0013.7227/92/1305-2931$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 5 Printed in U.S.A.
Society
Binding of an Extracellular Steroid-Binding Globulin to Membranes and Soluble Receptors from Human Breast Cancer Cells (MCF-7 Cells) C. S. PORTO, The Population
N. A. MUSTO,
C. W. BARDIN,
G. L. GUNSALUS
AND
Council, New York, NY 10021
ABSTRACT. strated ized by onstrated isolated studies
Studies of MCF-7 breast cancer cells demonthat sex hormone-binding globulin (SHBG) is internalreceptor-mediated endocytosis. The present study demspecific binding of SHBG to receptor on membranes from MCF-7 cells. Scatchard analysis of these binding suggested that SHBG binds to a single class of sites on
from MCF-7 membranes used buffers containing protease inhibitors, 10% glycerol, and 10 mM 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (246 f 14 pmol/mg protein) and a lo-fold decrease in binding affinity (Kd at 37 C = 2 x lo-’ M). (Endocrinology 130: 29312936, 1992)
membranes. The analysis yielded a dissociation constant (Kd) at 37 C of 3 x lo-* M and a binding capacity of 48 + 0.12 pmol/ mg protein.
A procedure
for solubilizing
the
SHBG
receptor
I
N THE blood of many species androgens and estrogens are bound to a specific sex hormone-binding globulin (SHBG), which is also referred to in the literature as testosterone-estradiol-binding globulin (TeBG) and sex steroid-binding protein (SBP) (l-3). This protein is synthesized by the liver (4) and secreted into the vascular compartment. It is commonly thought that extracellular steroid-binding proteins act physiologically to protect the circulating steroid from metabolism, thus serving as a reservoir to replenish the biologically active free pool. This view has been challenged by the demonstration that this protein can leave the vascular compartment (2, 5) and can be bound to as well as internalized by androgen-responsive cells (6-9) and by the finding that this same protein is produced in testis, where it is termed ABP, for transport to the epididymis where it is bound and internalized by principal cells (10, 11). Recently, binding sites for SHBG, which have been termed SHBG receptors, were demonstrated on the surface of human prostatic and human prostatic tumor cells (12-14) and solubilized with a variety of detergents (13). We have studied the binding of SHBG to androgenresponsive MCF-7 cells (15). This study showed that Received December 12, 1991. Address requests for reprints and all correspondence to: C. W. Bardin, The Population Council, 1230 York Avenue, New York, NY 10021. *These studies were supported by National Institutes of Health Grant HD-13541, Fogarty International Center (1 F05 TW04065-Ol), and Conselho National Desenvolvimento Cientifico e TecnologicoBrazil (203049-87) Fellowship (to C.S.P).
binding of SHBG was followed by its uptake into membrane vesicles of MCF-7 cells by receptor-mediated endocytosis (16). We now report specific binding of SHBG to MCF-7 cell membranes and solubilization of SHBG receptor from these membranes. Materials
and Methods
Cell culture
MCF-7 cells obtained from the American Type Culture Collection (Rockville, MD) were plated in flasks (70 cm’, Corning GlassWorks, Corning, NY) at a density of approximately 6-7 x lo5 cells per flask. Cells were cultured in Ham’s F-12Dulbecco’smodified Eagle’s medium (F-lB/DME, 1:l; Gibco Laboratories, Grand Island, NY) containing 1.2 g/liter sodium bicarbonate, 15 mM HEPES (Calbiochem, La Jolla, CA), 20 mg/liter
gentamycin
(Gibco Laboratories),
and 5% horse serum
plus 2.5% newborn calf serum (Hazelton, Lenexa, KS). The cells were grown in a humidified atmosphereof 95% air-5% CO, at 37 C for 4 days. Forty-eight hours before each experiment the cells were incubated in serum-freemediumcontaining: insulin, 6.25 pg/ml; transferrin, 6.25 pg/ml; selenium,6.25 rig/ml; BSA, 1.25 mg/ml; linoleic acid, 5.35 pg/ml (ITS+; Collaborative Research, Bedford, MA); epidermal growth factor (EGF; Biomedical Technologies, Stoughton, MA), 100 rig/ml; and prostaglandin F?,, (Sigma Chemical Co., St. Louis, MO), 100 rig/ml (17). Ligand Rabbit SHBG was purified by affinity chromatography as described previously (18) and radiolabeled with radioiodinated tyramine-cellobiose (TC; Sigma) adduct as described by Pitt-
2931
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2932
SHBG RECEPTORS
man et al. (19) and the labeled protein purified by size exclusion on Bio-Gel P-6 (200-400 mesh, Bio-Rad Laboratories, Richmond, CA). The radiolabeled preparations were greater than 97% precipitable by 10% trichloroacetic acid, 2% phosphotungstic acid precipitation and bind quantitatively to an androgen affinity column (16). Specific activities were approximately 1.2-2.0 &i/rg protein. The ligand was used without added testosterone. Preparation
and solubilization
of membranes
After incubation in serum-freemedium, cells were cooledto 0 C, rinsed with ice-cold PBS, detachedby rubber policeman, resuspendedin PBS, and sedimentedby centrifugation (200 x g for 5 min). All subsequentprocedureswere carried out at 4 C, and all solutionscontained freshly added5 mM N-ethylmaleimide (Sigma), and 10 mM phenylmethylsulfonyl fluoride (Sigma)to inhibit proteolysis. Cell pellets were resuspendedin buffer A (0.3 M sucrosein 20 mM Tris-HCl, pH 7.4; Sigma)and disrupted in a glasshomogeneizerwith a tightly fitting Teflon pestle. The homogenatewas centrifuged at 10,000x g for 15 min, and the resulting pellet was resuspendedin buffer A, homogeneized,and centrifuged asbefore. The combinedsupernatants were centrifuged at 100,000x g for 1 h. The resulting pellet was resuspendedin buffer B (25 mM Tris-HCl, pH 7.4, 10 mM CaCl,) and either stored at -70 C for binding experimentsor usedimmediately for preparation of solubilizedSHBG receptor. In preliminary studiesmembranesweretreated with various concentrations of Triton X-100, NP-40, and 3-[(3cholamidopropyl)dimethylammonio]-I-propanesulfonate (CHAPS). Both Triton X-100 and NP-40 (1% vol/vol) released30-40% of total membraneproteins, but receptor binding activity was lost in 24 h at -70 C; lower concentrations (0.1 and 0.5%) released only 5-10% of membraneprotein. By contrast CHAPS (Sigma) solubilizedprotein and preservedbinding activity (see Results). The optimal procedurefor solubilizing the receptor usedmembranes(6-8 mg protein/ml) resuspendedin buffer B containing enzyme inhibitors, 10% (vol/vol) glycerol (Fisher, Waltham, MA) and 10 mM CHAPS (Sigma Chemical Co.). The suspensionswere stirred at 4 C for 1 h and centrifuged at 100,000x g for 1 h. The supernatants,containing the SHBG receptor, could be stored at -70 C for 24 h without lossof activity. Membrane
binding
assay
Binding experiments were conducted in 1.5 ml Eppendorf polypropylene centrifuge tubes that had beenpretreated for 24 h at 37 C with 1 ml buffer C plus enzyme inhibitors (25 mM Tris-HCl, pH 7.4, 10 mM CaC12and 0.1% BSA) (Sigma) to minimize tube blanks. [iz51]TC-SHBG (62.5 ng; 6.9 nM) was incubated with membranes(0.3 mg protein/ml) in buffer C for periods of l-6 hours at 37 C, in the presenceor absenceof unlabeledSHBG, in a final volume of 0.3 ml. All assayswere performed in duplicate. Free and bound [‘251]TC-SHBG were separatedby centrifugation at 13,000x g for 10 min at 4 C. The supernatant wasaspirated, and the pellet waswashedwith buffer B and recentrifuged for 10 additional minutes. The final pellet was counted in a Packard y-scintillation counter. Nonspecificbinding wasabout 20% of total binding.
ON MCF-7 CELLS Assay of solubilized
Endo. 1992 Vol 130. No 5
receptor
Immediately before the assay,the solubleextracts were diluted to 0.5 mg protein/ml with buffer B at 4 C containing 10% (vol/vol) glycerol and 10 mM CHAPS supplementedwith proteaseinhibitors. Aliquots of the diluted extracts (0.1 ml) were addedto assaymixtures identical to those usedfor the membrane binding assay.After a l-6 h incubation at 37 C, the free and bound [‘251]TC-SHBG were separated(at 4 C) by precipitating the receptor-SHBG complex by addition of 0.5 ml buffer B containing 1 mg/ml bovine-y-globulins (Sigma) and 0.5 ml 12% (wt/vol) polyethylene glycol (PEG-8000) (Fisher Scientific, Pittsburgh, PA). After mixing, the tubes were incubated for 10 min and centrifuged at 13,000x g for 10 min at 4 C. The supernatantswere aspiratedand the pellets were suspendedin 1 ml buffer B containing 10% (vol/vol) glycerol and 10 mM CHAPS. Polyethylene glycol (0.5 ml) wasaddedand the tubes vortexed. After 10 min the tubes were centrifuged, the supernatant aspirated,and radioactivity in the pellets wasmeasured. Under theseconditions the nonspecificbinding wasabout 17% of total binding. Protein
determination
Protein concentration in the membranepreparations and soluble extracts was determined according to Bradford (20) usingBSA as a standard. Binding
analysis
Binding analysiswasperformed by weighted nonlinear least squarescurve fitting asdescribedpreviously (21,22).
Results Binding of r”“IlTC-SHBG cell membrane
to SHBG receptor in MCF-7
The binding of SHBG to MCF-7 cell membranes is shown in Fig. 1. Specific binding was the difference between total binding, which was 16-18% of added [1251] TC-SHBG at 37 C, and nonspecific binding determined in the presence of a 500-fold excess of unlabeled SHBG. The binding reached a plateau by 4 h at 37 C. A comparison of binding of SHBG at 37 C and 4 C showed a distinct temperature dependence. In four experiments in which each point was analyzed in duplicate the specific binding at 37 C was 3-fold higher than at 4 C (Fig. 1). A Scatchard plot of SHBG binding to MCF-7 cell membranes at 37 C is shown in Fig. 2. The results are consistent with SHBG binding to a single set of sites. An analysis of five experiments, each with duplicate determination, yielded a dissociation constant (KJ of 3 x lo-’ M and a binding capacity of 48 f 0.12 pmol/mg membrane protein (means f SEM). The specificity of binding was established by comparing the abilities of unlabeled SHBG, transferrin, cq-acid glycoprotein, and fetuin to compete with [lz51]TC-SHBG for binding to MCF-7 cell membranes (Fig. 3). The
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SHBG
RECEPTORS
8-
ON MCF-7
CELLS
2933
16
6-
1
2
4
6
TIME (h) binding of [‘251]TC-SHBG to MCF-7
FIG. 1. Time course of specific cell membranes. MCF-7 cell membranes (0.3 mg membrane protein/ ml) were incubated with [‘251]TC-SHBG (62.5 ng) with or without a 500-fold excess of unlabeled SHBG at 37 C and 4 C. Specific binding is the difference between total binding (16-18% of added [YJTCSHBG) and nonspecific binding determined in the presence of 500-fold excess of unlabeled SHBG. Values expressed as mean f SEM (n = 4 experiments).
specific binding of the radioactive ligand was reduced 80% by SHBG. The other proteins did not compete for SHBG binding sites. Solubilization of the SHBG receptor The ability of a variety of detergents (Triton X-100, NP-40, and CHAPS) to solubilize SHBG receptors from MCF-7 cell membranes was evaluated. CHAPS was found to be both efficient in solubilizing the receptor and did not interfere with binding. To determine the optimum concentrations of CHAPS for solubilization of SHBG receptors, MCF-7 cell membranes (6-8 mg protein/ml) were incubated at 4 C for 1 h with various concentrations of this detergent. The results showed that solubilization of membrane proteins and receptors depended on the concentration of CHAPS (Fig. 4). There was a linear increase in released MCF-7 cell membrane proteins until a concentration of about 5 mM. Further increases in the detergent concentration did not lead to a proportional release of membrane protein (Fig. 4A). The SHBG binding activity in the supernatant fractions increased almost linearly up to 10 mM CHAPS with a corresponding decrease in the binding activity of the pellet fraction (Fig. 4B). When the CHAPS concentration was further increased SHBG binding activity was reduced in both the supernatant fraction and the pellet. Based on these studies 10 mM CHAPS was used in all studies on the soluble receptor. Various concentrations
4
5
10 BOUND
15
20
25
(nM)
FIG. 2. Displacement by unlabeled SHBG of [iz51]TC-SHBG (62.5 ng) bound to MCF-7 cell membranes. Labeled ligand (6.9 nM) was incubated with membranes (0.3 mg membrane protein/ml) in the presence of various amounts of unlabeled SHBG for 6 h at 37 C. Five experiments were performed. Data from a single experiment are plotted in Scatchard coordinates. Curved line is the computer-generated best fit of raw data (0) to a high affinity, low capacity binding species (specific binding, solid straight line) and a low affinity, high capacity species (nonspecific binding, dashed straight line) (21). Total binding was 16-18% of added counts; nonspecific binding was less than 4% of total counts which is shown by the horizontal dashed line.
of glycerol (10, 20, 30%) were tested in order to improve the stability of the solubilized receptors. The SHBG binding activity of the soluble extract in 10 mM CHAPS was increased 1.5fold with 10% and 2-fold with 20% and 30%. The two highest concentrations of glycerol interfered with the polyethylene glycol precipitation. Thus 10% glycerol was use in all experiments on the solubilized receptor. Storage for 24 h at -70 C resulted in no loss of activity. The binding of [lz51]TC-SHBG to the solubilized receptor was studied. Analysis of the competition data with SHBG revealed one population of binding sites (Fig. 5). A Kd of 2 x 10e7 M and a binding capacity of 246 f 14 pmol/mg protein (mean f SEM) were estimated. Solubilization of the receptor lowered its binding affinity lofold but increased its specific activity 5-fold. Discussion The presence of specific membrane-binding sites for SHBG was first proposed by Strel’chyonok et al. (23)
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2934
SHBG
RECEPTORS
ON MCF-7
Endo - 1992 Voll30. No 5
CELLS
0.8
0 c! m
0.6
0.4
0.2 10
20
30
CHAPS (mM) 0
5 COMPETITOR
50
500
(pmol)
FIG. 3. Specificity of the SHBG binding. MCF-7 cell membranes were incubated with [iz51]TC-SHBG (62.5 ng) and various concentrations of rabbit SHBG (O), transferrin (0), al-acid glycoprotein (A), and fetuin (0) for 6 h at 37 C. B,, Binding of [Y]TC-SHBG in the absence of competitor; B, binding of [Y]TC-SHBG in the presence of competitor. Values expressed as mean + SEM (n = 5 experiments).
using human decidual endometrium. These authors found that the Kd at 4 C was 3 X lo-‘* M. Subsequently, Hryb et al. identified a SHBG receptor in human prostate membranes. High and low affinity sites were identified with Kd of 1.5 X 10e8 and 8.1 X 10e6 M, respectively. Solubilization of these receptors lowered the Kd to 1.4 x lo-’ and 1.0 X 10m7~ (12, 13). The present report demonstrates one class of specific sites binding for SHBG on MCF-7 cell membranes. The binding sites are saturable, specific, and have a affinity for SHBG (Kd = 3 X 10-s M) and a concentration (48 pmol/mg protein), similar to those reported for the corticosteroid-binding globulin (CBG) in MCF-7 cells (24, 25). Solubilization lowers the affinity of receptors on MCF-7 cell membranes which is in contrast to the increased affinity of receptors on prostate membranes after solubilization. The reason for this large variation in Kd for the soluble SHBG receptors noted above could relate either to tissue differences in factors that stabilize receptor or technical differences between laboratories. Evidence in favor of the former is that both soluble and membrane-associated forms of SHBG receptor in testes had affinities 10 times greater than that of the receptor from MCF-7 cells using exactly the same methods (Porto, C. S., unpublished observations) . High binding affinities have been observed for other membrane receptors and their respective serum protein ligands. For example, the transferrin receptor (26) and
B
o
10
20 CHAPS
30
(mM)
FIG. 4. Effects of CHAPS on the distribution of proteins and SHBG binding activity. MCF-7 cell membranes were solubilized in buffer A containing 10% (vol/vol) glycerol and various concentrations of CHAPS. Protein content (A) and binding activity (B) were determined in supernatant (M) and pellet (M) fractions obtained after centrifugation. Values expressed as mean f SEM (n = 3 experiments).
the low density lipoprotein receptor have Kd values of 6 x lo-’ M and 33 x lo-’ M, respectively (27). The difference between these protein-receptor systems and the one for SHBG is that the plasma concentrations of transferrin and low density lipoprotein exceed their receptor dissociation constants by several orders of magnitude, whereas the plasma concentrations of SHBG (2.5 x lOma M in men and 5.0 X 1O-8 M in women) (28) are similar to receptor dissociation constant (Kd = 1.5 X 10e8 M in human prostatic membrane and 3 x 10m8 M in MCF-7 cell membranes). Similarly, another serum steroid binding protein, CBG, and its membrane receptor have a Kd of 8.7 X 10e7 M and a serum concentration of 6.5 X 10e7 M. Therefore, the receptors for SHBG and CBG are more
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SHBG
RECEPTORS
ON MCF-7
CELLS
2935
the membrane. In conclusion, our results provide evidence for the presence of SHBG receptors on MCF-7 cells. The role this specific binding plays in the biology of these cells remains to be determined. It could be involved in regulation of androgen delivery, thus serving as a transport mechanism, or, alternatively, it could subserve a classical hormone-receptor signaling function. With regard to this latter possibility Nakhla et al. (31) observed that receptor-mediated uptake of another extracellular steroid binding protein (CBG) by MCF-7 cells was associated with increases in adenylate cyclase activity. This observation suggests that CBG may have a “hormone” activity in MCF-7 cells. This possibility remains to be evaluated for SHBG.
\ \
“0
5
Acknowledgments We gratefully acknowledge Richard Hale in the preparation 20
40
60
60
the assistence of Nichelle of this manuscript.
Edwards
and
100
References BOUND
(WA)
FIG. 5. Displacement
by unlabeled SHBG of [Y]TC-SHBG (62.5 ng) bound to the soluble receptor. Labeled ligand (6.9 nM) was incubated with the CHAPS-solubilized receptor in the presence of increasing amounts of unlabeled SHBG for 6 h at 37 C. Data from a single experiment are plotted in Scatchard coordinates. Curved line is the computer-generated best fit of raw data (0) to a high affinity, low capacity binding species (specific binding, solid straight line) and a low affinity, high capacity species (nonspecific binding, dashed straight line) (21). Four experiments were performed; total binding without unlabeled SHBG was 19-21% of total added counts. Nonspecific binding was less than 3% of total counts. In the absence of receptor l-2% of added counts were precipitated with polyethylene glycol.
like those for hormones in that the Kd values for cellsurface hormone receptors approximate the concentration of the hormone in circulation such that changes in hormone concentration will be reflected in proportional changes in the fraction of receptor occupied. The present study also demonstrated that SHBG receptors from MCF-7 cells can be quantitatively solubilized in both an active and a stable form using the zwitterionic detergent CHAPS in the presence of glycerol. The same detergent was also used to solubilize receptor in prostate membranes (13). The presence of 10% (vol/vol) glycerol during the detergent extraction, centrifugation, and storage of the receptors is an important element in the recovery and maintenance of active soluble SHBG receptors, a finding in agreement with others (29, 30). This observation should be useful for future investigation of the structure and properties of these receptors. The factors causing a decrease in receptor binding affinity after solubilization are not yet known but may result from conformational changes in the receptor secondary to its removal from the environment in
1. Bardin CW, Musto N, Gunsalus G, Kotite N, Cheng S-L, Larrea F, Becker R 1981 Extracellular androgen binding proteins. Annu Rev Physiol 43:189-198 2. Siiteri PK, Murai JT, Hammond GH, Nisker JA, Raymore WJ, Kuhn RW 1982 The serum transport of steroid hormones. Recent Prog Horm Res 38:457-510 3. Westphal U 1983 Steroid-protein interaction: from past to present. J Steroid Biochem 19:1-15 4. Khan MS, Knowles BB, Aden DP, Rosner W 1981 J Clin Endocrinol Metab 53:448-449 5. Pardridge WM 1981 Transport of protein bound hormones into tissues in uiuo. Endocr Rev 2:103-123 6. Bordin S, Petra PH 1980 Immunocytochemical localization of the sex steroid-binding protein (SBP) of plasma in tissues of the adult monkey, Mocaca nemestrina. Proc Nat1 Acad Sci USA 77:56785682 7. Tardivel-Lacombe J, Egloff M, Mazabraud A, Degrelle H 1984 Immunohistochemical detection of the sex steroid-binding plasma protein in human mammary carcinoma cells. Biochem Biophys Res Commun 118:488-494 8. Rosen V, Jung I, Baulieu EE, Robe1 P 1975 Androgen-binding proteins in human benign prostatic hypertrophy. J Clin Endocrinol Metab 41:761-770 9. Sinnecker G, Hiort 0, Kwan PW, DeLellis RA 1990 Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue. Horm Metab Res 22:47-50 10. Gerard A, Egloff M, El Harate A, Domingo M, Gueant JL, Dang CD, Degrelle H 1988 Internalization of human sex steroid-binding protein in the monkey epididymis. J Mol Endocrinol5:239-251 11. Gerard A, Khanfri J, Gueant JL, Fremont S, Nicolas JP, Grignon G, Gerard H 1988 Electron microscope radioautographic evidence of in uiuo androgen-binding protein internalization in the rat epididymis principal cells. Endocrinology 122:1297-1307 12. Hryb DJ, Khan MS, Rosner W 1985 Testosterone-estradiol binding globulin binds to human prostatic cell membranes. Biochem Biophys Res Commun 128432-440 13. Hryb DJ, Khan MS, Romas NA, Rosner W 1989 Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate. J Biol Chem 264:5378-5383 14. Nakhla AM, Khan MS, Rosner W 1990 Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3’,5’-monophosphate. J Clin Endocrinol Metab 71:398-404
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2936
SHBG
RECEPTORS
JH, Woods GR, Lee FJ 1981 The specific binding of 15. MacIndoe androgens and the subsequent distribution of androgen-receptor complexes within MCF-7 human breast cancer cells. Steroids 38:439-452 16. Porto CS, Gunsalus GL, Bardin CW, Phillips DM, Musto NA 1991 Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells. Endocrinology 129:436-445 17. Barnes D, Sato G 1979 Growth of a human mammary tumor cell line in a serum-free medium. Nature 281:388-389 18. Kotite NJ, Musto NA 1982 Subunit structure of rabbit testosterone estradiol-binding globulin. J Biol Chem 257:5118-5124 RC, Carew TE, Glass CK, Green SR, Taylor CAJ, Attie 19. Pittman AD 1983 A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in uiuo. Biochem J 212:791-800 20. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 21. Rolland R, Gunsalus GL, Hammond JM 1976 Demonstration of specific binding of prolactin by porcine corpora lutea. Endocrinology 98:1083-1091 22. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239 23. Strel’chyonok OA, Avvakumov GV, Survilo LI 1984 A recognition
ON MCF-7
24.
25.
26.
27.
28.
29.
30. 31.
CELLS
Endo. 1992 Vol 130. No 5
system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes. Biochim Biophys Acta 802:459-466 Hryb DJ, Khan MS, Romas NA, Rosner W 1986 Specific binding of human corticosteroid-binding globulin to cell membranes. Proc Nat1 Acad Sci USA 83:3253-3256 Singer CJ, Khan MS, Rosner W 1988 Characteristics of the binding of corticosteroid-binding globulin to rat cell membranes. Endocrinology 122:89-86 Dautry-Varsat A, Ciechanover A, Lodish HF 1983 pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Nat1 Acad Sci USA 80~2258-2262 Brown MS, Goldstein JL 1979 Binding site of macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Nat1 Acad Sci USA 76:333-337 Rosner W, Hryb DJ, Khan MS, Singer CJ, Nakhla AM 1988 Are corticosteroid-binding globulin and sex hormone-binding globulin hormones? Ann NY Acad Sci 538137-145 Dias JA, Huston JJ, Reichert Jr LE 1981 Effect of the structurestabilizing agent glycerol on detergent-solubilized follicle-stimulating hormone receptors from calf testis. Endocrinology 109:736-742 Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134 Nakhla AM, Khan MS, Rosner W 1988 Induction of adenylate cyclase in a mammary carcinoma cell line by human corticosteroidbinding globulin. Biochem Biophys Res Commun 153:1012-1018
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Vol. 130, No. 5 Printed in U.S.A.
Society
Binding of an Extracellular Steroid-Binding Globulin to Membranes and Soluble Receptors from Human Breast Cancer Cells (MCF-7 Cells) C. S. PORTO, The Population
N. A. MUSTO,
C. W. BARDIN,
G. L. GUNSALUS
AND
Council, New York, NY 10021
ABSTRACT. strated ized by onstrated isolated studies
Studies of MCF-7 breast cancer cells demonthat sex hormone-binding globulin (SHBG) is internalreceptor-mediated endocytosis. The present study demspecific binding of SHBG to receptor on membranes from MCF-7 cells. Scatchard analysis of these binding suggested that SHBG binds to a single class of sites on
from MCF-7 membranes used buffers containing protease inhibitors, 10% glycerol, and 10 mM 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (246 f 14 pmol/mg protein) and a lo-fold decrease in binding affinity (Kd at 37 C = 2 x lo-’ M). (Endocrinology 130: 29312936, 1992)
membranes. The analysis yielded a dissociation constant (Kd) at 37 C of 3 x lo-* M and a binding capacity of 48 + 0.12 pmol/ mg protein.
A procedure
for solubilizing
the
SHBG
receptor
I
N THE blood of many species androgens and estrogens are bound to a specific sex hormone-binding globulin (SHBG), which is also referred to in the literature as testosterone-estradiol-binding globulin (TeBG) and sex steroid-binding protein (SBP) (l-3). This protein is synthesized by the liver (4) and secreted into the vascular compartment. It is commonly thought that extracellular steroid-binding proteins act physiologically to protect the circulating steroid from metabolism, thus serving as a reservoir to replenish the biologically active free pool. This view has been challenged by the demonstration that this protein can leave the vascular compartment (2, 5) and can be bound to as well as internalized by androgen-responsive cells (6-9) and by the finding that this same protein is produced in testis, where it is termed ABP, for transport to the epididymis where it is bound and internalized by principal cells (10, 11). Recently, binding sites for SHBG, which have been termed SHBG receptors, were demonstrated on the surface of human prostatic and human prostatic tumor cells (12-14) and solubilized with a variety of detergents (13). We have studied the binding of SHBG to androgenresponsive MCF-7 cells (15). This study showed that Received December 12, 1991. Address requests for reprints and all correspondence to: C. W. Bardin, The Population Council, 1230 York Avenue, New York, NY 10021. *These studies were supported by National Institutes of Health Grant HD-13541, Fogarty International Center (1 F05 TW04065-Ol), and Conselho National Desenvolvimento Cientifico e TecnologicoBrazil (203049-87) Fellowship (to C.S.P).
binding of SHBG was followed by its uptake into membrane vesicles of MCF-7 cells by receptor-mediated endocytosis (16). We now report specific binding of SHBG to MCF-7 cell membranes and solubilization of SHBG receptor from these membranes. Materials
and Methods
Cell culture
MCF-7 cells obtained from the American Type Culture Collection (Rockville, MD) were plated in flasks (70 cm’, Corning GlassWorks, Corning, NY) at a density of approximately 6-7 x lo5 cells per flask. Cells were cultured in Ham’s F-12Dulbecco’smodified Eagle’s medium (F-lB/DME, 1:l; Gibco Laboratories, Grand Island, NY) containing 1.2 g/liter sodium bicarbonate, 15 mM HEPES (Calbiochem, La Jolla, CA), 20 mg/liter
gentamycin
(Gibco Laboratories),
and 5% horse serum
plus 2.5% newborn calf serum (Hazelton, Lenexa, KS). The cells were grown in a humidified atmosphereof 95% air-5% CO, at 37 C for 4 days. Forty-eight hours before each experiment the cells were incubated in serum-freemediumcontaining: insulin, 6.25 pg/ml; transferrin, 6.25 pg/ml; selenium,6.25 rig/ml; BSA, 1.25 mg/ml; linoleic acid, 5.35 pg/ml (ITS+; Collaborative Research, Bedford, MA); epidermal growth factor (EGF; Biomedical Technologies, Stoughton, MA), 100 rig/ml; and prostaglandin F?,, (Sigma Chemical Co., St. Louis, MO), 100 rig/ml (17). Ligand Rabbit SHBG was purified by affinity chromatography as described previously (18) and radiolabeled with radioiodinated tyramine-cellobiose (TC; Sigma) adduct as described by Pitt-
2931
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2932
SHBG RECEPTORS
man et al. (19) and the labeled protein purified by size exclusion on Bio-Gel P-6 (200-400 mesh, Bio-Rad Laboratories, Richmond, CA). The radiolabeled preparations were greater than 97% precipitable by 10% trichloroacetic acid, 2% phosphotungstic acid precipitation and bind quantitatively to an androgen affinity column (16). Specific activities were approximately 1.2-2.0 &i/rg protein. The ligand was used without added testosterone. Preparation
and solubilization
of membranes
After incubation in serum-freemedium, cells were cooledto 0 C, rinsed with ice-cold PBS, detachedby rubber policeman, resuspendedin PBS, and sedimentedby centrifugation (200 x g for 5 min). All subsequentprocedureswere carried out at 4 C, and all solutionscontained freshly added5 mM N-ethylmaleimide (Sigma), and 10 mM phenylmethylsulfonyl fluoride (Sigma)to inhibit proteolysis. Cell pellets were resuspendedin buffer A (0.3 M sucrosein 20 mM Tris-HCl, pH 7.4; Sigma)and disrupted in a glasshomogeneizerwith a tightly fitting Teflon pestle. The homogenatewas centrifuged at 10,000x g for 15 min, and the resulting pellet was resuspendedin buffer A, homogeneized,and centrifuged asbefore. The combinedsupernatants were centrifuged at 100,000x g for 1 h. The resulting pellet was resuspendedin buffer B (25 mM Tris-HCl, pH 7.4, 10 mM CaCl,) and either stored at -70 C for binding experimentsor usedimmediately for preparation of solubilizedSHBG receptor. In preliminary studiesmembranesweretreated with various concentrations of Triton X-100, NP-40, and 3-[(3cholamidopropyl)dimethylammonio]-I-propanesulfonate (CHAPS). Both Triton X-100 and NP-40 (1% vol/vol) released30-40% of total membraneproteins, but receptor binding activity was lost in 24 h at -70 C; lower concentrations (0.1 and 0.5%) released only 5-10% of membraneprotein. By contrast CHAPS (Sigma) solubilizedprotein and preservedbinding activity (see Results). The optimal procedurefor solubilizing the receptor usedmembranes(6-8 mg protein/ml) resuspendedin buffer B containing enzyme inhibitors, 10% (vol/vol) glycerol (Fisher, Waltham, MA) and 10 mM CHAPS (Sigma Chemical Co.). The suspensionswere stirred at 4 C for 1 h and centrifuged at 100,000x g for 1 h. The supernatants,containing the SHBG receptor, could be stored at -70 C for 24 h without lossof activity. Membrane
binding
assay
Binding experiments were conducted in 1.5 ml Eppendorf polypropylene centrifuge tubes that had beenpretreated for 24 h at 37 C with 1 ml buffer C plus enzyme inhibitors (25 mM Tris-HCl, pH 7.4, 10 mM CaC12and 0.1% BSA) (Sigma) to minimize tube blanks. [iz51]TC-SHBG (62.5 ng; 6.9 nM) was incubated with membranes(0.3 mg protein/ml) in buffer C for periods of l-6 hours at 37 C, in the presenceor absenceof unlabeledSHBG, in a final volume of 0.3 ml. All assayswere performed in duplicate. Free and bound [‘251]TC-SHBG were separatedby centrifugation at 13,000x g for 10 min at 4 C. The supernatant wasaspirated, and the pellet waswashedwith buffer B and recentrifuged for 10 additional minutes. The final pellet was counted in a Packard y-scintillation counter. Nonspecificbinding wasabout 20% of total binding.
ON MCF-7 CELLS Assay of solubilized
Endo. 1992 Vol 130. No 5
receptor
Immediately before the assay,the solubleextracts were diluted to 0.5 mg protein/ml with buffer B at 4 C containing 10% (vol/vol) glycerol and 10 mM CHAPS supplementedwith proteaseinhibitors. Aliquots of the diluted extracts (0.1 ml) were addedto assaymixtures identical to those usedfor the membrane binding assay.After a l-6 h incubation at 37 C, the free and bound [‘251]TC-SHBG were separated(at 4 C) by precipitating the receptor-SHBG complex by addition of 0.5 ml buffer B containing 1 mg/ml bovine-y-globulins (Sigma) and 0.5 ml 12% (wt/vol) polyethylene glycol (PEG-8000) (Fisher Scientific, Pittsburgh, PA). After mixing, the tubes were incubated for 10 min and centrifuged at 13,000x g for 10 min at 4 C. The supernatantswere aspiratedand the pellets were suspendedin 1 ml buffer B containing 10% (vol/vol) glycerol and 10 mM CHAPS. Polyethylene glycol (0.5 ml) wasaddedand the tubes vortexed. After 10 min the tubes were centrifuged, the supernatant aspirated,and radioactivity in the pellets wasmeasured. Under theseconditions the nonspecificbinding wasabout 17% of total binding. Protein
determination
Protein concentration in the membranepreparations and soluble extracts was determined according to Bradford (20) usingBSA as a standard. Binding
analysis
Binding analysiswasperformed by weighted nonlinear least squarescurve fitting asdescribedpreviously (21,22).
Results Binding of r”“IlTC-SHBG cell membrane
to SHBG receptor in MCF-7
The binding of SHBG to MCF-7 cell membranes is shown in Fig. 1. Specific binding was the difference between total binding, which was 16-18% of added [1251] TC-SHBG at 37 C, and nonspecific binding determined in the presence of a 500-fold excess of unlabeled SHBG. The binding reached a plateau by 4 h at 37 C. A comparison of binding of SHBG at 37 C and 4 C showed a distinct temperature dependence. In four experiments in which each point was analyzed in duplicate the specific binding at 37 C was 3-fold higher than at 4 C (Fig. 1). A Scatchard plot of SHBG binding to MCF-7 cell membranes at 37 C is shown in Fig. 2. The results are consistent with SHBG binding to a single set of sites. An analysis of five experiments, each with duplicate determination, yielded a dissociation constant (KJ of 3 x lo-’ M and a binding capacity of 48 f 0.12 pmol/mg membrane protein (means f SEM). The specificity of binding was established by comparing the abilities of unlabeled SHBG, transferrin, cq-acid glycoprotein, and fetuin to compete with [lz51]TC-SHBG for binding to MCF-7 cell membranes (Fig. 3). The
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SHBG
RECEPTORS
8-
ON MCF-7
CELLS
2933
16
6-
1
2
4
6
TIME (h) binding of [‘251]TC-SHBG to MCF-7
FIG. 1. Time course of specific cell membranes. MCF-7 cell membranes (0.3 mg membrane protein/ ml) were incubated with [‘251]TC-SHBG (62.5 ng) with or without a 500-fold excess of unlabeled SHBG at 37 C and 4 C. Specific binding is the difference between total binding (16-18% of added [YJTCSHBG) and nonspecific binding determined in the presence of 500-fold excess of unlabeled SHBG. Values expressed as mean f SEM (n = 4 experiments).
specific binding of the radioactive ligand was reduced 80% by SHBG. The other proteins did not compete for SHBG binding sites. Solubilization of the SHBG receptor The ability of a variety of detergents (Triton X-100, NP-40, and CHAPS) to solubilize SHBG receptors from MCF-7 cell membranes was evaluated. CHAPS was found to be both efficient in solubilizing the receptor and did not interfere with binding. To determine the optimum concentrations of CHAPS for solubilization of SHBG receptors, MCF-7 cell membranes (6-8 mg protein/ml) were incubated at 4 C for 1 h with various concentrations of this detergent. The results showed that solubilization of membrane proteins and receptors depended on the concentration of CHAPS (Fig. 4). There was a linear increase in released MCF-7 cell membrane proteins until a concentration of about 5 mM. Further increases in the detergent concentration did not lead to a proportional release of membrane protein (Fig. 4A). The SHBG binding activity in the supernatant fractions increased almost linearly up to 10 mM CHAPS with a corresponding decrease in the binding activity of the pellet fraction (Fig. 4B). When the CHAPS concentration was further increased SHBG binding activity was reduced in both the supernatant fraction and the pellet. Based on these studies 10 mM CHAPS was used in all studies on the soluble receptor. Various concentrations
4
5
10 BOUND
15
20
25
(nM)
FIG. 2. Displacement by unlabeled SHBG of [iz51]TC-SHBG (62.5 ng) bound to MCF-7 cell membranes. Labeled ligand (6.9 nM) was incubated with membranes (0.3 mg membrane protein/ml) in the presence of various amounts of unlabeled SHBG for 6 h at 37 C. Five experiments were performed. Data from a single experiment are plotted in Scatchard coordinates. Curved line is the computer-generated best fit of raw data (0) to a high affinity, low capacity binding species (specific binding, solid straight line) and a low affinity, high capacity species (nonspecific binding, dashed straight line) (21). Total binding was 16-18% of added counts; nonspecific binding was less than 4% of total counts which is shown by the horizontal dashed line.
of glycerol (10, 20, 30%) were tested in order to improve the stability of the solubilized receptors. The SHBG binding activity of the soluble extract in 10 mM CHAPS was increased 1.5fold with 10% and 2-fold with 20% and 30%. The two highest concentrations of glycerol interfered with the polyethylene glycol precipitation. Thus 10% glycerol was use in all experiments on the solubilized receptor. Storage for 24 h at -70 C resulted in no loss of activity. The binding of [lz51]TC-SHBG to the solubilized receptor was studied. Analysis of the competition data with SHBG revealed one population of binding sites (Fig. 5). A Kd of 2 x 10e7 M and a binding capacity of 246 f 14 pmol/mg protein (mean f SEM) were estimated. Solubilization of the receptor lowered its binding affinity lofold but increased its specific activity 5-fold. Discussion The presence of specific membrane-binding sites for SHBG was first proposed by Strel’chyonok et al. (23)
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2934
SHBG
RECEPTORS
ON MCF-7
Endo - 1992 Voll30. No 5
CELLS
0.8
0 c! m
0.6
0.4
0.2 10
20
30
CHAPS (mM) 0
5 COMPETITOR
50
500
(pmol)
FIG. 3. Specificity of the SHBG binding. MCF-7 cell membranes were incubated with [iz51]TC-SHBG (62.5 ng) and various concentrations of rabbit SHBG (O), transferrin (0), al-acid glycoprotein (A), and fetuin (0) for 6 h at 37 C. B,, Binding of [Y]TC-SHBG in the absence of competitor; B, binding of [Y]TC-SHBG in the presence of competitor. Values expressed as mean + SEM (n = 5 experiments).
using human decidual endometrium. These authors found that the Kd at 4 C was 3 X lo-‘* M. Subsequently, Hryb et al. identified a SHBG receptor in human prostate membranes. High and low affinity sites were identified with Kd of 1.5 X 10e8 and 8.1 X 10e6 M, respectively. Solubilization of these receptors lowered the Kd to 1.4 x lo-’ and 1.0 X 10m7~ (12, 13). The present report demonstrates one class of specific sites binding for SHBG on MCF-7 cell membranes. The binding sites are saturable, specific, and have a affinity for SHBG (Kd = 3 X 10-s M) and a concentration (48 pmol/mg protein), similar to those reported for the corticosteroid-binding globulin (CBG) in MCF-7 cells (24, 25). Solubilization lowers the affinity of receptors on MCF-7 cell membranes which is in contrast to the increased affinity of receptors on prostate membranes after solubilization. The reason for this large variation in Kd for the soluble SHBG receptors noted above could relate either to tissue differences in factors that stabilize receptor or technical differences between laboratories. Evidence in favor of the former is that both soluble and membrane-associated forms of SHBG receptor in testes had affinities 10 times greater than that of the receptor from MCF-7 cells using exactly the same methods (Porto, C. S., unpublished observations) . High binding affinities have been observed for other membrane receptors and their respective serum protein ligands. For example, the transferrin receptor (26) and
B
o
10
20 CHAPS
30
(mM)
FIG. 4. Effects of CHAPS on the distribution of proteins and SHBG binding activity. MCF-7 cell membranes were solubilized in buffer A containing 10% (vol/vol) glycerol and various concentrations of CHAPS. Protein content (A) and binding activity (B) were determined in supernatant (M) and pellet (M) fractions obtained after centrifugation. Values expressed as mean f SEM (n = 3 experiments).
the low density lipoprotein receptor have Kd values of 6 x lo-’ M and 33 x lo-’ M, respectively (27). The difference between these protein-receptor systems and the one for SHBG is that the plasma concentrations of transferrin and low density lipoprotein exceed their receptor dissociation constants by several orders of magnitude, whereas the plasma concentrations of SHBG (2.5 x lOma M in men and 5.0 X 1O-8 M in women) (28) are similar to receptor dissociation constant (Kd = 1.5 X 10e8 M in human prostatic membrane and 3 x 10m8 M in MCF-7 cell membranes). Similarly, another serum steroid binding protein, CBG, and its membrane receptor have a Kd of 8.7 X 10e7 M and a serum concentration of 6.5 X 10e7 M. Therefore, the receptors for SHBG and CBG are more
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SHBG
RECEPTORS
ON MCF-7
CELLS
2935
the membrane. In conclusion, our results provide evidence for the presence of SHBG receptors on MCF-7 cells. The role this specific binding plays in the biology of these cells remains to be determined. It could be involved in regulation of androgen delivery, thus serving as a transport mechanism, or, alternatively, it could subserve a classical hormone-receptor signaling function. With regard to this latter possibility Nakhla et al. (31) observed that receptor-mediated uptake of another extracellular steroid binding protein (CBG) by MCF-7 cells was associated with increases in adenylate cyclase activity. This observation suggests that CBG may have a “hormone” activity in MCF-7 cells. This possibility remains to be evaluated for SHBG.
\ \
“0
5
Acknowledgments We gratefully acknowledge Richard Hale in the preparation 20
40
60
60
the assistence of Nichelle of this manuscript.
Edwards
and
100
References BOUND
(WA)
FIG. 5. Displacement
by unlabeled SHBG of [Y]TC-SHBG (62.5 ng) bound to the soluble receptor. Labeled ligand (6.9 nM) was incubated with the CHAPS-solubilized receptor in the presence of increasing amounts of unlabeled SHBG for 6 h at 37 C. Data from a single experiment are plotted in Scatchard coordinates. Curved line is the computer-generated best fit of raw data (0) to a high affinity, low capacity binding species (specific binding, solid straight line) and a low affinity, high capacity species (nonspecific binding, dashed straight line) (21). Four experiments were performed; total binding without unlabeled SHBG was 19-21% of total added counts. Nonspecific binding was less than 3% of total counts. In the absence of receptor l-2% of added counts were precipitated with polyethylene glycol.
like those for hormones in that the Kd values for cellsurface hormone receptors approximate the concentration of the hormone in circulation such that changes in hormone concentration will be reflected in proportional changes in the fraction of receptor occupied. The present study also demonstrated that SHBG receptors from MCF-7 cells can be quantitatively solubilized in both an active and a stable form using the zwitterionic detergent CHAPS in the presence of glycerol. The same detergent was also used to solubilize receptor in prostate membranes (13). The presence of 10% (vol/vol) glycerol during the detergent extraction, centrifugation, and storage of the receptors is an important element in the recovery and maintenance of active soluble SHBG receptors, a finding in agreement with others (29, 30). This observation should be useful for future investigation of the structure and properties of these receptors. The factors causing a decrease in receptor binding affinity after solubilization are not yet known but may result from conformational changes in the receptor secondary to its removal from the environment in
1. Bardin CW, Musto N, Gunsalus G, Kotite N, Cheng S-L, Larrea F, Becker R 1981 Extracellular androgen binding proteins. Annu Rev Physiol 43:189-198 2. Siiteri PK, Murai JT, Hammond GH, Nisker JA, Raymore WJ, Kuhn RW 1982 The serum transport of steroid hormones. Recent Prog Horm Res 38:457-510 3. Westphal U 1983 Steroid-protein interaction: from past to present. J Steroid Biochem 19:1-15 4. Khan MS, Knowles BB, Aden DP, Rosner W 1981 J Clin Endocrinol Metab 53:448-449 5. Pardridge WM 1981 Transport of protein bound hormones into tissues in uiuo. Endocr Rev 2:103-123 6. Bordin S, Petra PH 1980 Immunocytochemical localization of the sex steroid-binding protein (SBP) of plasma in tissues of the adult monkey, Mocaca nemestrina. Proc Nat1 Acad Sci USA 77:56785682 7. Tardivel-Lacombe J, Egloff M, Mazabraud A, Degrelle H 1984 Immunohistochemical detection of the sex steroid-binding plasma protein in human mammary carcinoma cells. Biochem Biophys Res Commun 118:488-494 8. Rosen V, Jung I, Baulieu EE, Robe1 P 1975 Androgen-binding proteins in human benign prostatic hypertrophy. J Clin Endocrinol Metab 41:761-770 9. Sinnecker G, Hiort 0, Kwan PW, DeLellis RA 1990 Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue. Horm Metab Res 22:47-50 10. Gerard A, Egloff M, El Harate A, Domingo M, Gueant JL, Dang CD, Degrelle H 1988 Internalization of human sex steroid-binding protein in the monkey epididymis. J Mol Endocrinol5:239-251 11. Gerard A, Khanfri J, Gueant JL, Fremont S, Nicolas JP, Grignon G, Gerard H 1988 Electron microscope radioautographic evidence of in uiuo androgen-binding protein internalization in the rat epididymis principal cells. Endocrinology 122:1297-1307 12. Hryb DJ, Khan MS, Rosner W 1985 Testosterone-estradiol binding globulin binds to human prostatic cell membranes. Biochem Biophys Res Commun 128432-440 13. Hryb DJ, Khan MS, Romas NA, Rosner W 1989 Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate. J Biol Chem 264:5378-5383 14. Nakhla AM, Khan MS, Rosner W 1990 Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3’,5’-monophosphate. J Clin Endocrinol Metab 71:398-404
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2936
SHBG
RECEPTORS
JH, Woods GR, Lee FJ 1981 The specific binding of 15. MacIndoe androgens and the subsequent distribution of androgen-receptor complexes within MCF-7 human breast cancer cells. Steroids 38:439-452 16. Porto CS, Gunsalus GL, Bardin CW, Phillips DM, Musto NA 1991 Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells. Endocrinology 129:436-445 17. Barnes D, Sato G 1979 Growth of a human mammary tumor cell line in a serum-free medium. Nature 281:388-389 18. Kotite NJ, Musto NA 1982 Subunit structure of rabbit testosterone estradiol-binding globulin. J Biol Chem 257:5118-5124 RC, Carew TE, Glass CK, Green SR, Taylor CAJ, Attie 19. Pittman AD 1983 A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in uiuo. Biochem J 212:791-800 20. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 21. Rolland R, Gunsalus GL, Hammond JM 1976 Demonstration of specific binding of prolactin by porcine corpora lutea. Endocrinology 98:1083-1091 22. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239 23. Strel’chyonok OA, Avvakumov GV, Survilo LI 1984 A recognition
ON MCF-7
24.
25.
26.
27.
28.
29.
30. 31.
CELLS
Endo. 1992 Vol 130. No 5
system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes. Biochim Biophys Acta 802:459-466 Hryb DJ, Khan MS, Romas NA, Rosner W 1986 Specific binding of human corticosteroid-binding globulin to cell membranes. Proc Nat1 Acad Sci USA 83:3253-3256 Singer CJ, Khan MS, Rosner W 1988 Characteristics of the binding of corticosteroid-binding globulin to rat cell membranes. Endocrinology 122:89-86 Dautry-Varsat A, Ciechanover A, Lodish HF 1983 pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Nat1 Acad Sci USA 80~2258-2262 Brown MS, Goldstein JL 1979 Binding site of macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Nat1 Acad Sci USA 76:333-337 Rosner W, Hryb DJ, Khan MS, Singer CJ, Nakhla AM 1988 Are corticosteroid-binding globulin and sex hormone-binding globulin hormones? Ann NY Acad Sci 538137-145 Dias JA, Huston JJ, Reichert Jr LE 1981 Effect of the structurestabilizing agent glycerol on detergent-solubilized follicle-stimulating hormone receptors from calf testis. Endocrinology 109:736-742 Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134 Nakhla AM, Khan MS, Rosner W 1988 Induction of adenylate cyclase in a mammary carcinoma cell line by human corticosteroidbinding globulin. Biochem Biophys Res Commun 153:1012-1018
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