrenal cells bearing LH receptors, significant stimulation of extracellular cyclic AMP production was observed during incubation with 10 and 100 nM hCG, with increases of 25 and 50%, respectively, above control values. The corresponding steroidogenic responses in normal adrenal cells showed that maximal ACTH concentration increased corticosterone production about 20-fold and that no stimulation was observed with 10 or 100 nM hCG. In adrenal cells bearing transferred LH receptors, 10 and 100 nM hCG elicited significant increments in corticosterone production (P < 0.01). In parallel with the rise in extracellular cyclic AMP, increased levels of intracellular cyclic AMP were also observed during hormone stimulation (Fig. 7). Incubation with 10 nM of ACTH stimulated intracellular and extracellular cyclic AMP and corticosterone maximally. Stimulation of LH receptor-bearing cells with 1 nM hCG increased intracellular cyclic AMP values by 60%, extracellular values by 100%, and corticosterone responses by 60%. With 10 nM hCG (extreme right) all responses were again significantly increased. The increases in corticosterone and cyclic AMP observed during hCG stimulation of adrenal cells bearing LH receptors were similar to the responses attained during stimulation with 10-11M ACTH (10). Doserelated responses of corticosterone production in adrenal cells are elicited by concentrations of ACTH between 10-12 and 10-9M, with EDs5 of 10-10M ACTH. Also, significant increases in cyclic AMP production were elicited by 10-12 M ACTH. To observe gonadotropic stimulation of the small quantity of LH receptors transferred to adrenal cells, optimization of the adrenal cell preparation and pre-incubation were necessary to permit detection of the cyclic AMP and steroid responses evoked by activation of the heterologous receptors. In a recent report, transfer of catecholamine receptors to adrenal tumor cells by fusion with avian erythrocytes was described, with functional coupling to cyclic AMP production but evidently not to steroidogenesis (20). In the present work transfer of receptors for a large glycoprotein hormone, LH, was followed by coupling to both cyclic AMP formation and steroid biosynthesis. In these studies, we have demonstrated that detergent-solubilized testicular and ovarian LH receptors can be resolved from adenylate cyclase by concanavalin A-Sepharose and/or DEAE-cellulose chromatography. By the lectin-affinity chromatographic procedure, testicular LH receptors could be completely separated from adenylate cyclase in Leydig cell extracts. However, the ovary contained two populations of LH receptors, a major fraction that interacted with concanavalin A and did not possess adenylate cyclase activity and a smaller population that was not adsorbed to the lectin, similar to the adenylate cyclase activity. Furthermore, ion-exchange chromatography revealed that adenylate cyclase was not coincident with either of the receptor peaks, indicating that adenylate cyclase could be dissociated from the binding sites and that LH receptors and adenylate cyclase activity reside in separate
Proc. Natl. Acad. Sci. USA 75 (1978)
4773
molecular entities. Since the lipid-associated LH receptors did not adsorb to the lectin, and such receptors appear to be either cytosolic or loosely attached to the cell membrane, it is possible that these sites are incompletely glycosylated by comparison with the membrane-bound receptor molecules. Our studies have also demonstrated that these receptors can be incorporated into the plasma membrane of isolated adrenal cells, and that such transferred receptors are functionally coupled to adenylate cyclase, as indicated by the cyclic AMP and corticosterone responses to gonadotropin in LH receptor-bearing adrenal cells. These results are again consistent with the existence of hormone receptors and adenylate cyclase in the normal steroidogenic cell as independent and physically separate molecules that become associated to form an active complex during receptor occupancy by the homologous hormone. 1. Catt, K. J., Tsuruhara, T., Mendelson, C., Ketelslegers, J.-M. & Dufau, M. L. (1974) in Hormone Binding and Target Cell Activation in the Testis, eds. Dufau, M. L. & Means, A. R. (Plenum, New York), pp. 1-30. 2. Seong, S. H., Rajaniemi, H. J., Cho, M. O., Hirshfield, A. N. & Midgley, A. R. (1974) Endocrinology 95,589-598. 3. Dufau, M. L., Charreau, E. H. & Catt, K. J. (1973) J. Biol. Chem. 248,6973-6982. 4. Charreau, E. H., Dufau, M. L. & Catt, K. J. (1974) J. Biol. Chem. 249,4189-4195. 5. Conti, M., Dufau, M. L. & Catt, K. J. (1978) Biochim. Biophys. Acta 541, 35-44. 6. Dufau, M. L., Baukal, A. J., Ryan, D. & Catt, K. J. (1977) Mol. Cell Endocrinol. 6, 253-269. 7. Mendelson, C., Dufau, M. L. & Catt, K. J. (1975) J. Biol. Chem. 250,8818-8823. 8. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem.
58,541-548. 9. Douglas, J., Aguilera, G., Kondo, T. & Catt, K. J. (1978) Endo-
crinology 102,685-695. 10. Sala, G., Dufau, M. L. & Catt, K. J. (1978) Clin. Res. 26, 312A. 11. Dufau, M. L., Tsuruhara, T., Horner, K. A., Podesta, E. & Catt, K. J. (1977) Proc. Natl. Acad. Sci. USA 74,3419-3423. 12. Gross, H. A., Ruder, H. J., Brown, K. S. & Lipsett, M. B. (1972)
Steroids 20,681-695. 13. Dufau, M. L., Watanabe, K. & Catt, K. J. (1973) Endocrinology 92,6-11. 14. Steiner, A. L., Parker, C. W. & Kipnis, D. M. (1972) J. Biol. Chem. 247, 1106-1113. 15. Harper, J. F. & Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, 207-218. 16. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-470. 17. Dufau, M. L., Podesta, E. & Catt, K. J. (1975) Proc. Natl. Acad. Sci. USA 72, 1272-1275. 18. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-665. 19. Schramm, M., Orly, J., Eimerl, S. & Korner, J. (1977) Nature (London) 268, 310-313. 20. Schulster, D., Orly, J., Seidel, G. & Schramm, M. (1978) J. Biol. Chem. 253, 1201-1206. 21. Ross, E. M. & Gillman, A. G. (1977) J. Biol. Chem. 252,69666969. 22. Tullner, W. W. (1973) Natl. Cancer Inst. Monogr. 12, 211223.
Vol. 75, No. 10, pp. 4769-4773, October 1978 Biochemistry
Gonadal luteinizing hormone receptors and adenylate cyclase: Transfer of functional ovarian luteinizing hormone receptors to adrenal fasciculata cells (gonadotropin receptors/adrenal cells/cyclic AMP/steroidogenesis/receptor transfer)
MARIA L. DUFAU, KEIKO HAYASHI, GRACIELA SALA, ALBERT BAUKAL, AND KEVIN J. CATT Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, Maryland 20014
Communicated by Roy Hertz, July 17, 1978
ovary and testis by treatment with 0.5% Lubrol PX as described (6). In the present study, detergent solubilization was performed in the presence of 10 mM sodium fluoride to preserve enzyme activity throughout the fractionation procedures. The soluble extracts were fractionated by chromatography on 1.5 X 90 cm columns of Sepharose 6B (3, 6), 1 X 15 cm columns of DEAEcellulose, and 1 X 14 cm columns of Sepharose-concanavalin A. All chromatography columns were equilibrated with 0.5% Lubrol PX/5 mM sodium fluoride/5 mM magnesium chloride in the appropriate buffers before use (50 mM Tris-HCl, pH 7.4, for Sepharose 6B and concanavalin A-Sepharose; 5 mM TrisHC1, pH 7.4, for DEAE-cellulose). Aliquots from the original extract and eluate fractions after chromatography'were assayed for LH receptor binding (7) and adenylate cyclase activity
ABSTRACT Luteinized rat ovaries contain a high concentration of particulate luteinizing hormone (lutropin, LH) receptors and a small quantity of lipid-associated receptors that float in the 360,000 X g supernatant fraction of ovarian homogenates. During fractionation of Lubrol-solubilized LH receptors and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from the ovary an testis, LH rece tors and adenylate cyclase were coincident on gel filtration, but could be resolved during ion-exchange chromatography of soluble ovarian preparations and were completely separated by lectin-affinity chromatography on Sepharose-concanavalin A. For further analysis of receptor-adenylate cyclase coupling, the lipid-rich fraction of ovarian luteal cells was used to transfer gonadal LH receptors to isolated adrenal fasciculata cells. The lipid vesicles obtained from ovarian homogenates by flotation at 360,000 X g contained 5-10% of the ovarian LH receptors and were devoid of adenylate cyclase activity. During incubation of lipid-associated receptors with dispersed rat fasciculata cells at 16°C, progressive incorporation of LH binding sites into the adrenal cells was observed. When adrenal cells bearing heterotopic LH receptors were incubated with 1 nM human choriogonadotropin, cyclic AMP production was consistently stimulated, with an accompanying increase in corticosterone production. These results indicate that LH receptors exist as separate entities from adenylate cyclase in the gonadal cell membrane and can become functionally coupled to adenylate cyclase to evoke cyclic AMP production and steroidogenesis in the host adrenal cells to which they are transferred.
(8).
Adrenal Cell Preparation. Cells from the fasciculata-reticularis layer of adrenal glands from male Sprague-Dawley rats were dispersed essentially as described (9), by incubation of minced adrenal tissue at 370C for 15 min with 2 mg of crude collagenase (Worthington type II) per 25 .g of DNase (Sigma H-li) per ml in medium 199 containing 0.2% bovine serum albumin. After two cycles of this treatment, the dispersed adrenal cells were pooled, filtered through gauze into plastic centrifuge bottles, and sedimented at 120 X g for 10 min. The cell pellet, usually containing about 15 million cells from 100 adrenal glands, was suspended in medium 199/bovine serum albumin and incubated batchwise for 2 hr at 37°C (10). After this period of preincubation, the cells were collected by centrifugation and suspended in 6 ml of medium 199/bovine serum albumin containing the ovarian lipid-associated LH receptor sites. Transfer of LH Receptors. LH receptors were incorporated by incubation of the ovarian lipid fraction with adrenal cells at 16°C for up to 4 hr. After incubation, the cells were centrifuged and washed twice with 50 ml of medium 199/bovine serum albumin. The initial supernatant and washes containing the lipid material and nonincorporated receptors were saved for quantitation of receptor binding activity. The adrenal cells bearing LH receptors, or control cells previously incubated with lipid material without receptors (binding activity was irreversibly inactivated by heating the lipid preparation at 37°C for 30 min), were suspended in medium 199/bovine serum albumin to a final concentration of about 250,000 cells per ml. Adrenal Cell Incubation and Sample Preparation. Corticotropin (ACTH, porcine, Sigma, 150 international units/mg) or (hCG, 10,000 international units/mg) was added as 100-,ul
The majority of particulate high-affinity receptors for luteinizing hormone (lutropin, LH) in the testis and ovary are membrane bound, consistent with the initial action of gonadotropins upon the cell membrane (1, 2). These membrane receptors can be solubilized together with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by nonionic detergents such as Triton X-100 and Lubrol PX (3, 4). In addition to the membrane-bound receptors that require detergent for solubilization, the luteinized ovary contains up to 10% of receptors that are spontaneously "soluble" and can be recovered in the floating lipid fraction of the 360,000 X g supernatant fraction (5). In this report, we describe the chromatographic resolution of solubilized LH receptors and adenylate cyclase in detergent extracts of testis and ovary and the results of studies on the transfer of lipid-associated ovarian LH receptor to adrenal fasciculata cells. MATERIALS AND METHODS Solubilization and Chromatography of LH Receptors and Adenylate Cyclase. LH receptors and adenylate cyclase were extracted from particulate membrane-rich fractions of the The publication costs of this article were defrayed in part by page
Abbreviations: LH, luteinizing hormone (lutropin); Pi/NaCl, Dulbecco's phosphate-buffered saline (pH 7.4); hCG, human choriogonadotropin; ACTH, corticotropin.
charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4769
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aliquots to sample vials containing 1 ml of cells and 100 Ail of 1.25 mM 1-methyl 3-isobutylxanthine (Aldrich Chemical Co). Incubations were performed at 370C under 95% 02/5% CO2 with shaking at 100 cycles/min, usually for 2 hr, and terminated by transferring the vials to an ice bath. All subsequent steps were at 0-40C. The vials were decanted into 16-ml polyethylene tubes and centrifuged at 250 X g for 10 min. The supernatant solution was assayed for corticosterone and extracellular cyclic AMP released into the incubation medium. The cells were washed twice with ice-cold medium 199 containing 0.1% bovine serum albumin and resuspended in 1 ml of Dulbecco's phosphate-buffered saline (pH 7.4) (Pi/NaCl) containing 1 mM theophylline. After sonication for 15 sec, the preparation was heated at 1000C for 12 min and analyzed for total and intracellular cyclic AMP (11). Assay of Corticosterone and Cyclic AMP. Corticosterone was measured by the radioimmunoassay procedure of Gross et al. (12) and cyclic AMP was measured by a modification (11, 13) of the method of Steiner et al. (14) with addition of the acetylation step as described by Harper and Brooker (15). Preparation of Ovarian Soluble Lipid-Associated Receptors. The lipid-associated soluble receptors were prepared as described (5) from luteinized ovaries of immature female rats killed 9 days after administration of gonadotropins. The ovaries from gonadotropin-primed rats were dissected free of adventitial tissue and frozen in liquid nitrogen prior to storage at -600C. For preparation of lipid-associated LH receptors, ovaries were minced, washed several times in P1/NaCl, then homogenized in 2-3 vol of Pi/NaCl by 20 strokes in a Dounce glass homogenizer. After dilution to the equivalent of one ovary per ml of Pi/NaCl, the homogenate was centrifuged at 1000 X g for 15 min. The pellet was discarded and the supernate was centrifuged again at 20,000 X g for 30 min. The supernatant fraction was then centrifuged in a Beckman model L75 preparative ultracentrifuge at 360,000 X g for 3 hr at 4°C, and the floating lipid layer- was collected and suspended in medium 199/bovine serum albumin. Aliquots of this preparation were assayed for LH/hCG receptor binding capacity, and the remainder of the lipid fraction was used for transfer of the LH receptors to adrenal fasciculata cells. Lipid was analyzed after removal of neutral lipids by silicic acid chromatography; phospholipids were fractionated by two-dimensional thin-layer chromatography on Silica gel H and quantitated by phosphorus analysis (16). Cholesterol (total and free) and fatty acid composition of triglycerides and cholesterol esters were determined by gas-liquid chromatography. Assay of Soluble and Cell-Bound LH Receptors. LH receptors were measured in equilibrium binding assays by incubation with increasing concentrations of unlabeled hormone in the presence of a constant amount of tracer '25I-labeled hCG (50,000 cpm, about 0.01 nM hCG). The labeled hormone was prepared by enzymatic radioiodination as described (17). Bound and free tracer were separated by centrifugation for binding assays in adrenal cells (7), and by double precipitation with polyethylene glycol (3) for the soluble ovarian lipid-associated receptors or detergent-solubilized testicular and ovarian receptors. Binding capacities were calculated by direct analysis of the binding curve and/or by Scatchard analysis (18), as described (5). RESULTS AND DISCUSSION Initial studies on the fractionation of detergent-solubilized receptors from testis and ovary on Sepharose 6B (6) showed three peaks of receptor binding activity, one which eluted at the void volume and two other peaks with Kav of 0.32 and 0.58. Adenylate cyclase activity coeluted only with the two higher mo-
Proc. Nati. Acad. Sci. USA 75 (1978)
lecular weight peaks of receptor binding activity. The recovery of enzymatic activity was relatively low during the initial experiments; considerable losses were observed during the fractionation step, with retention of only about 5% of the initial enzyme activity. In more recent analyses, solubilization of adenylate cyclase in the presence of 5mM sodium fluoride and 5 mM magnesium chloride was found to preserve the activity of the enzyme, and the recovery of adenylate cyclase activity after fractionation was between 75 and 98%. Consequently, the present studies were performed on solubilized ovarian fractions prepared and analyzed in the presence of sodium fluoride and magnesium chloride. Gel filtration analysis of Lubrol-solubilized ovarian receptors and adenylate cyclase on Sepharose 6B showed two peaks of LH receptor binding with coincident peaks of adenylate cyclase activity (Fig. 1). The major peak was eluted with Kav of 0.28, and there was a minor peak at the void volume. Identical results were obtained during fractionation of detergent-solubilized testicular extracts. In these studies, the small molecular weight species (Kav 0.58) of receptor binding activity (6) was not observed. These results suggest that sodium fluoride and magnesium chloride may stabilize the receptor molecule, preventing dissociation of a portion or subunit of the LH receptor containing the binding site. Similar results have been observed upon fractionation of the solubilized hormone-receptor complex in the absence of sodium fluoride and magnesium chloride. In this case, the hormone acts as the stabilizing agent, perhaps in a manner related to the ability of hormone to preserve the LH receptor from the rapid degradation that usually occurs after detergent solubilization (3, 4). The coincident elution of gonadotropin receptors and adenylate cyclase activity during gel filtration in this and previous experiments (6) suggested that the solubilized receptor molecule may exist as a loose complex containing both the hormone binding site and adenylate cyclase. Alternatively, the receptor binding site and adenylate cyclase activity could be physically separate but coincident during gel filtration on Sepharose 6B. However, further studies by group-specific affinity chromatography on Sepharose-concanavalin A clearly showed that testicular LH receptor sites were eluted separately from the corresponding adenylate cyclase activity (Fig. 2). The enzyme Kav
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Biochemistry: Dufau et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 4. Ion-exchange chromatography of detergent-soluble ovarian LH/hCG receptors and adenylate cyclase on a 1 X 14 cm column of DEAE-cellulose with elution by a linear gradient (0-0.5 M) of sodium chloride: Salt concentration (M) is shown in parentheses.
was not adsorbed by the lectin affinity column, and eluted with the majority of the protein in the soluble preparation, while all of the LH/hCG receptor binding activity was adsorbed to the column and could be subsequently eluted by 0.2 M a-methylmannoside. During chromatography of soluble ovarian preparations on Sepharose-concanavalin A, two peaks of receptor binding activity were observed (Fig. 3). The unadsorbed protein contained a minor receptor peak and all of the adenylate cyclase activity in the preparation. The major receptor peak was retained by the affinity column and could be subsequently eluted with 0.2 M methylmannoside as a broad peak of binding activity. The appearance of two species of ovarian receptors was also noted during fractionation of detergent-solubilized ovarian preparations on DEAE-cellulose chromatography (Fig. 4). The individual receptor peaks were eluted by 175 and 255 mM NaCl, while adenylate cyclase activity was eluted as a sharp peak at 220 mM NaCl (Fig. 4). In contrast, the receptor activity of the ovarian lipid-associated receptor did not adsorb to concanavalin A-Sepharose and eluted with most of the protein contained in the preparation. Unlike the detergent-solubilized ovarian preparation, the lipid fraction did not contain detectable adenylate cyclase activity when assayed in five separate- experiments. The preceding studies indicated that the gonadotropin receptor and adenylate
cyclase are physically separate entities that can be resolved during appropiate fractionation procedures. The finding that detergent-solubilized receptors and enzyme activities can be resolved by chromatography does not exclude the possibility that the two species are noncovalently associated in the lipid bilayer of the plasma membrane. However, recent studies using cell fusion (19, 20) and reconstitution of detergent-extracted adenylate cyclase components (21) have also indicated that hormone receptors and adenylate cyclase exist as separate entities within the cell membrane. The lipid-associated LH receptors of the ovary provided a further approach to analysis of the functional properties of LH receptors from gonadal tissue after incorporation into heterologous cells, in this case the steroidogenic cells of the adrenal gland. For this purpose it was important to optimize the conditions for cellular responses to hormone stimulation; preincubation of cells was found to be necessary to observe parallel stimulation of cyclic AMP and corticosterone by- ACTH over
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the most sensitive range of steroidogenic responses (10). Also, it was noted that in adrenal fasciculata cells, in contrast to Leydig cells (10, 11), extracellular cyclic AMP, intracellular cyclic AMP, and receptor-bound cyclic AMP rose in parallel with corticosterone. In the Leydig cells, cyclic AMP bound to protein kinase and intracellular cyclic AMP were the parameters that most clearly increased in parallel with steroidogenesis over the more sensitive range of the dose-related responses to hormone action (11). The ovarian lipid-associated receptor preparation used as the source of receptors for transfer in this study contained 78% -J
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triglyceride, 17% esterified cholesterol, 1.1% free cholesterol, and 3.5% phospholipid. Phospholipid analysis showed a major content of phosphatidylcholine (54%) and phosphatidylethanolamine (24%), with lower proportions of sphingomyelin (9.4%), phosphatidylinositol (5.8%), phosphatidylserine (1%), and phosphatidylcholine (1.8%). Morphological analysis of the dispersed lipid receptor preparation, performed after negative staining with phosphotungstic acid on carbonized Formvar-300 mesh copper-covered grids, showed fat globules with a size range of 85-350 nm. When adrenal cells were incubated for 4 hr at 160C with the ovarian lipid-associated receptor preparation, incorporation of vesicles within the cytoplasm could be subsequently observed by electron microscopy. It is likely that the fat globules undergo endocytosis and/or fusion with the plasma membrane and that the lipophilic molecules in the vesicle are incorporated into the cell's plasma membrane. During studies on the uptake of ovarian LH receptors by adrenal cells (Fig. 5), it was found that these receptors were incorporated into the cell membrane and were subsequently available to bind '25I-labeled hCG. The number of LH receptors increased with time; at 4 hr, the cell bound an average of 200 fmol of hCG per 106 cells, or about 120 receptors per cell. The efficiency of uptake of the LH receptors was relatively high, and 80-90% of the added receptors were incorporated by the cells, evidently into the cell membrane since they were accessible for binding of 125I-labeled hCG. No specific binding of '25I-labeled hCG to control adrenal cells was observed, consistent with the absence of LH receptors in the adrenal gland. The absence of a functional response of the rat adrenal to gonadotropin was previously demonstrated by the inability of hCG to increase the weight of the ventral prostate of castrated animals, whereas high doses of ACTH stimulated adrenal androgen production and caused growth of the accessory organs (22). Stimulation of control adrenal cells without LH receptors by a maximum ACTH concentration (1 nM) provoked a 7-fold
Biochemistry:
Dufau et al.
increase of cyclic AMP production over control values (Fig. 6). In contrast, no stimulation of cyclic AMP or steroid production'. was elicited by 10-100 nM hCG in control cells. In adrenal cells bearing LH receptors, significant stimulation of extracellular cyclic AMP production was observed during incubation with 10 and 100 nM hCG, with increases of 25 and 50%, respectively, above control values. The corresponding steroidogenic responses in normal adrenal cells showed that maximal ACTH concentration increased corticosterone production about 20-fold and that no stimulation was observed with 10 or 100 nM hCG. In adrenal cells bearing transferred LH receptors, 10 and 100 nM hCG elicited significant increments in corticosterone production (P < 0.01). In parallel with the rise in extracellular cyclic AMP, increased levels of intracellular cyclic AMP were also observed during hormone stimulation (Fig. 7). Incubation with 10 nM of ACTH stimulated intracellular and extracellular cyclic AMP and corticosterone maximally. Stimulation of LH receptor-bearing cells with 1 nM hCG increased intracellular cyclic AMP values by 60%, extracellular values by 100%, and corticosterone responses by 60%. With 10 nM hCG (extreme right) all responses were again significantly increased. The increases in corticosterone and cyclic AMP observed during hCG stimulation of adrenal cells bearing LH receptors were similar to the responses attained during stimulation with 10-11M ACTH (10). Doserelated responses of corticosterone production in adrenal cells are elicited by concentrations of ACTH between 10-12 and 10-9M, with EDs5 of 10-10M ACTH. Also, significant increases in cyclic AMP production were elicited by 10-12 M ACTH. To observe gonadotropic stimulation of the small quantity of LH receptors transferred to adrenal cells, optimization of the adrenal cell preparation and pre-incubation were necessary to permit detection of the cyclic AMP and steroid responses evoked by activation of the heterologous receptors. In a recent report, transfer of catecholamine receptors to adrenal tumor cells by fusion with avian erythrocytes was described, with functional coupling to cyclic AMP production but evidently not to steroidogenesis (20). In the present work transfer of receptors for a large glycoprotein hormone, LH, was followed by coupling to both cyclic AMP formation and steroid biosynthesis. In these studies, we have demonstrated that detergent-solubilized testicular and ovarian LH receptors can be resolved from adenylate cyclase by concanavalin A-Sepharose and/or DEAE-cellulose chromatography. By the lectin-affinity chromatographic procedure, testicular LH receptors could be completely separated from adenylate cyclase in Leydig cell extracts. However, the ovary contained two populations of LH receptors, a major fraction that interacted with concanavalin A and did not possess adenylate cyclase activity and a smaller population that was not adsorbed to the lectin, similar to the adenylate cyclase activity. Furthermore, ion-exchange chromatography revealed that adenylate cyclase was not coincident with either of the receptor peaks, indicating that adenylate cyclase could be dissociated from the binding sites and that LH receptors and adenylate cyclase activity reside in separate
Proc. Natl. Acad. Sci. USA 75 (1978)
4773
molecular entities. Since the lipid-associated LH receptors did not adsorb to the lectin, and such receptors appear to be either cytosolic or loosely attached to the cell membrane, it is possible that these sites are incompletely glycosylated by comparison with the membrane-bound receptor molecules. Our studies have also demonstrated that these receptors can be incorporated into the plasma membrane of isolated adrenal cells, and that such transferred receptors are functionally coupled to adenylate cyclase, as indicated by the cyclic AMP and corticosterone responses to gonadotropin in LH receptor-bearing adrenal cells. These results are again consistent with the existence of hormone receptors and adenylate cyclase in the normal steroidogenic cell as independent and physically separate molecules that become associated to form an active complex during receptor occupancy by the homologous hormone. 1. Catt, K. J., Tsuruhara, T., Mendelson, C., Ketelslegers, J.-M. & Dufau, M. L. (1974) in Hormone Binding and Target Cell Activation in the Testis, eds. Dufau, M. L. & Means, A. R. (Plenum, New York), pp. 1-30. 2. Seong, S. H., Rajaniemi, H. J., Cho, M. O., Hirshfield, A. N. & Midgley, A. R. (1974) Endocrinology 95,589-598. 3. Dufau, M. L., Charreau, E. H. & Catt, K. J. (1973) J. Biol. Chem. 248,6973-6982. 4. Charreau, E. H., Dufau, M. L. & Catt, K. J. (1974) J. Biol. Chem. 249,4189-4195. 5. Conti, M., Dufau, M. L. & Catt, K. J. (1978) Biochim. Biophys. Acta 541, 35-44. 6. Dufau, M. L., Baukal, A. J., Ryan, D. & Catt, K. J. (1977) Mol. Cell Endocrinol. 6, 253-269. 7. Mendelson, C., Dufau, M. L. & Catt, K. J. (1975) J. Biol. Chem. 250,8818-8823. 8. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem.
58,541-548. 9. Douglas, J., Aguilera, G., Kondo, T. & Catt, K. J. (1978) Endo-
crinology 102,685-695. 10. Sala, G., Dufau, M. L. & Catt, K. J. (1978) Clin. Res. 26, 312A. 11. Dufau, M. L., Tsuruhara, T., Horner, K. A., Podesta, E. & Catt, K. J. (1977) Proc. Natl. Acad. Sci. USA 74,3419-3423. 12. Gross, H. A., Ruder, H. J., Brown, K. S. & Lipsett, M. B. (1972)
Steroids 20,681-695. 13. Dufau, M. L., Watanabe, K. & Catt, K. J. (1973) Endocrinology 92,6-11. 14. Steiner, A. L., Parker, C. W. & Kipnis, D. M. (1972) J. Biol. Chem. 247, 1106-1113. 15. Harper, J. F. & Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, 207-218. 16. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-470. 17. Dufau, M. L., Podesta, E. & Catt, K. J. (1975) Proc. Natl. Acad. Sci. USA 72, 1272-1275. 18. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-665. 19. Schramm, M., Orly, J., Eimerl, S. & Korner, J. (1977) Nature (London) 268, 310-313. 20. Schulster, D., Orly, J., Seidel, G. & Schramm, M. (1978) J. Biol. Chem. 253, 1201-1206. 21. Ross, E. M. & Gillman, A. G. (1977) J. Biol. Chem. 252,69666969. 22. Tullner, W. W. (1973) Natl. Cancer Inst. Monogr. 12, 211223.
