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Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors
33
Molecular and Cellular Endocrinology, 53 (1987) 33-44 Elsevier Scientific Publishers Ireland. Ltd.
MCE 01706
Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors U. Gehring, K. Mugele, H. Arndt and W. Busch Institut
ftirBiologische Chemie der Unioersitiit, Im Neuenheimer Feld 501, 6900 Heidelberg, F.R.G. (Received 22 December 1986; accepted 9 April 1987)
Key work
Heteromeric receptor structure; Receptor activation;
Receptor dissociation;
Sodium molybdate
Summary Apparent molecular weights of wild-type and nt’ (‘increased nuclear transfer’) mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of M, 328000 and 298000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of M, 130 000 and 63 000 of the wild-type and mutant, respectively, were obtained at 300 mM KC1 and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nt’ receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nt’ receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nt’ receptors. We conclude that the large forms of wild-type and nt’ receptors are heteromeric and contain only one hormone-binding polypeptide per complex.
Introduction The molecular actions of glucocorticoids and other steroid hormones depend on specific receptors which function as intracellular mediators within responsive cells. Different molecular forms of such receptors have been detected in extracts of target cells depending largely on the conditions of extraction and analysis. The hormone-binding polypeptide of the wild-type glucocorticoid receptor of various species and cell types has a molecular weight of about 95 000 as revealed by affinity labelling and gel electrophoresis in the presence of Address for correspondence: U. Gehring, Institut fur Biologische Chemie der Universitat, Im Neuenheimer Feld 501, 6900 Heidelberg, F.R.G. 0303-7207/87/$03.50
sodium dodecyl sulfate (for reviews, see Gronemeyer and Govindan, 1986; Gehring, 1986). On the other hand, receptor forms of molecular weights as large as 320 000 to 350000 have been detected under conditions which carefully avoid denaturation and subunit dissociation (Holbrook et al., 1983; Norris and Kohler, 1983; Sherman et al., 1983; Stevens et al., 1983; Vedeckis, 1983, 1985; Sherman and Stevens, 1984). These differences in physicochemical properties bring up the question in which way the hormone-binding polypeptide is organized within the large receptor form. It has been postulated that the complexes of molecular weight > 300000 may either be homotetramers of steroid-binding subunits (Norris and Kohler, 1983; Raaka and Samuels, 1983; Sherman et al., 1983; Vedeckis, 1983, 1985; Sherman and
0 1987 Elsevier Scientific Publishers Ireland, Ltd.
34
Stevens, 1984), may contain two such hormonebinding polypeptides associated with two somehow related subunits (Sherman et al., 1983; Sherman and Stevens, 1984), or may consist of an intermediate number of hormone-binding subunits plus an unknown number of other macromolecules (Vedeckis, 1985). Other investigations point to a heteromeric receptor structure (Joab et al., 1984; Economidis and Rousseau, 1985; Gehring and Amdt, 1985; Okret et al., 1985) in which the steroid-binding polypeptide is associated with non-hormone-binding subunits of largely unknown function. We have been interested in comparing the biochemical properties of wild-type glucocorticoid receptors of mouse lymphoma cells with defective receptors unable to elicit biological responses within the cells that harbor them (for reviews, see Gehring, 1980a, 1986). One such receptor mutant type that arose in cells selected for glucocorticoid unresponsiveness elicits increased affinity for DNA and increased nuclear binding (‘nuclear transfer increased’: nt’) compared to wild-type (Yamamoto et al., 1976). This abnormal receptor is of particular interest given its truncated steroid-binding polypeptide of M, 40 000 (Nordeen et al., 1981; Dellweg et al., 1982; Gehring and Hotz, 1983; Northrop et al., 1985). In the present study we determined the hydrodynamic properties of wild-type and nt’ receptors under different salt conditions. The data suggest heteromeric structures for the large forms of both wild-type and nt’ mutant receptors containing only one hormonebinding polypeptide per complex. Increasing salt concentrations evoke subunit dissociation of these large receptor forms which can be prevented by adding sodium molybdate to the buffers. We also show that activation of wild-type and nt’ mutant receptors to DNA binding forms involves subunit dissociation.
with the nt’ mutant receptor were carried out with extracts of S49.1TB.4.55R cells; in some instances we also used the cell clone S49.1TB.4.143R and obtained identical results. Cells were grown at 37°C under 10% CO, in RPMI-1640 medium supplemented with 2.5 g glucose per liter, 4 mM glutamine, and 10% horse serum or 5% fetal calf serum. Cells were harvested and stored frozen as previously described (Gehring, 1980b). Cell extracts Frozen cell pellets were homogenized at 0” C with 20 mM iV-tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.8 at 20” C) containing 2 mM mercaptoethanol, 1 mM EDTA and 10% glycerol by use of a Teflon pestle tissue grinder, and centrifuged at 100000 X g for 1 h. The supernatant was filtered through a glass fibre filter (Whatman GF/F) and incubated ‘for 2-3 h at 0 o C with 30 mM [3H]triamcinolone acetonide (New England Nuclear, 1 TBq/mmol). Excess unbound hormone was removed prior to use of receptor-hormone complexes in experiments by incubating cell extracts for 10 min with 1.9 mg charcoal per ml. Radioactivity was assessed by liquid scintillation spectrometry at 40% efficiency. Protein concentrations were 8-12 mg per ml. Activation of receptor complexes was either by warming to 20°C for 30 min followed by chilling and the addition of 10 mM sodium molybdate or by treatment with 300 mM KC1 for 1 h in the cold followed by dilution to a final concentration of 20 mM KCl. If receptors were to be preserved in the non-activated state we added 10 mM sodium molybdate to cell extracts simultaneously with the steroid. For partial proteolysis, wild-type receptor preparations were incubated for 10 min in the cold with 10 pg/ml bovine a-chymotrypsin (Serva) followed by the addition of 100 pg/ml chymostatin (Sigma).
Materials and methods Cell lines and cell culture The S49.1 mouse lymphoma sublines S49.1G.3 (wild-type), S49.1TB.4.55R (nt’ type) and S49.1TB.4.143R (nt’ type) were those previously used (Gehring and Hotz, 1983). Most experiments
Filtration through DNA-cellulose DNA-cellulose columns of 1 ml (containing 0.8-1.0 mg bound calf thymus DNA) were equilibrated with 10 mM Tricine buffer, pH 7.8, containing 10 mM sodium molybdate. Receptor samples were applied and after 30 min incubation
35
unbound receptors were washed out with the above buffer. Control experiments showed that the presence of molybdate did not interfere with the binding of activated receptor complexes to DNA-cellulose.
TABLE
1
MOLECULAR
PARAMETERS
Marker protein
Sedimentation
(source)
coefficient
Thyroglobulin
DEAE-cellulose chromatography DEAE-cellulose columns of 2 ml (Whatman DE 52) were equilibrated with 10 mM Tricine buffer, pH 7.8, containing 20 mM KC1 and 10 mM sodium molybdate. Receptor samples were allowed to interact with the ion-exchange resin for 30 min. Columns were then rinsed with buffer and bound receptors were eluted with a linear gradient of 25 ml each of 20 mM and 450 mM KC1 in buffer containing 10 mM sodium molybdate. Fractions of 1.5 ml were collected and assayed for radioactivity and KC1 concentration by measuring the conductivity of diluted aliquots. Gel permeation chromatography Gel filtration on Sephacryl S-300 (Pharmacia) was carried out as previously described (Gehring and Arndt, 1985) in 20 mM potassium phosphate buffer (pH 7.4 at 20°C) containing 2 mM mercaptoethanol and 1 mM EDTA. Columns of 200-210 ml bed volume were used, l-2 ml of hormone-labelled receptor preparations were applied and the columns were developed at a flow rate of approximately 8 ml/h. Sodium molybdate (20 mM) was used in some experiments and KC1 was added in concentrations as indicated. In some experiments hormone-labelled cell extracts without prior charcoal treatment were applied. The void volume (V,) and the included volume (Vi) of gel columns were determined by use of Blue Dextran 2000 (Pharmacia) and [ 3H]valine (Amersham; 2 TBq,/ mmol), respectively. Distribution coefficients K, were computed from K, = (V, &)/Vi (Sherman, 1975) where V, is the elution volume of receptors or marker proteins. Stokes radii of receptors were obtained from plots of K, values vs. log of Stokes radii of marker proteins (Sherman, 1975). The molecular parameters of marker proteins are listed in Table 1. Since the Stokes radii were not directly available from the literature we calculated them (Sherman, 1975) from published values for diffusion coefficients or, as in
OF MARKER
P-Galactosidase
_ 15.93 b
68.5 b
11.29 c*d
52.3 d
(C)
(bovine liver) (A)
(rabbit muscle) Hemoglobin
a Edelhoch,
4.44 1960;
b Sund
d Sumner
Schachman, s Braunitzer
_
7.90 e
(H)
(human)
1976;
86.1 a
(G)
(E. coli)
Aldolase
Stokes radius (A)
(S)
(T)
(bovine thyroid)
Catalase
PROTEINS
1962;
and
f and
32.1 @ Weber,
GralCn,
1938;
’ Schumaker
and
et al., 1964;
1963;
’ Sund et al.,
e Stellwagen Schachman,
h Behlke and Wandt,
and 1957;
1973.
the case of hemoglobin, from molecular weight, sedimentation coefficient and partial specific volume. Thyroglobulin was used only as external marker. Sedimentation analysis Sedimentation in linear glycerol gradients was carried out as described (Gehring and Arndt, 1985). Sodium molybdate (20 mM) and KC1 at various concentrations were added in some experiments. Fractions of about 100 ~1 were collected from the bottom of the tubes. Sedimentation coefficients of receptors were obtained from the linear correlation between fraction number and sedimentation coefficient of marker proteins (Table 1). Molecular weights were calculated from Stokes radii and sedimentation coefficients (Sherman, 1975; Niu et al., 1981), assuming an average partial specific volume v for proteins of 0.732 ml/g (Niu et al., 1981; Sherman and Stevens, 1984). For estimating the shape of receptor complexes, prolate ellipsoids were assumed and their axial ratios were calculated from Stokes radii and molecular weights (Schachman, 1959; Niu et al., 1981). Results Stabilized high molecular weight receptor forms It has been known for several years that ad-
36
dition of sodium molybdate to buffer solutions stabilizes steroid hormone receptors (for reviews, see Dahmer et al., 1984; Housley et al., 1984). We made use of this stabilizing effect in order to compare wild-type and nt’ mutant glucocorticoid receptors. The hydrodynamic properties of these receptor types were determined by gel permeation chromatography and sedimentation analysis. With both analytical techniques we obtained sharp and symmetrical peaks suggesting that non-specific aggregation of receptors is negligible (Gehring and Arndt, 1985). Gel filtration experiments a,t low salt concentrations gave Stokes radii of 83 A and 72 A for wild-type and nt’ receptors, respectively (Table 2). Despite this significant difference both receptor types had the same sedimentation behaviour (Table 2). Stokes radii and sedimentation coefficients were used to compute the apparent molecular weights of these large receptor forms and M, values of 335000 and 291000 were obtained for wild-type and nt’ receptors, respectively. As shown in Table 2, the addition of KC1 in concentrations up to 300 mM to these molybdatestabilized receptors did not significantly change TABLE
their hydrodynamic properties while in the absence of molybdate KC1 caused dissociation of receptor complexes (see below). In order to find out whether molybdate is unique and specific in stabilizing high molecular weight receptor forms we also tested related transition metal ox&anions on wild-type receptors in the presence of 300 mM KCl. As shown in Fig. 1, tungstate at 20 mM had a somewhat similar effect, although in addition to the prominent peak of Stokes radius 820A there was dissociated receptor material at 62 A. Even at ,50 mM tungstate the large receptor form of 82 A was not completely protected against dissociation by 300 mM KC1 (data not shown). Interestingly, vanadate at similar concentrations did not stabilize the high molecular weight receptor form at all (Fig. 1). Effect of salt on receptors
At very low salt concentrations the physicochemical properties of wild-type and nt’ mutant receptors were quite similar whether or not sodium molybdate was included in the buffers. The gel filtration experiments of Fig. 2 with no added KC1 and molybdate produced major receptor peaks
2
MOLECULAR
PROPERTIES
Stokes radii and sedimentation Receptor
Conditions
OF RECEPTORS coefficients
were determined
of analysis
in 3-6 independent
experiments;
mean values f SD are given.
Stokes radius
Sedimentation
Molecular
(A)
coefficient
weight
(S)
Axial ratio
type
KC1 (mM)
Na,MoO,
Wild-type
0 150 300
20 20 20
83.6k2.8 82.3 + 1.9 19.5 +_2.6
9.5 + 0.3 9.1+ 0.6 9.lkO.3
335 000 316000 306 000
13 13 12
0 50 150 300 600
_ _ _ _
80.6k1.8 69.1 k 2.2 70.2 + 0.8 62.0 k 2.6 61.4i2.9
9.6 f 0.3 9.7kO.2 6.0+-0.1 4.9 f 0.3 5.OkO.2
328000 286 000 178 000 128 000 130000
12 9 14 13 13
0 150 300
20 20 20
12.5 i 2.5 70.5 + 0.4 69.0 + 0.4
9.5 * 0.5 9.5 + 0.4 9.0+0.1
291000 283 000 262000
10 9 9
0 50 150 300 600
_ _ _ _ _
71.9i1.9 70.6 + 1.2 40.7 * 2.0 38.4k1.5 40.0 + 2.6
9.8 + 0.3 10.2 f 0.4 5.1+0.1 3.9kO.6 3.8 + 0.1
298 000 304000 88000 63 000 64000
9 9 5 7 7
nt’ mutant
(mM)
0.1
02
03 Distribution
114
05
0.6
0.7
Coefficient
Fig. 1. Gel filtration of receptors in the presence of transition metal oxi-anions. Wild-type receptor complexes were chromatographed as described under Materials and Methods in the presence of 20 mM each of sodium molybdate (o), sodium tungstate (0), or sodium vanadate (0). Receptor preparations containing 15 mg protein and approximately lo6 dpm of bound [ ‘H]triamcinolone acetonide were applied to each co1 umn.
Stokes radii of 81 and 72 A for the wild-type and the nt’ mutant, respectively. With both receptor types we observed in addition to these main peaks some heterodisperse material of smaller size (Fig. 2) which probably arose by dissociation during the course of the chromatography and possibly also to some extent by proteolytic degradation. The sedimentation coefficients of the main components again were the same for both receptor types (Table 2) thus leading to apparent molecular weights of 328000 and 298000 for wild-type and nt’ receptors. Table 2 also summarizes the results of experiments in which KC1 at various concentrations was added to receptor preparations in the absence of molybdate. At 300 mM salt the receptors ap,peared to be fully dissociated into forms of 62 A and 39 A, respectively, with molecular weights of about 130000 and 63000 for the wild-type and nt’ mutant. Further increase of the KC1 concentration to 600 mM did not produce any further changes. At intermediate salt concentrations, however, some intermediate forms appeared (Table 2), most notably khe 70 A forms of the wild-type receptor and a 40 A form of the nt’ receptor with an intermediate sedimentation coefficient. These receptor forms
with
Dlstrlbutlon
Caefficienl
Fig. 2. Gel filtration of receptors in the presence or absence of salt. Wild-type (A) and nti mutant (B) receptor complexes were chromatogmphed as described under Materials and Methods without added salt (0) or in the presence of 300 mM KC1 (0). Receptor preparations containing 15 mg protein (A) or 10 mg protein (B), each corresponding to approximately lo6 dpm bound [ 3H]triamcinolone acetonide were applied to each column.
have apparent molecular weights of 178000286 000 and 88000. In the gel filtration experiments with 50 mM KC1 we again observed in addition to the major peaks at 70 A some additional heterodispezse receptor material which peaked at about 60 A in the case of the wild-type and at around 40 A with the mutant receptor. These results indicate that receptor dissociation caused by salt does not necessarily occur in a single step. However, it is difficult at present to unequivocally describe the molecular properties of
38
these intermediate receptor forms. With 20 mM KC1 we obtained the same gel filtration and sedimentation profiles as in the absence of added salt (cf. Fig. 2). In several experiments we first exposed receptor preparations to high salt concentrations and subsequently diluted them with low salt buffers which contained sodium molybdate. Analysis by gel filtration and sedimentation revealed in each case the dissociated form of the wild-type and nt’ mutant receptors (data not shown). This further corroborates the conclusion that molybdate by itself does not produce the high molecular weight receptor forms but only inhibits their dissociation. Activation and dissociation of receptors
Steroid hormone receptor complexes in target cell extracts which had been prepared at low temperatures and in buffers of low ionic strength need to be activated in order to elicit DNA binding ability (for reviews, see Milgrom, 1981; Grody et al., 1982; Schmidt and Litwack, 1982). Activation can be brought about, for example, by increasing the salt concentration or by warming receptor preparations. Fig. 3 shows an experiment in which
A
I
z
8
i
20-
a
we achieved partial activation of wild-type receptors by warming to 20°C for 30 min. The sample was then split into two aliquots. One part was directly analyzed by gel filtration and sedimentation in a glycerol gradient and two distinct peaks were detected cor;esponding to the Stokes radii of about 80 and 60 A and sedimentation coefficients of 9.5 and 4-5 S, respectively (closed symbols in Fig. 3). The other aliquot was first run through DNA-cellulose in order to adsorb the activated receptor complexes and subsequently analyzed in the same way. We now observed Oonly the high molecular weight form of about 80 A and 9.5 S. In other experiments we first exposed the wild-type receptor to 300 mM KCI, diluted the sample to a final concentration of 20 mM KC1 and carried out a similar analysis in the presence of 20 mM KCl. In this way we only observed the receptor peak of about 62 A which could be fully removed by prior filtration through DNA-cellulose (data not shown). Similar experiments were also carried out with nt’ mutant receptors. The complex of 72 A was unable to bind to DNA and was consequently not adsorbed by DNA-cellulose. Partially activated nt’ receptor preparations obtained by warming to
E.
P
155
x k D ,IO-
A
\
0:2 Dlstrlbutlon
0.3
0.L
Coefflclent
Fraction
Number
Fig. 3. Gel filtration and sedimentation analysis of partially activated receptors. Wild-type receptor complexes were activated at 20 “C and chromatographed on Sephacryl S-300 (A) or sedimented in glycerol gradients (B) as described under Materials and Methods. Samples were analyzed either without further treatment (0) or after passing through DNA-cellulose (0). Approximately 400000 dpm and 50000 dpm of receptor-bound [3H]triamcinolone acetonide were used for each of the gel filtration and sedimentation experiments, respectively. In the case of the material previously filtered through DNA-cellulose only an aliquot could be applied to the glycerol gradient; the results were corrected accordingly.
A
B
40.
J
-400 x x x
30-
/
-300
z
20-
-200
lo-
-100
10 Fraction
r
3 2
20
30
40
Number
250
,, -400
200-
,300 150T
t ,200 ; loo-
Y
2,100
50-
k
10
20
30
40
Fraction Number Fig. 4. DEAE-cellulose chromatography of receptors. Wild-type (A,B) and nt’ mutant (C,D) receptor complexes were chromatographed as described under Materials and Methods either in the non-activated state (A,C) or after activation at 20 o C (B,D). Activated samples were either loaded directly onto DEAE-cellulose (closed symbols in B and D) or after passing through DNA-cellulose (open symbols). Approximately 250000 dpm of receptor-bound [3H]triamcinolone acetonide were used for each experiment. Small crosses represent KC1 concentrations.
40
20 o C gave rise to gel filtration and sedimentation profiles with two peaks. Again the receptor form of lower molecular weight was eliminated by passing the pr~p~ation through DNA-cellulose (data not shown). Pretreatment of nt” receptors with 3000mM KC1 followed by dilution produced the 40 A form which was fully retained by DNA-cellulose. Taken together, these data show that the activated wild-type and nt’ receptors are the dissociated forms with molecular weights of approximately 130000 and 63000, respectively. By contrast, the high molecular weight complexes with apparent molecular weights of 330 000 and 290 Ooo are unable to bind to DNA. It has been shown that activated and nonactivated glucocorticoid receptors from rat liver can be separated by chromatography on DEAEcellulose or DEAE-Sephadex (Parchman and Litwack, 1977; Sakaue and Thompson, 1977). The non-activated receptor complex is more negatively charged and consequently requires higher salt concentrations for elution from DEAE-cellulose than the activated receptor. We applied this chromatographic technique to the mouse lymphoma cell receptor system in order to further distinguish between activated and non-activated wild-type and nt’ mutant receptors. Fig. 4 depicts representative experiments. Non-activated wild-type (Fig. 4A) and nt’ receptors (Fig. 4C) eluted with the same high salt concentration, i.e. 200 t 10 mM KCl. Upon partial activation by warming to 20 * C we obtained profiles with two peaks. Non-activated receptor complexes again eluted with 200 mM salt; the activated wild-type receptor eluted with 45-50 mM KC1 (Fig. 4B) while the activated nt’ receptor was contained in the flow-through of the column, i.e. eluted with I 20 mM KC1 (Fig. 4D). Which peaks correspond to the activated receptor species became very clear when receptor aliquots were again filtered through DNA-cellulose prior to their chromatography on DEAE-cellulose thus resulting in the removal of the activated receptors (open symbols in Fig. 4B and D). The activated wild-type and nt’ receptors thus differ not only in molecular weights but also in their ionic properties. In a previous study we had been unable to detect this difference in chromato-
graphic behaviour on DEAE-cellulose (Dellweg et al., 1982) because buffers of pH 8.5 had been used rather than pH 7.8 as in the present investigation. Partial ~roteo~~i~ of wind-type receptors
Mild treatment of wild-type receptors with chymotrypsin and other proteases has previously been shown to generate receptor species which are very similar to the nt’ mutant receptor with respect to increased affinity for DNA and the size of the ho~one-binding polypeptide as revealed by photoaffi~ty labelling and SDS-gel electrophoresis (Dellweg et al., 1982; Gehring and Hotz, 1983; Gehring, 1986). It was therefore of interest to determine the physicoche~c~ properties of partially proteolyzed receptors under bob-denatu~ng conditions. The molybdat~stabi~zed high molecular weight form of the wild-type receptor treated with a;;chymotrypsin had a Stokes radius of 68.9 t 2.7 A (n = 4) and a sedimentation coefficient of 10.0 & 0.1 S (n = 6) corresponding to an apparent molecular weight of 291000. Treatment with 300 mM KC1 in the absence of molybdate produced the dissociated form of 40.7 A and 3.8 S with a molecular weight of 65000. These data further emphasize the similarity with the nt” mut~t receptor (cf. Table 2). Activated and non-activated forms of the chymotrypsin-treated wild-type receptor could again be discriminated by chromatography on DEAE-cellulose. The profiles obtained were indistinguishable from those of Fig. 4C and D. The non-activated receptor again bound to the column and was eluted with 200 mM KC1 while the activated form, resembling the nt’ mutant, was not retained by DEAE-cellulose and appeared in the flow-through (data not shown). In some experiments with untreated wild-type receptors (cf. Fig. 4B) we also noted a small amount of receptor material in the flow-through; this probably arose by endogenous proteolysis. Discussion In the present co~~ication we give a detailed account of the molecular properties of the large forms of wild-type and nt’ mutant receptors from murine lymphoma cells. We obtained apparent
41
molecular weights of 330 000 and 295 000 for these receptor types, respectively, under conditions which carefully avoid dissociation and denaturation. The difference in molecular mass of about 35000 between wild-type and mutant reflects the size difference of the hormone-binding polypeptides of these receptors. Molecular weights of 94000 and 40000 had previously been obtained under denaturing conditions for the wild-type and nt’ receptors, i.e. a difference of about 55000 (Gehring, 1986). Considering that fundamentally different analytical techniques have been used for investigating native and denatured receptors we regard wild-type and nt’ mutant receptors different by the same amount of molecular mass independent of the conditions of investigation. The results therefore suggest that the large receptor forms of the wild-type and the nt’ mutant are heteromers and contain only one steroid-binding subunit per large complex. We expect the associated macromolecular components to be the same in the wild-type and in the mutant. This view is strengthened by experiments in which we treated wild-type receptors with chymot~psin and obtained molecular parameters which are indistingui~able from those of nt’ receptors. It is interesting to note that another mutant receptor devoid of hormone-binding ability likewise exist: in a high molecular weight form of about 81 A (Westphal et al., 1984), i.e. the molecular size resembles that of the wild-type receptor. Using an immunochemical approach Okret et al. (1985) also suggest a heteromeric structure for the rat liver glucocorticoid receptor. Adding salt to receptor preparations results in progressive dissociation of the large complexes (Table 2). The wild-type and nt’ receptor species of M, 130 000 and 63 000, respectively, which we detected at 300600 mM KC1 are presumably fully dissociated. On the other hand these receptors have polypeptide molecular weights of 94000 and 40000 (see above). These differences in molecular weights appear to be due again to different analytical procedures, as discussed above. Alternatively, but perhaps less likely, the hormone-binding polypeptides are associated under non-denaturing conditions with some as yet u~dentified component(s}.
The dissociated wild-type and nt’ mutant receptors of M; 130000 and 63 000 are activated forms which are able to bind to DNA. They are less negatively charged than the non-activated, non-dissociated receptors and consequently stick less tightly to DEAE-cellulose. The fact that the activated wild-type and nt’ receptors behave differently on DEAE-cellulose clearly shows that the amino terminal half of the hormone-binding polypeptide that is missing from the nt’ mutant (Gehring, 1986), does contribute significantly to the ionic properties of the activated receptors. With recent cloning of receptor-specific cDNAs of full length the amino acid sequences of the human, mouse and rat glucocorticoid receptors are now known (Hollenberg et al., 1985; Danielsen et al., 1986; Miesfeld et al., 1986); these polypeptides consist of 777, 783 and 795 amino acids, respectively, and are quite homologous in sequence. The most striking difference is a stretch of 8 and 19 glutamine residues within the amino terminal half of the mouse and rat receptors which is absent from the human. The biological significance of this sequence, if any, is not known. The domains for hormone binding and for interacting with DNA are almost identical. Similar to the other steroid hormone receptors, the DNA binding domain of about 70 amino acids is located in the middle of the polypeptide chain and the hormone binding domain occupies the carboxy terminal part. A sequence comparison of the DNA binding domains of the known steroid and thyroid hormone receptors shows complete conservation of 9 cysteine residues as well as several basic and hydrophobic amino acids. The DNA binding domain is thought to fold in such a way that two finger-like structures are formed which resemble those detected in the Xenopus transcription factor IIIA (Miller et al., 1985) and several other DNA binding proteins. This structure may be stabilized by zinc ions which are tetrahedrally coordinated with cysteine residues. The sequence analysis of two receptor mutants of mouse lymphoma cells led to the identification of two amino acids in the hormone binding domain which are directly or indirectly involved in steroid binding: glutamic acid at position 546 and tyrosine at 770 (Danielsen et al., 1986). In addition, cysteine 644 (corre-
42
sponding to position 656 in the rat receptor) was found to react with the affinity label dexamethasane 21-mesylate (Simons et al., 1987). This shows that a fairly long part of the polypeptide chain consisting of at least 225 residues is involved in forming the hormone binding domain. As to the nt’ mutant receptor its molecular origin is still not yet clear. What is known is that this amino terminally truncated polypeptide is synthesized from 5’ truncated mRNAs (Miesfeld et al., 1984) which probably arise through abnormal splicing. Very recent experiments show sequence divergence on the cDNA level upstream of amino acid position 404 (in the murine wild-type receptor). Synthesis of the nt’ polypeptide may either start with methionine 406 (position of the wild-type) or from an upstream AUG originating from intronic sequences (Miesfeld et al., 1987). We observed that non-activated wild-type and nt’ receptors bind quite tightly to DEAE-cellulose and elute under identical conditions. Thus, the macromolecular components which are associated with the hormone-binding polypeptides appear to be highly charged. This brings up the question as to the identity of these associated macromolecules. A non-hormone-binding polypeptide of M, 90 000 has been detected which is common to the large forms of various steroid hormone receptors (Joab et al., 1984; Housley et al., 1985; Riehl et al., 1985; Mendel et al., 1986) which has been identified as heat-shock protein hsp 90 (Catelli et al., 1985; Sanchez et al., 1985). The charge properties of the high molecular weight receptor complexes appear to be largely determined by hsp 90 which is an acidic protein of pI 5.2 and which elutes from DEAE-cellulose or DEAE-Sephadex with about 200 mM salt (Welch and Feramisco, 1982; Lai et al., 1984). The function of hsp 90 in relation to steroid hormone receptors, however, remains unknown. There is also convincing evidence for RNA being associated with large-sized glucocorticoid receptors (Economidis and Rousseau, 1985; KovaCiE-Milivojevic et al., 1985; Webb and Litwack, 1986). This RNA can be covalently cross-linked to the hormone-binding polypeptide and is of low molecular weight similar to tRNA. Since RNAs are much more dense than proteins
they have significantly lower partial specific volumes (in the order of 0.50 ml/g). Therefore, the value for v (0.732 ml/g) assumed in the present study for large receptor complexes is probably too high; it had previously also been used in a series of other investigations (Niu et al., 1981; Sherman et al., 1983; Stevens et al., 1983; Vedeckis, 1983,1985; Sherman and Stevens, 1984). Supposing an RNA content of roughly 10% we would expect v values of about 0.715 ml/g resulting in molecular weights for the undissociated wild-type and nt’ receptors of about 310 OO!, and 280000, respectively, as computed from 81 A, 9.6 S and 72 A, 9.8 S (Table 2). These sizes fit quite well to what one would expect for complexes consisting of one hormone-binding polypeptide, two molecules of hsp 90 and one molecule of tRNA. It needs to be emphasized that a receptor constitution of this type is but a hypothetical structure at present. Unequivocal proof for the molecular composition of the high molecular weight receptors will have to come from reconstitution experiments. Such experiments are now in progress. The size properties of the non-activated large steroid receptor forms reported in the literature are remarkably similar especially when molybdate was used as a stabilizing agent (see, for example, Sherman and Stevens, 1984; Vedeckis, 1985). This is certainly not so for the various dissociated receptor forms and we find it difficult to compare our results with some of the published data. In particular, we have not been able to obtain evidence for an intermediate wild-type receptor form of 83 A and 5.0-5.2 S (M, 176000) as has been described (Vedeckis, 1983; Reker et al., 1985). Interestingly, this partially dissociated receptor species was obtained in the presence of 20 mM molybdate and 300 mM KCl, i.e. under conditions which in our hands do not result in dissociation (cf. Table 2). On the whole our data agree well with those of Stevens et al. (1983) who used wildreceptors of the mouse type and nt’ mutant lymphoma strain F1798. They reported Stokes radii of 81 and 70 A for the molybdate-stabilized wild-type and nt’ receptors, respectively, and 60 and 28 A for the salt-treated receptors. Even though lymphoid cells are generally believed to be poor in proteolytic activities the 28 A form of the
43
P1798 nt’ receptor is likely to be a partially pro-
teolyzed species. In fact, we observed in several experiments S49.1 n,t’ receptors with a Stokes radius of about 30 A, especially under low salt conditions and following a 20 o C treatment. Dissociated nt’ receptors appear to be particularly susceptible to endogenous proteases. Acknowledgement This work was supported by grants from the Deutsche Forschungsgemeinschaft. References Beblke, J. and Wand& I. (1973) Acta Biol. Med. Ger. 31, 383-388. Braunitzer, G., Hilse, K., Rudloff, V. and Hilschmarm, N. (1964) Adv. Protein Chem. 19, l-65. Catelh, M.G., Binart, N., Jung-Testas, I., Renoir, J.M., Baulieu, E.E., Feramisco, J.R. and Welch, W.J. (1985) EMBO J. 4, 3131-3135. Dabmer, M.K., Housley, P.R. and Pratt, W.B. (1984) Annu. Rev. Pbysiol. 46, 67-81. Danielsen, M., Northrop, J.P. and Ringold, GM. (1986) EMBO J. 5,2513-2522. Dellweg, H.-G., Hotz, A., Mugele, R. and Gebring, U. (1982) EMBG J. 1, 285-289. Economidis, I.V. and Rousseau, G.G. (1985) FEBS L&t. 181, 47-52. Edelhoch, H. (1960) J. Biol. Chem. 235,1326-1334. Gehring, U. (1980a) In: Biochemical Actions of Hormones, Vol. 7, Ed.: G. Litwack (Academic Press, New York) pp. 205-232. G&ring, U. (1980b) Mol. Cell. Endocrinol. 20, 261-274. Gehring, U. (1986) Mol. Cell. Endocrinol. 48, 89-96. Gebring, U. and Arndt, H. (1985) FEBS Lett. 179, 138-142. G&ring, U. and Hotz, A. (1983) Biochemistry 22, 4013-4018. Grady, W.W., Schrader, W.T. and O’Malley, B.W. (1982) Endocr. Rev. 3,141-163. Gro~emeyer, H. and Govindan, M.V. (1986) Mol. Cell. Endocrinol. 46, 1-19. Holbrook, N.J., Bodwell, J.E., Jeffries, M. and Munck, A. (1983) 3. Biol. Chem. 258, 6477-6485. Hollenberg, SM., We~berger, C., Ong, ES., Cerelli, G., Ore, A., Lebo, R., Thompson, E.B., Rosenfeld, M.G. and Evans, R.M. (1985) Nature 318, 635-641. Housley, P.R., Grippo, J.F., Dahmer, M.K. and Pratt, W.B. (1984) In: Biochemical Action of Hormones, Vol. 11, Ed.: G. Litwack (Academic Press, New York} pp. 347-376. Hot&y, P.R., Sanchez, E.R., Westphal, H.M., Beato, M. and Pratt, W.B. (1985) J. Biol. Cbem. 260, 13810-13817. Joab, I., Radanyi, C., Renoir, M., Buchou, T., Catelli, M.-G., Binart, N., Mester, J. and Baulieu, E.E. (1984) Nature 308, 850-853.
Kova~i~-Milivoje~~, B., LaPointe, MC., Reker, C.E. and Vedeckis, W.V. (1985) Bi~he~st~ 24, 7357-7366. Lai, B.-T., Chin, N.W., Stanek, A.E., Keh, W. and Lanks, K.W. (1984) Mol. Cell. Biol. 4, 2802-2810. Mendel, D.B., BodwelI, J.E., Gametchu, B., Harrison, R.W. and Munck, A. (1986) J. Biol. Chem. 261,3758-3763. Miesfeld, R., Okret, S., Wikstrijm, A.-C., Wrange, (i., Gustafsson, J.-A, and Yamamoto, K.R. (1984) Nature 312,779-781. Miesfeld, R., Rusconi, S., Godowski, P.J., Maler, B.A., Okret, S., Wikstrom, A.-C., Gustafsson, J.-A. and Yamamoto, K.R. (1986) Cell 46, 389-399. Miesfeld, R., Sakai, D., Inoue, A., Schena, M., Godowski, P.J. and Yamamoto, K.R. (1987) In: Steroid Hormone Action, UCLA Symposium on Molecular and Cellular Biology, Ed.: G.M. Ringold (Alan R. Liss, New York} in press. Milgrom, E. (1981) In: Biodbemical Actions of Hormones, Vol. 8, Ed.: G. Litwack (Academic Press, New York) pp. 465-492. Miller, J., McLachlan, A.D. and I&g, A. (1985) EMBG J. 4, 1609-1614. Niu, E.-M., Neal, R.M., Pierce, V.K. and Sherman, M.R. (1981) J. Steroid Biochem. 15, l-10. Nordeen, SK., Lan, N.C., Showers, M.O. and Baxter, J.D. (1981) .I. Biol. Chem. 256,10503-10508. Norris, J.S. and Kohler, P.0, (1983) J. Biol. Chem. 258, 2350-2356. Northrop, J.P., Gametchu, B., Harrison, R.W. and Ringoid, G.M. (1985) J. Biol. Chem. 260,6398-6403. C&ret, S., W~str~m, A.-C. and Gustafsson, J.-A. (1985) Biochemistry 24, 6581-6586. Par&man, L.C. and Litwack, G. (1977) Arch. Biocbem. Biophys. 183,374-382. Raaka, B. and Samuels, H.H. (1983) J. Biol. Chem. 258, 417-425. Reker, C.E., KovariE-MilivojeviC, B., Eastman-Reks, S.B. and Vedeckis, W.V. (1985) Biochemistry 24, 196-204. Riebl, R.M., Sullivan, W.P., Vroman, B.T., Bauer, V.J., Pearson, G.R. and Toft, D.O. (1985) Biochemistry 24, 6586-6591. Sakaue, Y. and Thompson, E.B. (1977) B&hem. Biophys. Res. Commun. 77,533-541. Sanchez, E.R., Toft, D.O., Schlesinger, M.J. and Pratt, W.B. (1985) J. Biol. Chem. 260,12398-12401. Schachman, H.K. (1959) Ultracent~fugation in ~~~hernist~ (Academic Press, New York). Schmidt, T.J. and Litwack, G. (1982) Physiol. Rev. 62, 1131-1192. Schumaker, V.N. and Schachman, H.K. (1957) B&him. Biophys. Acta 23, 628-639. Sherman, M.R. (1975) Methods Enzymol. 36,211-234. Sherman, M.R. and Stevens, J. (1984) Annu. Rev. Physiol. 46, 83-105. Sherman, M.R., Moran, MC., Tuazon, F.B. and Stevens, Y.-W. (1983) J. Biol. Chem. 258, 10366-10377. Simons, S.S., Pumphrey, J.G., Rudikoff, S. and Eisen, H.J. (1987) J. Cell. Biocbem. Suppl. llA, 111. Stellwagen, E. and Schac~~, H.K. (1962) Bi~hemis~ 1, 1056-1069.
44 Stevens, J., Stevens, Y.-W. and Haubenstock, H. (1983) In: Biochemical Actions of Hormones, Vol. 10, Ed.: G. Litwack (Academic Press, New York) pp. 383-446. Sumner, J.B. and GralCn, N. (1938) J. Biol. Chem. 125, 33-36. Sund, H. and Weber, K. (1963) B&hem. Z. 337,24-34. Sund, H., Weber, K. and Molbert, E. (1967) Eur. J. Biochem. 1, 400-410. Vedeckis, W.V. (1983) Biochemistry 22, 1983-1989. Vedeckis, W.V. (1985) In: Hormonally Responsive Tumors, Ed.: V.P. Hollander (Academic Press, New York) pp. 3-61.
Webb, M.L. and Litwack, G. (1986) In: Biochemical Actions of Hormones, Vol. 13, Ed.: G. Litwack (Academic Press, New York) pp. 379-402. Welch, W.J. and Feramisco, J.R. (1982) J. Biol. Chem. 257, 14949-14959. Westphal, H.M., Mugele, K., Beato, M. and Gehring, U. (1984) EMBO J. 3, 1493-1498. Yamamoto, K.R., Gehring, U., Stampfer, M.R. and Sibley, C.H. (1976) Recent Prog. Horm. Res. 32, 2-32.
Molecular and Cellular Endocrinology, 53 (1987) 33-44 Elsevier Scientific Publishers Ireland. Ltd.
MCE 01706
Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors U. Gehring, K. Mugele, H. Arndt and W. Busch Institut
ftirBiologische Chemie der Unioersitiit, Im Neuenheimer Feld 501, 6900 Heidelberg, F.R.G. (Received 22 December 1986; accepted 9 April 1987)
Key work
Heteromeric receptor structure; Receptor activation;
Receptor dissociation;
Sodium molybdate
Summary Apparent molecular weights of wild-type and nt’ (‘increased nuclear transfer’) mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of M, 328000 and 298000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of M, 130 000 and 63 000 of the wild-type and mutant, respectively, were obtained at 300 mM KC1 and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nt’ receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nt’ receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nt’ receptors. We conclude that the large forms of wild-type and nt’ receptors are heteromeric and contain only one hormone-binding polypeptide per complex.
Introduction The molecular actions of glucocorticoids and other steroid hormones depend on specific receptors which function as intracellular mediators within responsive cells. Different molecular forms of such receptors have been detected in extracts of target cells depending largely on the conditions of extraction and analysis. The hormone-binding polypeptide of the wild-type glucocorticoid receptor of various species and cell types has a molecular weight of about 95 000 as revealed by affinity labelling and gel electrophoresis in the presence of Address for correspondence: U. Gehring, Institut fur Biologische Chemie der Universitat, Im Neuenheimer Feld 501, 6900 Heidelberg, F.R.G. 0303-7207/87/$03.50
sodium dodecyl sulfate (for reviews, see Gronemeyer and Govindan, 1986; Gehring, 1986). On the other hand, receptor forms of molecular weights as large as 320 000 to 350000 have been detected under conditions which carefully avoid denaturation and subunit dissociation (Holbrook et al., 1983; Norris and Kohler, 1983; Sherman et al., 1983; Stevens et al., 1983; Vedeckis, 1983, 1985; Sherman and Stevens, 1984). These differences in physicochemical properties bring up the question in which way the hormone-binding polypeptide is organized within the large receptor form. It has been postulated that the complexes of molecular weight > 300000 may either be homotetramers of steroid-binding subunits (Norris and Kohler, 1983; Raaka and Samuels, 1983; Sherman et al., 1983; Vedeckis, 1983, 1985; Sherman and
0 1987 Elsevier Scientific Publishers Ireland, Ltd.
34
Stevens, 1984), may contain two such hormonebinding polypeptides associated with two somehow related subunits (Sherman et al., 1983; Sherman and Stevens, 1984), or may consist of an intermediate number of hormone-binding subunits plus an unknown number of other macromolecules (Vedeckis, 1985). Other investigations point to a heteromeric receptor structure (Joab et al., 1984; Economidis and Rousseau, 1985; Gehring and Amdt, 1985; Okret et al., 1985) in which the steroid-binding polypeptide is associated with non-hormone-binding subunits of largely unknown function. We have been interested in comparing the biochemical properties of wild-type glucocorticoid receptors of mouse lymphoma cells with defective receptors unable to elicit biological responses within the cells that harbor them (for reviews, see Gehring, 1980a, 1986). One such receptor mutant type that arose in cells selected for glucocorticoid unresponsiveness elicits increased affinity for DNA and increased nuclear binding (‘nuclear transfer increased’: nt’) compared to wild-type (Yamamoto et al., 1976). This abnormal receptor is of particular interest given its truncated steroid-binding polypeptide of M, 40 000 (Nordeen et al., 1981; Dellweg et al., 1982; Gehring and Hotz, 1983; Northrop et al., 1985). In the present study we determined the hydrodynamic properties of wild-type and nt’ receptors under different salt conditions. The data suggest heteromeric structures for the large forms of both wild-type and nt’ mutant receptors containing only one hormonebinding polypeptide per complex. Increasing salt concentrations evoke subunit dissociation of these large receptor forms which can be prevented by adding sodium molybdate to the buffers. We also show that activation of wild-type and nt’ mutant receptors to DNA binding forms involves subunit dissociation.
with the nt’ mutant receptor were carried out with extracts of S49.1TB.4.55R cells; in some instances we also used the cell clone S49.1TB.4.143R and obtained identical results. Cells were grown at 37°C under 10% CO, in RPMI-1640 medium supplemented with 2.5 g glucose per liter, 4 mM glutamine, and 10% horse serum or 5% fetal calf serum. Cells were harvested and stored frozen as previously described (Gehring, 1980b). Cell extracts Frozen cell pellets were homogenized at 0” C with 20 mM iV-tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.8 at 20” C) containing 2 mM mercaptoethanol, 1 mM EDTA and 10% glycerol by use of a Teflon pestle tissue grinder, and centrifuged at 100000 X g for 1 h. The supernatant was filtered through a glass fibre filter (Whatman GF/F) and incubated ‘for 2-3 h at 0 o C with 30 mM [3H]triamcinolone acetonide (New England Nuclear, 1 TBq/mmol). Excess unbound hormone was removed prior to use of receptor-hormone complexes in experiments by incubating cell extracts for 10 min with 1.9 mg charcoal per ml. Radioactivity was assessed by liquid scintillation spectrometry at 40% efficiency. Protein concentrations were 8-12 mg per ml. Activation of receptor complexes was either by warming to 20°C for 30 min followed by chilling and the addition of 10 mM sodium molybdate or by treatment with 300 mM KC1 for 1 h in the cold followed by dilution to a final concentration of 20 mM KCl. If receptors were to be preserved in the non-activated state we added 10 mM sodium molybdate to cell extracts simultaneously with the steroid. For partial proteolysis, wild-type receptor preparations were incubated for 10 min in the cold with 10 pg/ml bovine a-chymotrypsin (Serva) followed by the addition of 100 pg/ml chymostatin (Sigma).
Materials and methods Cell lines and cell culture The S49.1 mouse lymphoma sublines S49.1G.3 (wild-type), S49.1TB.4.55R (nt’ type) and S49.1TB.4.143R (nt’ type) were those previously used (Gehring and Hotz, 1983). Most experiments
Filtration through DNA-cellulose DNA-cellulose columns of 1 ml (containing 0.8-1.0 mg bound calf thymus DNA) were equilibrated with 10 mM Tricine buffer, pH 7.8, containing 10 mM sodium molybdate. Receptor samples were applied and after 30 min incubation
35
unbound receptors were washed out with the above buffer. Control experiments showed that the presence of molybdate did not interfere with the binding of activated receptor complexes to DNA-cellulose.
TABLE
1
MOLECULAR
PARAMETERS
Marker protein
Sedimentation
(source)
coefficient
Thyroglobulin
DEAE-cellulose chromatography DEAE-cellulose columns of 2 ml (Whatman DE 52) were equilibrated with 10 mM Tricine buffer, pH 7.8, containing 20 mM KC1 and 10 mM sodium molybdate. Receptor samples were allowed to interact with the ion-exchange resin for 30 min. Columns were then rinsed with buffer and bound receptors were eluted with a linear gradient of 25 ml each of 20 mM and 450 mM KC1 in buffer containing 10 mM sodium molybdate. Fractions of 1.5 ml were collected and assayed for radioactivity and KC1 concentration by measuring the conductivity of diluted aliquots. Gel permeation chromatography Gel filtration on Sephacryl S-300 (Pharmacia) was carried out as previously described (Gehring and Arndt, 1985) in 20 mM potassium phosphate buffer (pH 7.4 at 20°C) containing 2 mM mercaptoethanol and 1 mM EDTA. Columns of 200-210 ml bed volume were used, l-2 ml of hormone-labelled receptor preparations were applied and the columns were developed at a flow rate of approximately 8 ml/h. Sodium molybdate (20 mM) was used in some experiments and KC1 was added in concentrations as indicated. In some experiments hormone-labelled cell extracts without prior charcoal treatment were applied. The void volume (V,) and the included volume (Vi) of gel columns were determined by use of Blue Dextran 2000 (Pharmacia) and [ 3H]valine (Amersham; 2 TBq,/ mmol), respectively. Distribution coefficients K, were computed from K, = (V, &)/Vi (Sherman, 1975) where V, is the elution volume of receptors or marker proteins. Stokes radii of receptors were obtained from plots of K, values vs. log of Stokes radii of marker proteins (Sherman, 1975). The molecular parameters of marker proteins are listed in Table 1. Since the Stokes radii were not directly available from the literature we calculated them (Sherman, 1975) from published values for diffusion coefficients or, as in
OF MARKER
P-Galactosidase
_ 15.93 b
68.5 b
11.29 c*d
52.3 d
(C)
(bovine liver) (A)
(rabbit muscle) Hemoglobin
a Edelhoch,
4.44 1960;
b Sund
d Sumner
Schachman, s Braunitzer
_
7.90 e
(H)
(human)
1976;
86.1 a
(G)
(E. coli)
Aldolase
Stokes radius (A)
(S)
(T)
(bovine thyroid)
Catalase
PROTEINS
1962;
and
f and
32.1 @ Weber,
GralCn,
1938;
’ Schumaker
and
et al., 1964;
1963;
’ Sund et al.,
e Stellwagen Schachman,
h Behlke and Wandt,
and 1957;
1973.
the case of hemoglobin, from molecular weight, sedimentation coefficient and partial specific volume. Thyroglobulin was used only as external marker. Sedimentation analysis Sedimentation in linear glycerol gradients was carried out as described (Gehring and Arndt, 1985). Sodium molybdate (20 mM) and KC1 at various concentrations were added in some experiments. Fractions of about 100 ~1 were collected from the bottom of the tubes. Sedimentation coefficients of receptors were obtained from the linear correlation between fraction number and sedimentation coefficient of marker proteins (Table 1). Molecular weights were calculated from Stokes radii and sedimentation coefficients (Sherman, 1975; Niu et al., 1981), assuming an average partial specific volume v for proteins of 0.732 ml/g (Niu et al., 1981; Sherman and Stevens, 1984). For estimating the shape of receptor complexes, prolate ellipsoids were assumed and their axial ratios were calculated from Stokes radii and molecular weights (Schachman, 1959; Niu et al., 1981). Results Stabilized high molecular weight receptor forms It has been known for several years that ad-
36
dition of sodium molybdate to buffer solutions stabilizes steroid hormone receptors (for reviews, see Dahmer et al., 1984; Housley et al., 1984). We made use of this stabilizing effect in order to compare wild-type and nt’ mutant glucocorticoid receptors. The hydrodynamic properties of these receptor types were determined by gel permeation chromatography and sedimentation analysis. With both analytical techniques we obtained sharp and symmetrical peaks suggesting that non-specific aggregation of receptors is negligible (Gehring and Arndt, 1985). Gel filtration experiments a,t low salt concentrations gave Stokes radii of 83 A and 72 A for wild-type and nt’ receptors, respectively (Table 2). Despite this significant difference both receptor types had the same sedimentation behaviour (Table 2). Stokes radii and sedimentation coefficients were used to compute the apparent molecular weights of these large receptor forms and M, values of 335000 and 291000 were obtained for wild-type and nt’ receptors, respectively. As shown in Table 2, the addition of KC1 in concentrations up to 300 mM to these molybdatestabilized receptors did not significantly change TABLE
their hydrodynamic properties while in the absence of molybdate KC1 caused dissociation of receptor complexes (see below). In order to find out whether molybdate is unique and specific in stabilizing high molecular weight receptor forms we also tested related transition metal ox&anions on wild-type receptors in the presence of 300 mM KCl. As shown in Fig. 1, tungstate at 20 mM had a somewhat similar effect, although in addition to the prominent peak of Stokes radius 820A there was dissociated receptor material at 62 A. Even at ,50 mM tungstate the large receptor form of 82 A was not completely protected against dissociation by 300 mM KC1 (data not shown). Interestingly, vanadate at similar concentrations did not stabilize the high molecular weight receptor form at all (Fig. 1). Effect of salt on receptors
At very low salt concentrations the physicochemical properties of wild-type and nt’ mutant receptors were quite similar whether or not sodium molybdate was included in the buffers. The gel filtration experiments of Fig. 2 with no added KC1 and molybdate produced major receptor peaks
2
MOLECULAR
PROPERTIES
Stokes radii and sedimentation Receptor
Conditions
OF RECEPTORS coefficients
were determined
of analysis
in 3-6 independent
experiments;
mean values f SD are given.
Stokes radius
Sedimentation
Molecular
(A)
coefficient
weight
(S)
Axial ratio
type
KC1 (mM)
Na,MoO,
Wild-type
0 150 300
20 20 20
83.6k2.8 82.3 + 1.9 19.5 +_2.6
9.5 + 0.3 9.1+ 0.6 9.lkO.3
335 000 316000 306 000
13 13 12
0 50 150 300 600
_ _ _ _
80.6k1.8 69.1 k 2.2 70.2 + 0.8 62.0 k 2.6 61.4i2.9
9.6 f 0.3 9.7kO.2 6.0+-0.1 4.9 f 0.3 5.OkO.2
328000 286 000 178 000 128 000 130000
12 9 14 13 13
0 150 300
20 20 20
12.5 i 2.5 70.5 + 0.4 69.0 + 0.4
9.5 * 0.5 9.5 + 0.4 9.0+0.1
291000 283 000 262000
10 9 9
0 50 150 300 600
_ _ _ _ _
71.9i1.9 70.6 + 1.2 40.7 * 2.0 38.4k1.5 40.0 + 2.6
9.8 + 0.3 10.2 f 0.4 5.1+0.1 3.9kO.6 3.8 + 0.1
298 000 304000 88000 63 000 64000
9 9 5 7 7
nt’ mutant
(mM)
0.1
02
03 Distribution
114
05
0.6
0.7
Coefficient
Fig. 1. Gel filtration of receptors in the presence of transition metal oxi-anions. Wild-type receptor complexes were chromatographed as described under Materials and Methods in the presence of 20 mM each of sodium molybdate (o), sodium tungstate (0), or sodium vanadate (0). Receptor preparations containing 15 mg protein and approximately lo6 dpm of bound [ ‘H]triamcinolone acetonide were applied to each co1 umn.
Stokes radii of 81 and 72 A for the wild-type and the nt’ mutant, respectively. With both receptor types we observed in addition to these main peaks some heterodisperse material of smaller size (Fig. 2) which probably arose by dissociation during the course of the chromatography and possibly also to some extent by proteolytic degradation. The sedimentation coefficients of the main components again were the same for both receptor types (Table 2) thus leading to apparent molecular weights of 328000 and 298000 for wild-type and nt’ receptors. Table 2 also summarizes the results of experiments in which KC1 at various concentrations was added to receptor preparations in the absence of molybdate. At 300 mM salt the receptors ap,peared to be fully dissociated into forms of 62 A and 39 A, respectively, with molecular weights of about 130000 and 63000 for the wild-type and nt’ mutant. Further increase of the KC1 concentration to 600 mM did not produce any further changes. At intermediate salt concentrations, however, some intermediate forms appeared (Table 2), most notably khe 70 A forms of the wild-type receptor and a 40 A form of the nt’ receptor with an intermediate sedimentation coefficient. These receptor forms
with
Dlstrlbutlon
Caefficienl
Fig. 2. Gel filtration of receptors in the presence or absence of salt. Wild-type (A) and nti mutant (B) receptor complexes were chromatogmphed as described under Materials and Methods without added salt (0) or in the presence of 300 mM KC1 (0). Receptor preparations containing 15 mg protein (A) or 10 mg protein (B), each corresponding to approximately lo6 dpm bound [ 3H]triamcinolone acetonide were applied to each column.
have apparent molecular weights of 178000286 000 and 88000. In the gel filtration experiments with 50 mM KC1 we again observed in addition to the major peaks at 70 A some additional heterodispezse receptor material which peaked at about 60 A in the case of the wild-type and at around 40 A with the mutant receptor. These results indicate that receptor dissociation caused by salt does not necessarily occur in a single step. However, it is difficult at present to unequivocally describe the molecular properties of
38
these intermediate receptor forms. With 20 mM KC1 we obtained the same gel filtration and sedimentation profiles as in the absence of added salt (cf. Fig. 2). In several experiments we first exposed receptor preparations to high salt concentrations and subsequently diluted them with low salt buffers which contained sodium molybdate. Analysis by gel filtration and sedimentation revealed in each case the dissociated form of the wild-type and nt’ mutant receptors (data not shown). This further corroborates the conclusion that molybdate by itself does not produce the high molecular weight receptor forms but only inhibits their dissociation. Activation and dissociation of receptors
Steroid hormone receptor complexes in target cell extracts which had been prepared at low temperatures and in buffers of low ionic strength need to be activated in order to elicit DNA binding ability (for reviews, see Milgrom, 1981; Grody et al., 1982; Schmidt and Litwack, 1982). Activation can be brought about, for example, by increasing the salt concentration or by warming receptor preparations. Fig. 3 shows an experiment in which
A
I
z
8
i
20-
a
we achieved partial activation of wild-type receptors by warming to 20°C for 30 min. The sample was then split into two aliquots. One part was directly analyzed by gel filtration and sedimentation in a glycerol gradient and two distinct peaks were detected cor;esponding to the Stokes radii of about 80 and 60 A and sedimentation coefficients of 9.5 and 4-5 S, respectively (closed symbols in Fig. 3). The other aliquot was first run through DNA-cellulose in order to adsorb the activated receptor complexes and subsequently analyzed in the same way. We now observed Oonly the high molecular weight form of about 80 A and 9.5 S. In other experiments we first exposed the wild-type receptor to 300 mM KCI, diluted the sample to a final concentration of 20 mM KC1 and carried out a similar analysis in the presence of 20 mM KCl. In this way we only observed the receptor peak of about 62 A which could be fully removed by prior filtration through DNA-cellulose (data not shown). Similar experiments were also carried out with nt’ mutant receptors. The complex of 72 A was unable to bind to DNA and was consequently not adsorbed by DNA-cellulose. Partially activated nt’ receptor preparations obtained by warming to
E.
P
155
x k D ,IO-
A
\
0:2 Dlstrlbutlon
0.3
0.L
Coefflclent
Fraction
Number
Fig. 3. Gel filtration and sedimentation analysis of partially activated receptors. Wild-type receptor complexes were activated at 20 “C and chromatographed on Sephacryl S-300 (A) or sedimented in glycerol gradients (B) as described under Materials and Methods. Samples were analyzed either without further treatment (0) or after passing through DNA-cellulose (0). Approximately 400000 dpm and 50000 dpm of receptor-bound [3H]triamcinolone acetonide were used for each of the gel filtration and sedimentation experiments, respectively. In the case of the material previously filtered through DNA-cellulose only an aliquot could be applied to the glycerol gradient; the results were corrected accordingly.
A
B
40.
J
-400 x x x
30-
/
-300
z
20-
-200
lo-
-100
10 Fraction
r
3 2
20
30
40
Number
250
,, -400
200-
,300 150T
t ,200 ; loo-
Y
2,100
50-
k
10
20
30
40
Fraction Number Fig. 4. DEAE-cellulose chromatography of receptors. Wild-type (A,B) and nt’ mutant (C,D) receptor complexes were chromatographed as described under Materials and Methods either in the non-activated state (A,C) or after activation at 20 o C (B,D). Activated samples were either loaded directly onto DEAE-cellulose (closed symbols in B and D) or after passing through DNA-cellulose (open symbols). Approximately 250000 dpm of receptor-bound [3H]triamcinolone acetonide were used for each experiment. Small crosses represent KC1 concentrations.
40
20 o C gave rise to gel filtration and sedimentation profiles with two peaks. Again the receptor form of lower molecular weight was eliminated by passing the pr~p~ation through DNA-cellulose (data not shown). Pretreatment of nt” receptors with 3000mM KC1 followed by dilution produced the 40 A form which was fully retained by DNA-cellulose. Taken together, these data show that the activated wild-type and nt’ receptors are the dissociated forms with molecular weights of approximately 130000 and 63000, respectively. By contrast, the high molecular weight complexes with apparent molecular weights of 330 000 and 290 Ooo are unable to bind to DNA. It has been shown that activated and nonactivated glucocorticoid receptors from rat liver can be separated by chromatography on DEAEcellulose or DEAE-Sephadex (Parchman and Litwack, 1977; Sakaue and Thompson, 1977). The non-activated receptor complex is more negatively charged and consequently requires higher salt concentrations for elution from DEAE-cellulose than the activated receptor. We applied this chromatographic technique to the mouse lymphoma cell receptor system in order to further distinguish between activated and non-activated wild-type and nt’ mutant receptors. Fig. 4 depicts representative experiments. Non-activated wild-type (Fig. 4A) and nt’ receptors (Fig. 4C) eluted with the same high salt concentration, i.e. 200 t 10 mM KCl. Upon partial activation by warming to 20 * C we obtained profiles with two peaks. Non-activated receptor complexes again eluted with 200 mM salt; the activated wild-type receptor eluted with 45-50 mM KC1 (Fig. 4B) while the activated nt’ receptor was contained in the flow-through of the column, i.e. eluted with I 20 mM KC1 (Fig. 4D). Which peaks correspond to the activated receptor species became very clear when receptor aliquots were again filtered through DNA-cellulose prior to their chromatography on DEAE-cellulose thus resulting in the removal of the activated receptors (open symbols in Fig. 4B and D). The activated wild-type and nt’ receptors thus differ not only in molecular weights but also in their ionic properties. In a previous study we had been unable to detect this difference in chromato-
graphic behaviour on DEAE-cellulose (Dellweg et al., 1982) because buffers of pH 8.5 had been used rather than pH 7.8 as in the present investigation. Partial ~roteo~~i~ of wind-type receptors
Mild treatment of wild-type receptors with chymotrypsin and other proteases has previously been shown to generate receptor species which are very similar to the nt’ mutant receptor with respect to increased affinity for DNA and the size of the ho~one-binding polypeptide as revealed by photoaffi~ty labelling and SDS-gel electrophoresis (Dellweg et al., 1982; Gehring and Hotz, 1983; Gehring, 1986). It was therefore of interest to determine the physicoche~c~ properties of partially proteolyzed receptors under bob-denatu~ng conditions. The molybdat~stabi~zed high molecular weight form of the wild-type receptor treated with a;;chymotrypsin had a Stokes radius of 68.9 t 2.7 A (n = 4) and a sedimentation coefficient of 10.0 & 0.1 S (n = 6) corresponding to an apparent molecular weight of 291000. Treatment with 300 mM KC1 in the absence of molybdate produced the dissociated form of 40.7 A and 3.8 S with a molecular weight of 65000. These data further emphasize the similarity with the nt” mut~t receptor (cf. Table 2). Activated and non-activated forms of the chymotrypsin-treated wild-type receptor could again be discriminated by chromatography on DEAE-cellulose. The profiles obtained were indistinguishable from those of Fig. 4C and D. The non-activated receptor again bound to the column and was eluted with 200 mM KC1 while the activated form, resembling the nt’ mutant, was not retained by DEAE-cellulose and appeared in the flow-through (data not shown). In some experiments with untreated wild-type receptors (cf. Fig. 4B) we also noted a small amount of receptor material in the flow-through; this probably arose by endogenous proteolysis. Discussion In the present co~~ication we give a detailed account of the molecular properties of the large forms of wild-type and nt’ mutant receptors from murine lymphoma cells. We obtained apparent
41
molecular weights of 330 000 and 295 000 for these receptor types, respectively, under conditions which carefully avoid dissociation and denaturation. The difference in molecular mass of about 35000 between wild-type and mutant reflects the size difference of the hormone-binding polypeptides of these receptors. Molecular weights of 94000 and 40000 had previously been obtained under denaturing conditions for the wild-type and nt’ receptors, i.e. a difference of about 55000 (Gehring, 1986). Considering that fundamentally different analytical techniques have been used for investigating native and denatured receptors we regard wild-type and nt’ mutant receptors different by the same amount of molecular mass independent of the conditions of investigation. The results therefore suggest that the large receptor forms of the wild-type and the nt’ mutant are heteromers and contain only one steroid-binding subunit per large complex. We expect the associated macromolecular components to be the same in the wild-type and in the mutant. This view is strengthened by experiments in which we treated wild-type receptors with chymot~psin and obtained molecular parameters which are indistingui~able from those of nt’ receptors. It is interesting to note that another mutant receptor devoid of hormone-binding ability likewise exist: in a high molecular weight form of about 81 A (Westphal et al., 1984), i.e. the molecular size resembles that of the wild-type receptor. Using an immunochemical approach Okret et al. (1985) also suggest a heteromeric structure for the rat liver glucocorticoid receptor. Adding salt to receptor preparations results in progressive dissociation of the large complexes (Table 2). The wild-type and nt’ receptor species of M, 130 000 and 63 000, respectively, which we detected at 300600 mM KC1 are presumably fully dissociated. On the other hand these receptors have polypeptide molecular weights of 94000 and 40000 (see above). These differences in molecular weights appear to be due again to different analytical procedures, as discussed above. Alternatively, but perhaps less likely, the hormone-binding polypeptides are associated under non-denaturing conditions with some as yet u~dentified component(s}.
The dissociated wild-type and nt’ mutant receptors of M; 130000 and 63 000 are activated forms which are able to bind to DNA. They are less negatively charged than the non-activated, non-dissociated receptors and consequently stick less tightly to DEAE-cellulose. The fact that the activated wild-type and nt’ receptors behave differently on DEAE-cellulose clearly shows that the amino terminal half of the hormone-binding polypeptide that is missing from the nt’ mutant (Gehring, 1986), does contribute significantly to the ionic properties of the activated receptors. With recent cloning of receptor-specific cDNAs of full length the amino acid sequences of the human, mouse and rat glucocorticoid receptors are now known (Hollenberg et al., 1985; Danielsen et al., 1986; Miesfeld et al., 1986); these polypeptides consist of 777, 783 and 795 amino acids, respectively, and are quite homologous in sequence. The most striking difference is a stretch of 8 and 19 glutamine residues within the amino terminal half of the mouse and rat receptors which is absent from the human. The biological significance of this sequence, if any, is not known. The domains for hormone binding and for interacting with DNA are almost identical. Similar to the other steroid hormone receptors, the DNA binding domain of about 70 amino acids is located in the middle of the polypeptide chain and the hormone binding domain occupies the carboxy terminal part. A sequence comparison of the DNA binding domains of the known steroid and thyroid hormone receptors shows complete conservation of 9 cysteine residues as well as several basic and hydrophobic amino acids. The DNA binding domain is thought to fold in such a way that two finger-like structures are formed which resemble those detected in the Xenopus transcription factor IIIA (Miller et al., 1985) and several other DNA binding proteins. This structure may be stabilized by zinc ions which are tetrahedrally coordinated with cysteine residues. The sequence analysis of two receptor mutants of mouse lymphoma cells led to the identification of two amino acids in the hormone binding domain which are directly or indirectly involved in steroid binding: glutamic acid at position 546 and tyrosine at 770 (Danielsen et al., 1986). In addition, cysteine 644 (corre-
42
sponding to position 656 in the rat receptor) was found to react with the affinity label dexamethasane 21-mesylate (Simons et al., 1987). This shows that a fairly long part of the polypeptide chain consisting of at least 225 residues is involved in forming the hormone binding domain. As to the nt’ mutant receptor its molecular origin is still not yet clear. What is known is that this amino terminally truncated polypeptide is synthesized from 5’ truncated mRNAs (Miesfeld et al., 1984) which probably arise through abnormal splicing. Very recent experiments show sequence divergence on the cDNA level upstream of amino acid position 404 (in the murine wild-type receptor). Synthesis of the nt’ polypeptide may either start with methionine 406 (position of the wild-type) or from an upstream AUG originating from intronic sequences (Miesfeld et al., 1987). We observed that non-activated wild-type and nt’ receptors bind quite tightly to DEAE-cellulose and elute under identical conditions. Thus, the macromolecular components which are associated with the hormone-binding polypeptides appear to be highly charged. This brings up the question as to the identity of these associated macromolecules. A non-hormone-binding polypeptide of M, 90 000 has been detected which is common to the large forms of various steroid hormone receptors (Joab et al., 1984; Housley et al., 1985; Riehl et al., 1985; Mendel et al., 1986) which has been identified as heat-shock protein hsp 90 (Catelli et al., 1985; Sanchez et al., 1985). The charge properties of the high molecular weight receptor complexes appear to be largely determined by hsp 90 which is an acidic protein of pI 5.2 and which elutes from DEAE-cellulose or DEAE-Sephadex with about 200 mM salt (Welch and Feramisco, 1982; Lai et al., 1984). The function of hsp 90 in relation to steroid hormone receptors, however, remains unknown. There is also convincing evidence for RNA being associated with large-sized glucocorticoid receptors (Economidis and Rousseau, 1985; KovaCiE-Milivojevic et al., 1985; Webb and Litwack, 1986). This RNA can be covalently cross-linked to the hormone-binding polypeptide and is of low molecular weight similar to tRNA. Since RNAs are much more dense than proteins
they have significantly lower partial specific volumes (in the order of 0.50 ml/g). Therefore, the value for v (0.732 ml/g) assumed in the present study for large receptor complexes is probably too high; it had previously also been used in a series of other investigations (Niu et al., 1981; Sherman et al., 1983; Stevens et al., 1983; Vedeckis, 1983,1985; Sherman and Stevens, 1984). Supposing an RNA content of roughly 10% we would expect v values of about 0.715 ml/g resulting in molecular weights for the undissociated wild-type and nt’ receptors of about 310 OO!, and 280000, respectively, as computed from 81 A, 9.6 S and 72 A, 9.8 S (Table 2). These sizes fit quite well to what one would expect for complexes consisting of one hormone-binding polypeptide, two molecules of hsp 90 and one molecule of tRNA. It needs to be emphasized that a receptor constitution of this type is but a hypothetical structure at present. Unequivocal proof for the molecular composition of the high molecular weight receptors will have to come from reconstitution experiments. Such experiments are now in progress. The size properties of the non-activated large steroid receptor forms reported in the literature are remarkably similar especially when molybdate was used as a stabilizing agent (see, for example, Sherman and Stevens, 1984; Vedeckis, 1985). This is certainly not so for the various dissociated receptor forms and we find it difficult to compare our results with some of the published data. In particular, we have not been able to obtain evidence for an intermediate wild-type receptor form of 83 A and 5.0-5.2 S (M, 176000) as has been described (Vedeckis, 1983; Reker et al., 1985). Interestingly, this partially dissociated receptor species was obtained in the presence of 20 mM molybdate and 300 mM KCl, i.e. under conditions which in our hands do not result in dissociation (cf. Table 2). On the whole our data agree well with those of Stevens et al. (1983) who used wildreceptors of the mouse type and nt’ mutant lymphoma strain F1798. They reported Stokes radii of 81 and 70 A for the molybdate-stabilized wild-type and nt’ receptors, respectively, and 60 and 28 A for the salt-treated receptors. Even though lymphoid cells are generally believed to be poor in proteolytic activities the 28 A form of the
43
P1798 nt’ receptor is likely to be a partially pro-
teolyzed species. In fact, we observed in several experiments S49.1 n,t’ receptors with a Stokes radius of about 30 A, especially under low salt conditions and following a 20 o C treatment. Dissociated nt’ receptors appear to be particularly susceptible to endogenous proteases. Acknowledgement This work was supported by grants from the Deutsche Forschungsgemeinschaft. References Beblke, J. and Wand& I. (1973) Acta Biol. Med. Ger. 31, 383-388. Braunitzer, G., Hilse, K., Rudloff, V. and Hilschmarm, N. (1964) Adv. Protein Chem. 19, l-65. Catelh, M.G., Binart, N., Jung-Testas, I., Renoir, J.M., Baulieu, E.E., Feramisco, J.R. and Welch, W.J. (1985) EMBO J. 4, 3131-3135. Dabmer, M.K., Housley, P.R. and Pratt, W.B. (1984) Annu. Rev. Pbysiol. 46, 67-81. Danielsen, M., Northrop, J.P. and Ringold, GM. (1986) EMBO J. 5,2513-2522. Dellweg, H.-G., Hotz, A., Mugele, R. and Gebring, U. (1982) EMBG J. 1, 285-289. Economidis, I.V. and Rousseau, G.G. (1985) FEBS L&t. 181, 47-52. Edelhoch, H. (1960) J. Biol. Chem. 235,1326-1334. Gehring, U. (1980a) In: Biochemical Actions of Hormones, Vol. 7, Ed.: G. Litwack (Academic Press, New York) pp. 205-232. G&ring, U. (1980b) Mol. Cell. Endocrinol. 20, 261-274. Gehring, U. (1986) Mol. Cell. Endocrinol. 48, 89-96. Gebring, U. and Arndt, H. (1985) FEBS Lett. 179, 138-142. G&ring, U. and Hotz, A. (1983) Biochemistry 22, 4013-4018. Grady, W.W., Schrader, W.T. and O’Malley, B.W. (1982) Endocr. Rev. 3,141-163. Gro~emeyer, H. and Govindan, M.V. (1986) Mol. Cell. Endocrinol. 46, 1-19. Holbrook, N.J., Bodwell, J.E., Jeffries, M. and Munck, A. (1983) 3. Biol. Chem. 258, 6477-6485. Hollenberg, SM., We~berger, C., Ong, ES., Cerelli, G., Ore, A., Lebo, R., Thompson, E.B., Rosenfeld, M.G. and Evans, R.M. (1985) Nature 318, 635-641. Housley, P.R., Grippo, J.F., Dahmer, M.K. and Pratt, W.B. (1984) In: Biochemical Action of Hormones, Vol. 11, Ed.: G. Litwack (Academic Press, New York} pp. 347-376. Hot&y, P.R., Sanchez, E.R., Westphal, H.M., Beato, M. and Pratt, W.B. (1985) J. Biol. Cbem. 260, 13810-13817. Joab, I., Radanyi, C., Renoir, M., Buchou, T., Catelli, M.-G., Binart, N., Mester, J. and Baulieu, E.E. (1984) Nature 308, 850-853.
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