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Interaction of calf uterus estradiol receptor with erythrocyte cytoskeleton
0022.4731i81~010307.06SOX0/0 CopyrIght 0 1981 Pergamon Press Ltd
Jownul of Srrmid Bwchumi.s-rr~ Vol. IS. pp 307 10 312 1981 Prmtcd m Great Brttam All rights reserved
INTERACTION OF CALF UTERUS ESTRADIOL RECEPTOR WITH ERYTHROCYTE CYTOSKELETON G.
A. PUCA. E. NOLA, A. M. MOLINARI,I. ARMETTAand V. SICA
Istituto di Patologia Generale, I Medical School, University of Naples, 80138 Naples, Italy SUMMARY
The relationship of the cytosolic estradiol receptor with membranous and cytoskeletal structures of the cell matrix has been studied using a model system formed by the erythrocyte membrane. Extraction of erythrocyte ghosts with various procedures and also with the nonionic detergent Triton X-100 under conditions that yield a cytoskeletal matrix reveals the presence of binding sites for the soluble estradiol receptor of calf uterus. The interaction between the estradiol receptor and the cytoskeleton is critically dependent on temperature which is required for the interaction itself and not for a modification of the receptor molecule. Ionic strength and cations, with selectivity for Mg’*, also influence the interaction which has an optimum at pH 7.5. Saturation experiments reveal the presence of a limited number (less than 100) of sites per cell having high affinity (Kd 1 10e9 M) for the estradiol-receptor complex. The affinity of the
receptor for the steroid does not change after it has bound to the cytoskeleton. Limited proteolysis of the receptor leads to a loss of its binding capacity for the cytoskeleton without altering its estradiol binding properties, indicating that the cytoskeletal binding domain is different from the steroid binding domain. We suggest that the estradiol receptor, generally considered ‘soluble’ in the cytoplasm. might be physiologically associated with cytoskeletal components of the cell cytoplasm.
In the last few years in addition to the cytoplasmic receptors for estradiol, high affinity and specific estrogen binding sites have been identified by several authors in association with particulate fractions of target cells, such as microsomes and mithocondria [l J. nuclear membranes [Z, 33, plasma membranes [4-6], microsomal membranes[?]. These data raise the question of whether the high affinity estradial binding sites found in the particulate fractions of the cell have a specific function and are entities different from cytoplasmic receptor or whether the cytosol receptor might result from its release during the cell fractionation. After recent reports by Sheridan et a/.[81 and Pietras and Szego[5] it would seem that the whole question of the intracellular distribution of steroid receptor needs to be re-evaluated with special attention paid to the procedures used in preparing the cellular fractions. Increasing awareness of the very intricate and complex nature of the ultrastructural organization of eucariotic cell and the high degree of integration of the various cellular elements, suggest that steroid hormone receptors may be closely related to a highly organized microscopic network of structural elements [9. IO]. This network forms a morphological and functional units which plays a fundamental role in very important cellular functions [I I-15]. The present paper shows findings which provide evidence that the soluble estrogen receptor interacts in an integral manner with components of the erythrocyte cytoskeletal system; the latter may generally be considered as a simplified model system for exploring cytoskeletal properties of more complex eucariotic cells. SB
IS
L
EXPERIMENTALSECJ’JON
17@stradiol-2,4,6,7-t, (84 to 110 Ci/mmol) was from Amersham. Cow blood erythrocyte ghosts and cytoskeletons were prepared as described by Bennett and Branton[16]; these were resuspended with phosphate buffer at a protein concentration of 3 to 10 mg/ml. diluted 1: 1 (v:v) with 99% glycerol and stored at -70°C until used. Calf uterine cytosol was prepared as previously described [17] except that only phosphate buffer (7.5 mM phosphate buffer, pH 7.5) instead of Tris-HCl EDTA and dithithreitol buffer was used. The assay of specific binding activity of cytosol was peformed by the Dextran-coated charcoal method [18]. The binding of the [3H]-estradiolreceptor complex to erythrocyte ghosts and cytoskeletons was performed in duplicate. Calf uterine cytosol was preincubated for 10 min at 20°C in a total volume of 0.4ml of phosphate buffer with 2.5 pmoles of [3H]-estradiol, 12.5 PmoI KCI and 1.25 prnol MgCl*. The reaction was started by adding 0.1 ml of cytoskeIeton or ghost suspension (0.25-2 mg of protein) and followed either for 2 h at 2O’C or for 30 min at 3OjC. Parallel tests were performed in the presence of 1 x IOm6M unlabeled estradiol. The reaction was stopped by adding 10 ml of ice-cold phosphate buffer. Samples were centrifuged at 3O.OOOgfor 20 min at 4°C and the pellets were washed three times. The final pellet was resuspended with 1 ml of phosphate buffer and assayed for radioactivity. The values obtained in the presence of unlabeled hormone were subtracted from the total. Preparation and partial purification of receptor transforming factor (RTF) and the trypsinderived 4 S estradiol receptor were carried out as de-
307
G. A.
308
PUCA et al.
scribed elsewhere [17, 193. The native 8 S form of estradiol receptor was prepared by centrifugation on preparative sucrose gradients. The estradiol receptor was partially purified by heparin-agarose and purified to homogeneity as described elsewhere [20,21]. The ammonium sulphate-aggregated form of the receptor was prepared by ammonium sulphate precipitation cytosol labeled with (30%) of uterine Temperature5 x lo-’ M [3H]-estradiol [17]. induced aggregation of the receptor was performed by incubating calf uterine cytosol at 30°C for 30min in the presence of 5 x 10e9 M [“H]-estradiol. Both samples of the aggregated forms were applied to Sephadex G-200 columns and the radioactive peaks in the void volume collected.
I
m
s ii B s %
I
1
1
wLP-1 120 MINUTES
Erythrocyte ghosts do not show displaceable binding sites for estradiol. The amount of radioactivity which is associated with the ghosts is in fact the same in the presence or in the absence of a large excess of unlabeled estradiol. When calf uterine cytosol is added, some radioactivity appears associated to the ghost and it can be displaced by excess of unlabeled estradiol (Table 1). Extraction of erythrocyte ghosts with Triton X-100 increases the displaceable radioactivity bound by the residual shells (Table 1). Maximal binding occurs at 1% Triton X-100. a concentration used for the preparation of cytoskeleton from erythrocyte ghosts [22]. We therefore refer to the Triton X-100 extracted ghosts with the term “erythrocyte cytoskeleton”. The unlabeled estradiol displaceable sites appear only in the presence of uterine cytosol containing estradiol receptor but neither in the presence of albumin, a low afhnity estradiol binding proTable 1. Effect of extracting erythrocyte ghosts with Triton X-100 on the enhancement of binding of [‘Hl-estradiol receptor complex
Triton X-100 (Percent) 0
::: 0.5
1.0 1.5
d.p.m. Bound Cytosol added No eytosol C’HI-Ez [‘HI-E, [“HI-EI
17;Ez
C3H]-E,
17BtE*
3200 3100 3600 3010 3700 3180
3180 3600 3400 4000 3750 3250
4960 9120 15300 27100 32500 31400
4200 4050 3900 4400 3980 4350
Erythrocyte ghosts were extracted for 15 min at 0°C with the indicated concentrations of TritonX-100. After extraction ghosts were centrifuged and washed four times with phosphate buffer to eliminate detergent. Pellets were resuspended at a final protein concentration of 12 mg/ml with phosphate buffer. Aliquots of 0.1 ml of resuspended material were incubated 2 h at 20°C with 5 x 10e9 M [‘H]-estradiol ([‘HI-E,) in the presence or in the absence of 10e6 M unlabelled 17/3-est;adiol(l7fi-E,) with or without 200~1 of calf uterine cytosol. Total volume of the sample was 0.5 ml. Bound radioactivity was .-. measured as described under Ex-
perrmental Procedure Section.
I
40
30 RESULTS
I
Fig. 1. Time course of binding of estradiol-receptor complex to erythrocyte cytoskeleton. Erythrocyte cytoskeleton was incubated with 0.1 ml of calf uterine cytosol. Incubations were carried out at 0°C (m), 10°C (0), 20°C (A) and 30°C (A). At the indicated times the reaction was stopped by adding cold phosphate buffer and the binding was measured. The dotted line represents the binding at 4°C after preincubation of the estradiol-receptor complex for 2 h at 20°C.
tein, nor of uterine cytosol pretreated for 30min at 60°C (data not shown). We therefore consider this binding as due to the binding of the estradiol recep tor. Erythrocyte ghosts have been also extracted with high salt (KCl, 1 M), chaotropic salt (NaSCN, 0.5 M), acetic acid (0.1 M), sodium hydroxide (0.05 M), EDTA (5 mM). In all these cases an increase in the binding of [‘H]-estradiol receptor to the extracted ghosts is observed but always lower than that with the cytoskeleton. Incubation (6Omin at 30°C) of ghosts at low ionic strength, a procedure known to remove spectrin and actin [23], also increases the binding Inside out vesicles, obtained from spectrin and actin deprived ghosts, do bind [3H+estradiol receptor. Further studies were however carried out only with the erythrocyte cytoskeleton which operationally was easier to USe.
Binding of estradiol receptor to the cytoskeleton is time and temperature dependent (Fig. 1). Temperatures higher than 30°C were not used because of the inherent instability of the estradiol-receptor complex. Preincubation of calf uterine cytosol with the hormone for 2 h at 20°C does not change the subsequent binding of the [“HJestradiol-receptor complex to the cytoplasmic at 0°C (dotted line, Fig 1). The interaction is affected in important ways by KC1 or NaCl. Binding increases about three-fold in the presence of 25 mM KCl; above this concentration the binding decreases but not to values below those seen without KCl, even at ionic strengths higher than 1 M (Fig. 2A). In the presence of 25 mM KCl, Ca’* (up to 5 mM) has no stimulatory effect, while MnzC (5 mM) increases slightly the binding. Mg’+ stimulates the binding substantially, with an optimal concentration at 2.5 mM (Fig. 2B).
Estradiol receptor and erythrocyte cytoskeleton
100
200
300
400
2
Kc.1, mM
4
6
309
8
IO
MQCl2 .mM
Fig. 2. Effect of increasing concentrations of KC1 and MgClt on the binding of [3H)-estradiol-receptor complex to erythrocyte cytoskeleton. A. Erythrocyte cytoskcleton was incubated for 2 h at 20°C with 0.2 ml of calf uterine cytosol with the indicated concentration of KCI. B. Erythrocyte cytoskeleton and calf uterine cytosol were incubated in phosphate bulier containing 25 mM KCI at the indicated eoncentrations of MgC12.
The binding of [‘H]-estradiol-receptor complex is linearly related to the concentration of the cytoslceleton in the range of lOO-lCMlO* protein per mL provided the concentration of estradiol receptor is not limiting (Fig. 3A). The pH optimum of the cytoskeleton-receptor interaction is 7.5 (Fig 3B). When the cytoskeleton concentration is kept constant while the receptor concentration is increased the interaction appears to be saturable and of high afhnity (Fig. 4). Scatchard analyses of such saturation experiments suggest the presence of two classes of binding sites differing markedly in their affinity. The dissociation constant of the higher ffiity component is in the range of 10m9 M. From data obtained in saturation experiments, and independently determining that 1 mg of cytoskeletal protein corresponds to about 5 x lo-’ erythrocytes, it is possible to calculate that one erythrocyte cytoskeleton has a maximum of 50 to
CYTOSKELETON PROTEINS. IWJ~I
100 binding sites for the estradiol-receptor complex, that one mole of estradiol is bound per mole of receptor. The receptor is capable of binding strongly to the cytoskeleton in the absence of estradiol. The cytosoltreated and subsequently washed cytoskeleton can selectively bind estradiol and the affinity for the steroid of the cytoskeleton-bound receptor is unchanged (Fig. 5). Even at high concentration of the cytoskeleton relative to that of the cytosol only a maximum of 60-70% of the cytosol receptor is able to bind to the cytoskeleton. The receptor left in the supernatant under these conditions does not bind at all when added to fresh untreated cytoskeleton. Thus, this portion (3O+!k) must represent either denatured receptor or a subpopulation of receptor that is specifically incapable of binding to the cytoskeleton. When the various molassuming
PH
Fig. 3. Effect of cytoskeleton concentrations and of pH on the binding of [3H]-estradiol-receptor complex. A. The indicated concentrations of the cytoskeleton proteins were incubated for 30 min at 30°C with 0.1 ml calf uterine cytosol. B. Erythrocyte cytoskehon was resuspended with 7.5 mM phosphate buffers containing 25 mM KC1 and 2.5 mM MgC12, pH 6.5. 7, 7.5 or with 10 mM Tris-HCI buffers. pH 8 and 8.5. containing the same KC1 and MgC& concentrations, and incubated for 30min at 30°C with 0.2 ml calf uterine cytosol in the same buffer.
G. A.
310
PUCA
rt
ui.
8
t
I
2
CVTOSOL RECEPTOR
E
CYTOSOl RECEPTOR CSK COMPLEX
,’
A
,’
,
/
i
/’ I
J ‘H E,AECEPTOR tOMPLEXEOUNO
!
[lo-%] 1 1
3H-ESTRADIOL
I
I
I
2
3
4
~K~E;RE~EPTGRCOMPLEX [W"M]
Fig. 4. Effect of increasing the concentration of [3H]-estradiotreceptor complex on the binding to erythrocyte cytoskeleton. The indicated concentrations of [‘H]-estradiolreceptor complex partially purified by sepharose-heparin chromatography was incubated with 0.1 ml of erythrocyte cytoskeieton for 2 h at 20% Insert: Results plotted according to Scatchard.
ecular forms of estradiol receptor are prepared and studied separately, the native partially purified 8 S form, the highly purified 8 S receptor and the aggregated (whether by ammonium sulfate or by heating for 30min at 30°C) forms are all abie to bind with varying degrees of efficiency to the cytoskeleton (Table 2). Partial, controiled proteolysis of the estradioi receptor by trypsin or by RTF causes a loss in the ability to bind to the cytoskeleton despite the fact that the binding of estradiol to these receptor forms is unaltered (Table 2). DISCUSSION
Erythrocyte
ghosts display binding
sites for the
ADDED,
Fig. 5. Affinity for estradioi of cytosoi and cytoskeletonbound receptor. Aiiquots (0.1 ml) of fresh uterine cytosoi were preincubated 2 h at 2O’C and then incubated for I6 h at 4°C with the indicated concentrations of [3H]-estradioi in the absence (A) and in the presence (A) of IOOO-fold excess unlabeled estradiol. Final volume was 0.5 ml. Erythrocyte cytoskeieton (3 ml: 15 mg protein) was incubated with 6 ml of the same cytosoi in a total volume of 1S ml of phosphate buffer 25 mM KC1 containing and 2.5 mM MgCi,. After 2 h at 2O”C the mixture was centrifuged and pellet washed twice with phosphate buffer. Final pellet was resuspended with 15 ml phosphate buffer. Aiiquots (OS ml) were incubated 16 h at 4X with the indicated concentrations of [‘HI-estradioi in the absence (0) and presence (0) of IOOO-foldexcess unlabeled estradiol. At the end of the incubation samples were centrifuged. washed three times and radioactivity bound to cytoskeieton measured. estradiol
receptor
only when a portion
of the mem-
brane proteins are removed with various extraction methods, when ghosts are reduced to vesicles or when many of the proteins and most of the lipid bilayer are removed with the nonionic detergent Triton X-100. These sites are very strongly associated with the cytoskeleton of the erythrocyte membrane and are unmasked by the various extraction procedures.
Table 2. Binding of various estradioi receptor forms to erythrocyte cytoskeietons d.p.m. Added 8S 4s (Ca*+-RTF) 4S (Trypsin) Aggregates (NH,SOL) Aggregates (temperature) Pure receptor
[ lo-‘M^
27,700 10.900 11,6UO 21,900 7.400 16,OcQ
d.p.m. Bound 15,ocO 947 42 8,500 4.300 15,975
Percent 66 8 0 38 58 100
The various estradioi receptor forms were prepared as described under Experimental Procedure Section. Aiiquots of 0.1 ml of cytoskeieton (0.4mg of protein) were incubated 2 h at 22°C with 6 x lo-’ M [‘HI-estradiol in the presence or in the absence of 10e6 M unlabeled estradioi with the indicated amounts of estradioi receptor forms in a total volume of 03 ml of phosphate buffer containing 2.5 mM M&i, and 2.5mM KCI.
Bound [‘HI-estradiol-receptor complex was assessed as described.
Estradiol receptor and erythrocyte cytoskeleton The temperature dependence of the estradiol receptor binding suggests the requirement for an “open” cytoskeletal organization. Since prior heating of the cytosol does not facilitate the subsequent receptor binding to cytoskeletons at low temperatures (Fig. l), the temperature is not required for a modification or activation of the receptor. The temperature might reflect the need for increased fluidity of the residual membrane components, allowing the access of the receptor to its binding sites. Ionic strength influences the receptor-cytoskeleton interaction which appears to be enhanced by Mg2+ in a quite selective manner. Apparently, other than KC1 and Mg2+ no other cytoplasmic factors are able to significantly modify the interaction which is still observed after extensive dialysis and partial (Fig. 4) or complete purification of the estradiol receptor (Table 2). Saturation analysis of the receptor-cytoskeleton interaction reveals the presence of a very limited number (less than 100) of high affinity (Kd = 10e9 M) binding sites per erythrocyte ghost. This excludes the possibility that one of the major protein component of the cytoskeleton (e.g. band 3, spectrin, actin, ankyrin. band 4.1) may be responsible for the binding. The receptor domain which interacts with the cytoskeletal structure is different from the domain containing the steroid binding site. In fact. partial hydrolysis of the receptor by the Ca” dependent RTF Cl93 or by mild tryptic digestion lead to a loss in its ability to bind to the cytoskeleton without altering the hormone binding properties. In conclusion. findings presented in this paper are consistent with the presence of specific binding sites for the estrogen receptors in the cytoskeletal matrix of the erythrocyte membrane. Similar systems may exist in target cells, which could in part explain data apparently contradictory with the general model of action of steroid hormones [l-7.24-29]. The water soluble nature of the estradiol receptor found in cytosol might be an artefact of tissue homogenization and preparation as recently demonstrated by Pietras and Szego[S, 61. and the membrane receptor described by Parikh et a[.[73 and by others Cl-33 might be that fraction of receptor which has not been released during the cell fractionation.
Ackno*,ledgementsThis work was supported by Progetto Finalizzato: Controllo della Crescita Neoplastica. by a grant from The Wellcome Research Laboratories, RTP. North Carolina and the National Institute of Health contract No. NOI-CB-64074.
REFERENCES Noteboom W. D. and Gorski J.: Stereospecific binding of estrogens in the rat uterus. Arch. hi&em Biophrs. Ill (1965) 559-568. Jackson V. and Chalkley R.: The binding of estradiol-17/l to the bovine endometrial nuclear membrane. J. hiol. Chem. 249 (19741 1615-1626. Barrack E. R. and Coffey D. S.: The specific binding of
311
estrogens and androgens to the nuclear matrix of sex hormone responsive tissues. J. biol. Chem. 255 (1980) 7265-7275. 4. Pietras R. J. and Szego C. M.: Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature. Land. 265 (1977) 69-72. 5. Pietras R. J. and Szego C. M.: Estrogen receptors in uterine plasma membrane. J. steroid Biochem. 11 (1979) 1471-1483. 6. Pietras R. J. and !&ego C. M.: Partial purification and characterization of estrogen receptors in subfractions of hepatocyte plasma membranes. Biochem. J. 191 (1980) 743-760. 7. Parikh I., Lee Anderson W. and Neame P.: Identification of high affinity estrogen binding sites in calf uterine microsomal membranes. J. hiol. Chem. 255 (1980) 1026610270. 8. Sheridan P. J., Buchanan J. M.. Anselmo V. C. and Martin P. M.: Equilibrium: The intracellular distribution of the steroid receptors. Nature. Land. 282 (1979) 579-582. 9. Bretscher M. S. and Raff M. C.: Mammalian plasma membranes. Nature. Land. 258 (1975) 43-49. 10. Lazarides F.: Intermediate filaments as mechanical integrators of cellular space. Nature. Land. 283 (1980) 249-256. 11. Edelman G.: Surface modulation in cell recognition and cell growth. Science 192 (1976) 218-226. 13 Crossin K. L. and Carney D. H.: Evidence that microtubule depolymerization early in the cell cycle is sufficient to Initiate DNA synthesis. Ce// 23 (1981) 61-71. 13. Giannattasio G.. Zanini A., Rosa P., Meldolesi F.. A_.
Margolis R. K. and Margolis R. U.: Molecular organization of prolactin granules. III intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix. J. cell Biol. 86 (1980) 273279. 14. Cervera M.. Dreyfuss G. and Penman S.: Messanger RNA is translated when associated with the cyroskeleta1 framework in normal and VSV-infected HeLa cells. Cell 23 (1981) 113-120. 15. Willingham M. C. and Pastan I.: The receotosome: an intermediate organelle of receptor-mediated endocytosis in cultured fibroblasts. Cell 21 (1980) 67-77. 16. Bennett V. and Branton D.: Selective association of spectrin with cytoplasmic surface of human erythrocvte nlasma membranes. J. biol. Chem. 252 (1977) 2%3-'2763.
17. Puca G. A., Nola E.. Sica V. and Bresciani F.: Estrogen binding protein of calf uterus. Interrelationship between various forms and identification of a Receptor Factor. Biochemistry 11 (1972) Transforming 4157-4165. 18. Sanborn B. M.. Rao R. B. and Koreman S. G.: Interaction of 178.estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behavior. Biochemistry 10 (1971) 4955-4965. 19. Puca G. A., Nola E., Sica V. and Bresciani F.: Estrogen binding protein of calf uterus. Molecular and functional characterization of the Receptor Transforming Factor: a Cal’ activated protease. J. hiol. Chem. 252 (1977) 1358-1366. 20. Molinari A. M.. Medici N.. Moncharmont B. and Puca G. A.: Estradiol receptor of calf uterus: Interaction with heparin agarose and purification. Proc. narn. Acud. Sci. L’5.A. 74 (1977) 488&4890. 21. Puca G. A.. Sica V.. Nola E. and Bresciani F.: Purification and properties of native estradiol receptor. J. steroid Biochem. 11 (19791 301-306. ‘7 Lux S. E.: Dissecting the red cell membrane skeleton. __. h;urure. Lond. 281 (1979) 426-429. 23. Marchesi V. T.: Functional proteins of the human
312
G. A.
f%JCA
red blood cell membrane. Semin. Hemarol. 16 (1979) 3-20. 24. Baulieu E. E., Godeau F., Schorderet M. and Schorderet-Slatkine S.: Steroid-induced meiotic division in Xenopus luevis oocytes: Surface and calcium. Nature, Load. 275 (1978) 593-598. 25. Miigrom E., Atger M. and Bauheu E. E.: Studies on estrogen entry into uterine cells and an estradiolreceptor complex attachment to the nucleus. Is the entry of estrogen into uterine cells a protein-mediated process? Biochem. hiophys. Acta 320 (1973) 267-283. 26. Nenci I., Fabris G., Marchetti E. and Marzola A.: Intracetlular flow of particulate steroid-receptor com-
et at
plexes in steroid target cells. C’irchows Arch. B. Ceil Path 32 (1980) 139-145. 27. Munck A. and Brinck-Johnson T.: Specific and nonspecific physicochemical interactions of glucocorticoids and related steroids with rat thymus cells in uifro. J. biol. Chem. 243 (1968) 5556-5565. 28. Harrison R. W., Fairfield S. and Orth D. N.: Evidence for glucocorticoid transport through the target cell membrane. B&hem. biophys. Rex Comm. 61 (1974) 1262-1267. 29. Suyemitsu T. and Terayama H.: Specific binding sites for natural glucocorticoids in plasma membranes of rat liver. E~ocri~o~~y 96 (1975) 1499-1508.
Jownul of Srrmid Bwchumi.s-rr~ Vol. IS. pp 307 10 312 1981 Prmtcd m Great Brttam All rights reserved
INTERACTION OF CALF UTERUS ESTRADIOL RECEPTOR WITH ERYTHROCYTE CYTOSKELETON G.
A. PUCA. E. NOLA, A. M. MOLINARI,I. ARMETTAand V. SICA
Istituto di Patologia Generale, I Medical School, University of Naples, 80138 Naples, Italy SUMMARY
The relationship of the cytosolic estradiol receptor with membranous and cytoskeletal structures of the cell matrix has been studied using a model system formed by the erythrocyte membrane. Extraction of erythrocyte ghosts with various procedures and also with the nonionic detergent Triton X-100 under conditions that yield a cytoskeletal matrix reveals the presence of binding sites for the soluble estradiol receptor of calf uterus. The interaction between the estradiol receptor and the cytoskeleton is critically dependent on temperature which is required for the interaction itself and not for a modification of the receptor molecule. Ionic strength and cations, with selectivity for Mg’*, also influence the interaction which has an optimum at pH 7.5. Saturation experiments reveal the presence of a limited number (less than 100) of sites per cell having high affinity (Kd 1 10e9 M) for the estradiol-receptor complex. The affinity of the
receptor for the steroid does not change after it has bound to the cytoskeleton. Limited proteolysis of the receptor leads to a loss of its binding capacity for the cytoskeleton without altering its estradiol binding properties, indicating that the cytoskeletal binding domain is different from the steroid binding domain. We suggest that the estradiol receptor, generally considered ‘soluble’ in the cytoplasm. might be physiologically associated with cytoskeletal components of the cell cytoplasm.
In the last few years in addition to the cytoplasmic receptors for estradiol, high affinity and specific estrogen binding sites have been identified by several authors in association with particulate fractions of target cells, such as microsomes and mithocondria [l J. nuclear membranes [Z, 33, plasma membranes [4-6], microsomal membranes[?]. These data raise the question of whether the high affinity estradial binding sites found in the particulate fractions of the cell have a specific function and are entities different from cytoplasmic receptor or whether the cytosol receptor might result from its release during the cell fractionation. After recent reports by Sheridan et a/.[81 and Pietras and Szego[5] it would seem that the whole question of the intracellular distribution of steroid receptor needs to be re-evaluated with special attention paid to the procedures used in preparing the cellular fractions. Increasing awareness of the very intricate and complex nature of the ultrastructural organization of eucariotic cell and the high degree of integration of the various cellular elements, suggest that steroid hormone receptors may be closely related to a highly organized microscopic network of structural elements [9. IO]. This network forms a morphological and functional units which plays a fundamental role in very important cellular functions [I I-15]. The present paper shows findings which provide evidence that the soluble estrogen receptor interacts in an integral manner with components of the erythrocyte cytoskeletal system; the latter may generally be considered as a simplified model system for exploring cytoskeletal properties of more complex eucariotic cells. SB
IS
L
EXPERIMENTALSECJ’JON
17@stradiol-2,4,6,7-t, (84 to 110 Ci/mmol) was from Amersham. Cow blood erythrocyte ghosts and cytoskeletons were prepared as described by Bennett and Branton[16]; these were resuspended with phosphate buffer at a protein concentration of 3 to 10 mg/ml. diluted 1: 1 (v:v) with 99% glycerol and stored at -70°C until used. Calf uterine cytosol was prepared as previously described [17] except that only phosphate buffer (7.5 mM phosphate buffer, pH 7.5) instead of Tris-HCl EDTA and dithithreitol buffer was used. The assay of specific binding activity of cytosol was peformed by the Dextran-coated charcoal method [18]. The binding of the [3H]-estradiolreceptor complex to erythrocyte ghosts and cytoskeletons was performed in duplicate. Calf uterine cytosol was preincubated for 10 min at 20°C in a total volume of 0.4ml of phosphate buffer with 2.5 pmoles of [3H]-estradiol, 12.5 PmoI KCI and 1.25 prnol MgCl*. The reaction was started by adding 0.1 ml of cytoskeIeton or ghost suspension (0.25-2 mg of protein) and followed either for 2 h at 2O’C or for 30 min at 3OjC. Parallel tests were performed in the presence of 1 x IOm6M unlabeled estradiol. The reaction was stopped by adding 10 ml of ice-cold phosphate buffer. Samples were centrifuged at 3O.OOOgfor 20 min at 4°C and the pellets were washed three times. The final pellet was resuspended with 1 ml of phosphate buffer and assayed for radioactivity. The values obtained in the presence of unlabeled hormone were subtracted from the total. Preparation and partial purification of receptor transforming factor (RTF) and the trypsinderived 4 S estradiol receptor were carried out as de-
307
G. A.
308
PUCA et al.
scribed elsewhere [17, 193. The native 8 S form of estradiol receptor was prepared by centrifugation on preparative sucrose gradients. The estradiol receptor was partially purified by heparin-agarose and purified to homogeneity as described elsewhere [20,21]. The ammonium sulphate-aggregated form of the receptor was prepared by ammonium sulphate precipitation cytosol labeled with (30%) of uterine Temperature5 x lo-’ M [3H]-estradiol [17]. induced aggregation of the receptor was performed by incubating calf uterine cytosol at 30°C for 30min in the presence of 5 x 10e9 M [“H]-estradiol. Both samples of the aggregated forms were applied to Sephadex G-200 columns and the radioactive peaks in the void volume collected.
I
m
s ii B s %
I
1
1
wLP-1 120 MINUTES
Erythrocyte ghosts do not show displaceable binding sites for estradiol. The amount of radioactivity which is associated with the ghosts is in fact the same in the presence or in the absence of a large excess of unlabeled estradiol. When calf uterine cytosol is added, some radioactivity appears associated to the ghost and it can be displaced by excess of unlabeled estradiol (Table 1). Extraction of erythrocyte ghosts with Triton X-100 increases the displaceable radioactivity bound by the residual shells (Table 1). Maximal binding occurs at 1% Triton X-100. a concentration used for the preparation of cytoskeleton from erythrocyte ghosts [22]. We therefore refer to the Triton X-100 extracted ghosts with the term “erythrocyte cytoskeleton”. The unlabeled estradiol displaceable sites appear only in the presence of uterine cytosol containing estradiol receptor but neither in the presence of albumin, a low afhnity estradiol binding proTable 1. Effect of extracting erythrocyte ghosts with Triton X-100 on the enhancement of binding of [‘Hl-estradiol receptor complex
Triton X-100 (Percent) 0
::: 0.5
1.0 1.5
d.p.m. Bound Cytosol added No eytosol C’HI-Ez [‘HI-E, [“HI-EI
17;Ez
C3H]-E,
17BtE*
3200 3100 3600 3010 3700 3180
3180 3600 3400 4000 3750 3250
4960 9120 15300 27100 32500 31400
4200 4050 3900 4400 3980 4350
Erythrocyte ghosts were extracted for 15 min at 0°C with the indicated concentrations of TritonX-100. After extraction ghosts were centrifuged and washed four times with phosphate buffer to eliminate detergent. Pellets were resuspended at a final protein concentration of 12 mg/ml with phosphate buffer. Aliquots of 0.1 ml of resuspended material were incubated 2 h at 20°C with 5 x 10e9 M [‘H]-estradiol ([‘HI-E,) in the presence or in the absence of 10e6 M unlabelled 17/3-est;adiol(l7fi-E,) with or without 200~1 of calf uterine cytosol. Total volume of the sample was 0.5 ml. Bound radioactivity was .-. measured as described under Ex-
perrmental Procedure Section.
I
40
30 RESULTS
I
Fig. 1. Time course of binding of estradiol-receptor complex to erythrocyte cytoskeleton. Erythrocyte cytoskeleton was incubated with 0.1 ml of calf uterine cytosol. Incubations were carried out at 0°C (m), 10°C (0), 20°C (A) and 30°C (A). At the indicated times the reaction was stopped by adding cold phosphate buffer and the binding was measured. The dotted line represents the binding at 4°C after preincubation of the estradiol-receptor complex for 2 h at 20°C.
tein, nor of uterine cytosol pretreated for 30min at 60°C (data not shown). We therefore consider this binding as due to the binding of the estradiol recep tor. Erythrocyte ghosts have been also extracted with high salt (KCl, 1 M), chaotropic salt (NaSCN, 0.5 M), acetic acid (0.1 M), sodium hydroxide (0.05 M), EDTA (5 mM). In all these cases an increase in the binding of [‘H]-estradiol receptor to the extracted ghosts is observed but always lower than that with the cytoskeleton. Incubation (6Omin at 30°C) of ghosts at low ionic strength, a procedure known to remove spectrin and actin [23], also increases the binding Inside out vesicles, obtained from spectrin and actin deprived ghosts, do bind [3H+estradiol receptor. Further studies were however carried out only with the erythrocyte cytoskeleton which operationally was easier to USe.
Binding of estradiol receptor to the cytoskeleton is time and temperature dependent (Fig. 1). Temperatures higher than 30°C were not used because of the inherent instability of the estradiol-receptor complex. Preincubation of calf uterine cytosol with the hormone for 2 h at 20°C does not change the subsequent binding of the [“HJestradiol-receptor complex to the cytoplasmic at 0°C (dotted line, Fig 1). The interaction is affected in important ways by KC1 or NaCl. Binding increases about three-fold in the presence of 25 mM KCl; above this concentration the binding decreases but not to values below those seen without KCl, even at ionic strengths higher than 1 M (Fig. 2A). In the presence of 25 mM KCl, Ca’* (up to 5 mM) has no stimulatory effect, while MnzC (5 mM) increases slightly the binding. Mg’+ stimulates the binding substantially, with an optimal concentration at 2.5 mM (Fig. 2B).
Estradiol receptor and erythrocyte cytoskeleton
100
200
300
400
2
Kc.1, mM
4
6
309
8
IO
MQCl2 .mM
Fig. 2. Effect of increasing concentrations of KC1 and MgClt on the binding of [3H)-estradiol-receptor complex to erythrocyte cytoskeleton. A. Erythrocyte cytoskcleton was incubated for 2 h at 20°C with 0.2 ml of calf uterine cytosol with the indicated concentration of KCI. B. Erythrocyte cytoskeleton and calf uterine cytosol were incubated in phosphate bulier containing 25 mM KCI at the indicated eoncentrations of MgC12.
The binding of [‘H]-estradiol-receptor complex is linearly related to the concentration of the cytoslceleton in the range of lOO-lCMlO* protein per mL provided the concentration of estradiol receptor is not limiting (Fig. 3A). The pH optimum of the cytoskeleton-receptor interaction is 7.5 (Fig 3B). When the cytoskeleton concentration is kept constant while the receptor concentration is increased the interaction appears to be saturable and of high afhnity (Fig. 4). Scatchard analyses of such saturation experiments suggest the presence of two classes of binding sites differing markedly in their affinity. The dissociation constant of the higher ffiity component is in the range of 10m9 M. From data obtained in saturation experiments, and independently determining that 1 mg of cytoskeletal protein corresponds to about 5 x lo-’ erythrocytes, it is possible to calculate that one erythrocyte cytoskeleton has a maximum of 50 to
CYTOSKELETON PROTEINS. IWJ~I
100 binding sites for the estradiol-receptor complex, that one mole of estradiol is bound per mole of receptor. The receptor is capable of binding strongly to the cytoskeleton in the absence of estradiol. The cytosoltreated and subsequently washed cytoskeleton can selectively bind estradiol and the affinity for the steroid of the cytoskeleton-bound receptor is unchanged (Fig. 5). Even at high concentration of the cytoskeleton relative to that of the cytosol only a maximum of 60-70% of the cytosol receptor is able to bind to the cytoskeleton. The receptor left in the supernatant under these conditions does not bind at all when added to fresh untreated cytoskeleton. Thus, this portion (3O+!k) must represent either denatured receptor or a subpopulation of receptor that is specifically incapable of binding to the cytoskeleton. When the various molassuming
PH
Fig. 3. Effect of cytoskeleton concentrations and of pH on the binding of [3H]-estradiol-receptor complex. A. The indicated concentrations of the cytoskeleton proteins were incubated for 30 min at 30°C with 0.1 ml calf uterine cytosol. B. Erythrocyte cytoskehon was resuspended with 7.5 mM phosphate buffers containing 25 mM KC1 and 2.5 mM MgC12, pH 6.5. 7, 7.5 or with 10 mM Tris-HCI buffers. pH 8 and 8.5. containing the same KC1 and MgC& concentrations, and incubated for 30min at 30°C with 0.2 ml calf uterine cytosol in the same buffer.
G. A.
310
PUCA
rt
ui.
8
t
I
2
CVTOSOL RECEPTOR
E
CYTOSOl RECEPTOR CSK COMPLEX
,’
A
,’
,
/
i
/’ I
J ‘H E,AECEPTOR tOMPLEXEOUNO
!
[lo-%] 1 1
3H-ESTRADIOL
I
I
I
2
3
4
~K~E;RE~EPTGRCOMPLEX [W"M]
Fig. 4. Effect of increasing the concentration of [3H]-estradiotreceptor complex on the binding to erythrocyte cytoskeleton. The indicated concentrations of [‘H]-estradiolreceptor complex partially purified by sepharose-heparin chromatography was incubated with 0.1 ml of erythrocyte cytoskeieton for 2 h at 20% Insert: Results plotted according to Scatchard.
ecular forms of estradiol receptor are prepared and studied separately, the native partially purified 8 S form, the highly purified 8 S receptor and the aggregated (whether by ammonium sulfate or by heating for 30min at 30°C) forms are all abie to bind with varying degrees of efficiency to the cytoskeleton (Table 2). Partial, controiled proteolysis of the estradioi receptor by trypsin or by RTF causes a loss in the ability to bind to the cytoskeleton despite the fact that the binding of estradiol to these receptor forms is unaltered (Table 2). DISCUSSION
Erythrocyte
ghosts display binding
sites for the
ADDED,
Fig. 5. Affinity for estradioi of cytosoi and cytoskeletonbound receptor. Aiiquots (0.1 ml) of fresh uterine cytosoi were preincubated 2 h at 2O’C and then incubated for I6 h at 4°C with the indicated concentrations of [3H]-estradioi in the absence (A) and in the presence (A) of IOOO-fold excess unlabeled estradiol. Final volume was 0.5 ml. Erythrocyte cytoskeieton (3 ml: 15 mg protein) was incubated with 6 ml of the same cytosoi in a total volume of 1S ml of phosphate buffer 25 mM KC1 containing and 2.5 mM MgCi,. After 2 h at 2O”C the mixture was centrifuged and pellet washed twice with phosphate buffer. Final pellet was resuspended with 15 ml phosphate buffer. Aiiquots (OS ml) were incubated 16 h at 4X with the indicated concentrations of [‘HI-estradioi in the absence (0) and presence (0) of IOOO-foldexcess unlabeled estradiol. At the end of the incubation samples were centrifuged. washed three times and radioactivity bound to cytoskeieton measured. estradiol
receptor
only when a portion
of the mem-
brane proteins are removed with various extraction methods, when ghosts are reduced to vesicles or when many of the proteins and most of the lipid bilayer are removed with the nonionic detergent Triton X-100. These sites are very strongly associated with the cytoskeleton of the erythrocyte membrane and are unmasked by the various extraction procedures.
Table 2. Binding of various estradioi receptor forms to erythrocyte cytoskeietons d.p.m. Added 8S 4s (Ca*+-RTF) 4S (Trypsin) Aggregates (NH,SOL) Aggregates (temperature) Pure receptor
[ lo-‘M^
27,700 10.900 11,6UO 21,900 7.400 16,OcQ
d.p.m. Bound 15,ocO 947 42 8,500 4.300 15,975
Percent 66 8 0 38 58 100
The various estradioi receptor forms were prepared as described under Experimental Procedure Section. Aiiquots of 0.1 ml of cytoskeieton (0.4mg of protein) were incubated 2 h at 22°C with 6 x lo-’ M [‘HI-estradiol in the presence or in the absence of 10e6 M unlabeled estradioi with the indicated amounts of estradioi receptor forms in a total volume of 03 ml of phosphate buffer containing 2.5 mM M&i, and 2.5mM KCI.
Bound [‘HI-estradiol-receptor complex was assessed as described.
Estradiol receptor and erythrocyte cytoskeleton The temperature dependence of the estradiol receptor binding suggests the requirement for an “open” cytoskeletal organization. Since prior heating of the cytosol does not facilitate the subsequent receptor binding to cytoskeletons at low temperatures (Fig. l), the temperature is not required for a modification or activation of the receptor. The temperature might reflect the need for increased fluidity of the residual membrane components, allowing the access of the receptor to its binding sites. Ionic strength influences the receptor-cytoskeleton interaction which appears to be enhanced by Mg2+ in a quite selective manner. Apparently, other than KC1 and Mg2+ no other cytoplasmic factors are able to significantly modify the interaction which is still observed after extensive dialysis and partial (Fig. 4) or complete purification of the estradiol receptor (Table 2). Saturation analysis of the receptor-cytoskeleton interaction reveals the presence of a very limited number (less than 100) of high affinity (Kd = 10e9 M) binding sites per erythrocyte ghost. This excludes the possibility that one of the major protein component of the cytoskeleton (e.g. band 3, spectrin, actin, ankyrin. band 4.1) may be responsible for the binding. The receptor domain which interacts with the cytoskeletal structure is different from the domain containing the steroid binding site. In fact. partial hydrolysis of the receptor by the Ca” dependent RTF Cl93 or by mild tryptic digestion lead to a loss in its ability to bind to the cytoskeleton without altering the hormone binding properties. In conclusion. findings presented in this paper are consistent with the presence of specific binding sites for the estrogen receptors in the cytoskeletal matrix of the erythrocyte membrane. Similar systems may exist in target cells, which could in part explain data apparently contradictory with the general model of action of steroid hormones [l-7.24-29]. The water soluble nature of the estradiol receptor found in cytosol might be an artefact of tissue homogenization and preparation as recently demonstrated by Pietras and Szego[S, 61. and the membrane receptor described by Parikh et a[.[73 and by others Cl-33 might be that fraction of receptor which has not been released during the cell fractionation.
Ackno*,ledgementsThis work was supported by Progetto Finalizzato: Controllo della Crescita Neoplastica. by a grant from The Wellcome Research Laboratories, RTP. North Carolina and the National Institute of Health contract No. NOI-CB-64074.
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