42

Biochimica et Biophysica Acta, 500 (1977) 42--48

© Elsevier/North-Holland Biomedical Press

BBA 28325 H E T E R O G E N E I T Y OF ST E R O I D HORMONE RE CE PT O R IN A D U L T RAT LUNG CYTOSOL

M.K. AGARWAL and M. PHILIPPE I N S E R M U-36, 17, rue du Fer 5 Moulin, Paris 75005 (France)

(Received March 22nd, 1977) Summary C h r o m a t o g r a p h y on cellulose DEAE-52 columns revealed that the glucocorticoid r e c e p t o r from rat lung cytosol consisted of a c o m p o n e n t in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second c o m p o n e n t eluted with 0.04 M PO4, labelled with triamcinolone, dexamethasone, and a third m o i e t y in the 0.06 M PO4 region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, d e o x y c o r t i c o s t e r o n e ) . The third c o m p o n e n t coeluted with rat blood serum transcortin in double labelled experiments. Rat lung was devoid of a n o t h e r c o m p o n e n t in the 0.02 M PO4 found in rat liver supernate and o f the mineralocorticoid r e c ept or evident only in rat kidney. C h r o m a t o g r a p h y on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KC1. Collectively, these studies indicate the subunit nature of the lung recept or as evidenced in most tissues hi t her t o tested. Moreover, p o l y m o r p h i s m within a given subunit c o m p o n e n t can not be revealed by c o m p e t i t i o n alone, as a t t e m p t e d by others, but can be revealed under selected conditions of physical separation.

Introduction Association o f a steroid h o r m o n e with its specific cytoplasmic r e c e p t o r is said to constitute the first step whereby organ specific processes are m odul at ed in the appropriate target tissues [1,2]. In foetal rabbit lung, cortisol and d e x a m e t h a s o n e binding proteins have been det ect ed both in the cytoplasm and the nuclei and it is believed that translocation of the steroid-receptor complex from the cytoplasm to the nucleus initiates p r o d u c t i o n of the pul m onary surfactant [3,4]. Although in these and various other studies it is assumed that the receptor protein probably exists as a unitary vector capable of association with all

43 agonists of the active hormone, recent work from our laboratory has clearly shown heterogeneity and polymorphism in the nature of glucocorticoid receptors from the liver [5] and the mineralocorticoid receptor from the kidney [6,7]. It was furthermore observed that natural and synthetic steroids appear to bind to chromatographically distinct protein moieties [8]. This paper concerns a careful analysis of the nature of cytoplasmic glucocorticoid receptors from adult rat lung. Materials and Methods Adult male Wistar rats (150--200 g), maintained on laboratory food and tap water ad libitum, were bilaterally adrenalectomized at least 48 h prior to exsanguination under ether anaesthesia and were perfused with the initial buffer by aortic cannulation. Lung supernates (40%) were prepared in the initial buffer (0.001 M phosphate, pH 7.5 for DE-52; 0.01 M phosphate + 0.1 M NaC1 for G-200) by centrifugation at 105 000 X g (1 h) of which 4 ml were incubated (60 min, 4°C) with 2 • 10 -s M or 10 .7 M of tritiated steroid of choice. The free radioactivity was thereafter removed by further incubation (10 min, 4°C) with 100 mg/ml of activated charcoal (Sigma, C-5260), centrifugation (3000 X g) and passage through glass wool. Blood serum, obtained from the adrenalectomized rats was incubated, separately, with '4C-labelled corticosterone (0.25 pCi/ml) and treated in the same manner as lung cytosol in all cases. The cytosol alone (4 ml) or with serum (2 ml) was finally loaded on DEAEcellulose 52 columns (1 × 25 cm) (Whatman) equilibrated with 0.001 M phosphate, pH 7.5. After passage of 60--70 ml of this initial buffer (fraction volume 6--7 ml) protein was eluted with a linear gradient of 60 ml of 0.001 M and 60 ml of 0.2 M sodium phosphate, pH 7~5 at a flow rate of 60 ml/h at 4°C (fraction volume approx. 3 ml). For further details see refs. 5--7 and figure legends. Sephadex G-200 (Pharmacia) columns (1 × 130 cm) were equilibrated with 0.01 M phosphate, pH 7.4, containing 0.1 M NaC1. Cell sap (2 ml) alone or with serum (1 ml) was loaded on the column which was eluted with the initial buffer at a flow rate of 10--12 ml/h at 4°C (fraction volume 1.1--1.4 ml). Charcoal treatment was not employed when cytosol alone was fractionated. In other cases, 0.4 M KC1 was used in place of 0.1 M NaC1. Further details are given in legends and published earlier [ 5--7]. Fractions were processed for determination of absorbance at 280 nm in a spectrophotometer and a l-ml aliquot was mixed with 10 ml Unisolve (KochLight) for determination of radioactivity in a Packard Tricarb Scintillation spectrometer equipped with automatic background substraction and external standardization. [1,2-3H]Cortisol (56 Ci/mM; batch 5 8 0 3 5 ) a n d [1,2-3H]dexamethasone (15 Ci/mM, batch 66074) were obtained from C.E.A., Saclay, France; [1,2-4H] triamcinolone (16 Ci/mM; ZT 1423) was a product of Schwarzmann. [ 1,2,6,7-3H] Aldosterone (87 Ci/mM; batch 9), [ 1,2-3H] deoxycorticosterone (44 Ci/mM; batch 8), [4-14C]corticosterone (52 mCi/mM; batch 10) and [1,2-~H] corticosterone (82 Ci/mM; batch 13) were purchased from Amersham, U.K.

44 Radiochemical purity of the steroid exceeded 98% in thin layer chromatography; all other chemicals were high purity reagent grade from Merck. Results

With the two mineralocorticoids, binding to the 0.06 M component GRa of the glucocorticoid specific receptor (GR) was greater with deoxycorticosterone than with aldosterone, whereas the reverse was true of the component GRI in the 0.001 M prewash (Fig. la, b). In separate studies it was confirmed by both charcoal treatment and chromatography on Sephadex columns that the peak in the GR, region was protein bound. It is furthermore to be noted that transcortin (T) bound 14C-labelled corticosterone coeluted with the 0.06 M component in the double labelling experiment (Fig. la). There was no indication, however, of a mineralocorticoid specific receptor (MR), hitherto shown to be present only in the target tissue kidney [6,7] at any concentration of either steroid tested. With the two natural glucocorticoids (cortisol and cortieosterone) binding was limited to components eluted in 0.06 M phosphate region (GR4) and this increased quantitatively with 10 -7 M steroid concentration in place of 2 • 10 s M. (Fig. lc, d). No radioactivity was obtained in 0.02 M phosphate region (GR2) found in the liver [5,8]. With synthetic steroids, binding to the 0.001 M fraction was greater with dexamethasone than triamcinolone but no bound radioactivity could be eluted in the 0.06 M phosphate region as had been observed above with the natural glucocorticoids. Rather, both steroids (TA > DEX) were bound to a component eluted in the 0.04 M PO4 region (GR3) as previously described in the liver [8]. The specificity of binding was ascertained by the fact that bound radioactivity was totally wanting in the presence of a 100-fold excess of cold, homologous steroid; in cross competition studies, equimolar cortisol did not diminish dexamethasone binding to either the GRI or GR3 component and equimolar, cold dexamethasone did not influence cortisol binding to GR4 (not shown). In still other experiments, we attempted to affirm this sort of heterogeneity by chromatography of lung cytosol prior to binding analysis of various receptor populations in the corresponding fractions. However, absence of steroid during chromatography was found to labilize the receptor proteins, in keeping with the classical concept of substrate stabilization of the protein ligand. Qualitatively similar results were obtained when 10 -7 M steroid was used in place of the 2 • 10 -s M described here. Filtration through a Sephadex G-200 column, equilibrated at physiological isotonicity and pH, indicated a heavier component (H) in the 113 000 molecular weight range followed by another lighter (L) of approximately 67 000 daltons with either cortisol (Fig. 2a) or eorticosterone (Fig. 2b). Since these persisted upon rechromatography of isolated peaks it would appear unlikely that non specific aggregation could lead to polymorphic heterogeneity. In a double labelling experiment (Fig. 2a) the major, low molecular weight, component coeluted with serum transcortin-bound [ ~4C]cortieosterone (T). Thus, as with DE-52, it is not possible to say whether the former represents intraeellular transcortin or the receptor m o n o m e r since both entities appear to have the same molecular weight [ 5,8].

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42 Biochimica et Biophysica Acta, 500 (1977) 42--48 © Elsevier/North-Holland Biomedical Press BBA 28325 H E T E R O G E N E I T Y OF ST E R O I D H...
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