Characterization of a specific, high affinity binding macromolecule for ...

JOURNALOF BIOLOGICAL CHEMISTRY Vol. 255. No.21, Issue of November 10, pp. 10160-10166,1980 Printed in U.S.A. THE

Characterization of a Specific, High Affinity Binding Macromoleculefor lcx,25-Dihydroxyvitamin D, in Cultured Chick KidneyCells* (Received for publication, April 28, 1980)

Robert U. Simpson, RennyT. Franceschi, and HectorF. DeLucaS From the Departmentof Biochemistry. - . Collecre of Apricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

Cytosol prepared from vitamin D3-deficientkidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitaminD3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-hydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals of 5.6 X lo-” with an equilibriumbinding constant (KO) 57 fmol ofsites/mg of protein. The receptor-likeprotein has a M, = 72,000 andas with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitaminD3 >> 25-hydroxyvitamin D3 > la-hydroxyvitamin D3 > 24(R),25-dihydroxyvitaminDD;the receptor had no detectable affinity for vitamin D3.The kidney cells respond to 1,25-dihydroxyvitamin D3 bydiminishing 25hydroxyvitamin D3 la-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin DS metabolism.

have been detected in bone and intestine which bind 1,25( O H ) ~ Dwith B high affinity and low capacity and which have sedimentation coefficients of 3.0 to 3.7 S (2-5). Recent evidence suggests that kidney, the endocrine organ responsible for 1,25-(OH)2D3production via la-hydroxylation of 25-hydroxyvitamin D3, may be an additional target tissue for 1,25-(OH)2D3.Studies have shown that1,25-(OH)2D:3 can directly affect its own synthesis by decreasing renal la-hydroxylation and stimulating24-hydroxylation of 25-OH-D3 (6, 7). Supporting a nuclear mechanismof action in kidney, 1,25(OH)zD3 has been shown to stimulate renal RNA synthesis (8). In addition, Stumpf et al. (9) have presented autoradiographic evidence forthe nuclearlocalization of 1,25-(OH)z& in the distal tubulesof rat kidneys. Although these results suggest that a receptor protein for 1,25-(OH)zD3should be present in kidney, initial attempts to identify a renal 1,25-(OH)2D3binding protein were unsuccessful (10,ll). A recent report hasdescribed a 3.6 S 1,25-(OH)zDs binding protein in cytosol from rachitic chick kidney (12). This 3.6 S species accounted for a small percentage of the total1,251-(0H)~D~binding,however, andthepreparation showed a n uncharacteristically low affinity for ligand (Ku = 1.2 nM). In another study, murinekidney tubules were shown to contain a 3.2 S cytoplasmic 1,25-(OH)2Ds binding protein with a dissociation constant of0.2 nM (13). Since the mice used in this study were not vitamin D-deficient, the authors suggest that theaffinity constants andcapacity of the receptor preparation represent minimal estimates. The use of kidney cells in culture hasproven to be a reliable tool for studying control of 25-OH-D3 metabolism (14-16). by increasing 24-hydroxThere is convincing evidence that I ,25-dihydroxyvitamin These cells respond to 1,25-(OH)2DR D3 metabolite capableof ylase and decreasing la-hydroxylase activities (14, 15). In the D:3 is the hormonally active vitamin stimulating calcium and phosphate absorption by the intestine present communication, we have identified and characterized and the resorption of calcium and phosphate from bone (1). a receptor-like 1,25-(OH)zD:3binding protein in cultured kidSeveral lines of evidence suggest that 1,25-(OH)2Da1may act ney cells which may be involved in the regulation of 25-OHDBmetabolism. Unlike previous kidney preparations (12, 17), on its target tissues through amechanismsimilar to that proposed for classic steroid hormones (1).According to this cultured cell cytosol was found to be a more reliable source which is only minimally model, hormone first enters the target cell and binds to a for the 1,25-(OH)2D:3 binding protein contaminated with a 6 S 25-OH-D:, binding species. The use specific cytoplasmic receptor protein that carries the hormone to the cell nucleus where it induces the expression of those of cell culture techniquesprovides a renal cytosol preparation that hasallowed characterization of the 1,25-(OH)ZD3receptor genes involved in the hormonalresponse. Consistent with thishypothesis, putative receptor proteins and will facilitate studies aimed at examining this receptorlike protein and the role it plays in the regulation of renal * This work was supported by Program-Project Grant AM-14881 vitamin DRmetabolism. from the National Institutes of Health, NASA Contract NAS9-15580, and by the Harry SteenbockResearch Fund of the Wisconsin Alumni Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom all correspondence should be addressed. The abbreviations used are: 1,25-(OH)2D.j, 1,25-dihydroxyvitamin DZr;25-0H-D.1, 25-hydroxyvitamin D.I; 24,25-(OH)Jl1, 24,25-dihydroxyvitamin Dl.

+ ’

MATERIALS AND METHODS

Animals-One-day-old white Leghorn cockerels (Northern Hatcheries, Beaver Dam, Wis.) were fed a vitamin D-deficient soy protein diet (18)containing adequate calcium and phosphate for 4to 6 weeks. Animals were maintained at 25-26°C on an alternating 12 h light and dark cycle. Chernical~-25-OH-[26,27-:’H]D:~ (160 Ci/mmol) was prepared by the procedure of Napoli et al. (19). 1,25-(0H),-[26,27-”H]D,1(160 Ci/

10160

Receptor for la,25-Dihydroxyvitamin D:, in Chick Kidney Cells

10161

fuge (Beckman Instruments, Inc., Palo Alto, Cal.) using a T i 30 rotor. mmol) wasenzymatically prepared from25-0H-[26,27-"H]Ds (160 Ci/ mmol) by the methodsof Frolik and DeLuca (20).Nonradioactive 25- The supernatant was immediately frozen in an isopropyl alcohol-dry ice bath and stored at -70°C until use. Cytosol preparations stored OH-& was the gift of Drs. J. Allan Campbell and John Babcock of at -70OC were found to retain their initial binding capacity for up to the Upjohn Co. (Kalamazoo, Mich.). Nonradioactive 24(R),25-dihy60 days. droxyvitamin D.I and1,25-(OH)rD.1 werethe gift of Dr. M. Uskokovic For experimentswith chick kidney tissue homogenates, the kidneys of the Hoffmann-LaRoche Co. (Nutley, N. J.). Before use, all comwere minced to fragments no larger than 1.0 mm3 and rinsed three pounds were found to be greater than 96% pure by high pressure times in ice cold phosphate-bufferedsaline by centrifugation and liquid chromatography (21) and purity was confirmed by ultraviolet resuspension. One volume of washed kidney tissue was homogenized spectrophotometry. Compounds were stored in toluene under nitroin 2 volumesof the TKEDbuffer using a polytron homogenizer model gen at -20°C. PT-20(BrinkmannInstruments, Westbury, N. Y.).Isolation and Zsolation a n d Culture of Chick Kidney Celts-Vitamin D-deficient treatment of the cytosol was as described for cultured kidney cells. chicks were decapitated, their kidneys removed, and briefly blotted Equilibrium BindingStudies-Appropriate concentrations of 1,25with tissue paper saturated with 95% ethanol. They were then immediatelyplaced inWaymouth's mediacontaining penicillin ( I 0 0 (OH)z-[26,27-3H]D3 (160 Ci/mmol) in toluene were pipettedinto borosilicate glass tubes (13 X 100 mm), the solvent removed with units/ml), streptomycin (100 pg/ml), and fungizone (0.25 pg/ml) all nitrogen, and the labeled compound redissolved in 20 pl of absolute fromGIBCO(GrandIsland,N. Y.) and minced in this media to fragments approximately1.0 mm3 in size. The tissue mince was rinsed ethanol 10 min prior to cytosol addition. Nonsaturable 1,25-(OH)2D3 three times andplaced in a sterilized 25-ml Erlenmeyer flask contain- binding was assessedby parallel incubation containing 100-fold excess unlabeled 1,25-(OH)zD3.In these experimentsspecific binding was 50 ing a magnetic stir bar. A type 1 collagenase solution (2.5 mg/ml) from WorthingtonBiochemicals wasprepared in Hanks balanced salt to 80% of total binding. The binding reaction was initiated by the solution (GIBCO) and sterilefiltered. Four parts of collagenase solu- addition of approximately 0.25 mg of cytosol protein ina total volume tion were incubated with one part of kidney mince at 37°C with mild of 0.5 ml. Preliminary experimentsshowed that incubation of cytosol stirring from a magnetic mixer. Kidney digestion was monitored by with 1,25-(OH)2[26,27-3H]D3a t 25°C for 40 min was sufficient for binding equilibrium to be established. Bound hormone was separated light microscopy. After 15 to 25 min, digested tissue was vigorously pipetted 3 times with a 10-ml pipette and the digested tissue was from free hormone by absorption of free hormone to dextran-coated passed through two layers of sterile gauze removing undigested frag- charcoal as previously described (24) and 0.5-ml aliquots were then withdrawn and counted. For evaluation of 1,25-(OH)zD3equilibrium ments. The filtrate, consisting of free cells, tubules, and small tissue fragments was centrifuged at approximately 75 X g for 5 min and binding affinity and capacity, 1,25-(OH)~[26,27-~H]D3 concentrations resuspended in media plus antibiotics, This procedure was repeated varied from 0.01 to 10 m. Analysis of binding data was performed twice. The washed pelleted cells were resuspended in a final volume using the methodsof Scatchard (25), and linearregression analysis of of 0.25 to 0.50 ml in Waymouth's medium containing antibiotics as the data was used to obtain theline of best fit. Kinetic Estimation of Binding Consfants-The association rate noted and 5% fetal calf serum (GIBCO). The cell suspensionwas constant was estimated at 25°C by incubation of cytosol (0.5 mg of pipetted (15 ml) into75-cm' plastic Falcon T flasks and incubated a t protein/ml) with 0.5 nM 1,25-(OH)2[26,27-"H1D:,.Saturable binding 37°C in a 5% C02-95%air atmosphere. After 24 h the growth media with unattached cells was replaced with fresh media containing 5% was assessed by inclusion of parallel reaction tubes containing 100fold excess unlabeled hormone.At various time points,0.5-ml aliquots fetal calf serum andantibiotics. Cultures routinely reached confluency of cytosol were removedand added to 0.25 ml dextran-coated charcoal by 72 to 96 h a t which time experiments were begun. Microscopic kept on ice. The association rate constant was derived from the slope examination of confluent cultures displayed cells which had a flat polygonal or cuboidal shape typical of epithelial cells. Few elongated, of the plot of (1/T - S ) In [ T - X / S - x] uersus time of incubation, where X is the concentration of bound radioactive 1,25-(OH)2D:ja t spindle-shaped cells typical of fibroblasts were present. Assay of25-0H-D:1 Metabolism-Growth media was removed from time (t). T is the initial concentration of radiolabeled 1,25-(OH)eD:, confluent cultures24 h prior to experiments and replaced with serum- and S is the maximum concentration of binding sites. The dissociation rate constant was determined by incubation of free media containing the described antibiotics. At the time of assay the specific activity of stock 25-OH-["H-26,27]D:r(80 Ci/mmol) was cytosol (-0.5 mg of protein/ml) with 0.5 nM 1,25-(OH)z[26,27-"H1D:1. diluted with 25-OH-D:lto yield a final specific activity of 0.1 Ci/mmol. After the binding reaction proceeded for 30 min at 25OC, a 500-fold For these experiments, 1 nmol of 25-OH-['HH-26,27]Dawas added in excess of unlabeled 1,25-(OH)2Da was added so as to monitor the binding. At appropriate times, 0.5-ml 10 p l of absolute ethanol to 10 ml of fresh Waymouth's media. The reversibility of 1,25-(OH)2D:~ media was equilibrated with 95% 02-5%COz a t 37°C and then pipetted aliquots of cytosol were withdrawn and unbound hormone separated with dextran-coatedcharcoal. Saturable binding was determined from into a culture of cells. After 30 min at 37OC, the media was removed and the cells isolated from the culture flask by the addition of 10 mM a plot of In (1.25-OH~D3bound) uersus time and yielded a straight EDTA in 10 mM phosphate-buffered saline, pH 7.2. The cells and line with the slope equal to the dissociation rate constant. Lines of media were extracted by the methods of Bligh and Dyer (22). The best fit were obtained by linear regression. In the absence of excess unlabeled 1,25-(OH)zD:~, the binding of organic phase was collected, dried under nitrogen, and the residue labeled hormone was found to remain constant during the period of taken up in 0.5 ml of ch1oroform:hexane (65:35). Dihydroxyvitamin D:, metabolites were prepared for high pressure liquid chromatogra- study indicating that the observed dissociation process could not be phy by Sephadex LH-20 chromatography on a column (7 mm X 15 attributed to receptor degradation. cm) eluted with ch1oroform:hexane (65:35) as previouslydescribed Sucrose Gradient Analysis-Kidney cytosol preparations (-2 mg (23). A 25-cm Zorbax SIL column (DuPont) equilibrated with 10% of protein in 0.4 m l ) were incubated with2 nM 1,25-(OH)2[26,27isopropyl alcohol in 90% hexane on aDuPont model 830 high pressure :'HID3 dissolvedin absolute ethanolin the presence or absence of 100preparations were liquid chromatograph with UV detector was used. Nonradioactive 25- fold excess1,25-(OH)&.Alternatively,cytosol OH-D.1, 24,25-(OH)zDa, and 1,25-(OH)2D:I wererun with samples for incubated with 2 nM 25-OH-[26,27-"H]D.1. After 1 h a t 25'C. unbound hormone was removed with dextranidentification of relevant radioactive peaks; 1-ml fractions were collected and 1,25-(OH)~D3 and 24,25-(OH)*Dirpeaks were analyzed for coated charcoal and 0.2-ml aliquots were then analyzed on 4 to 20% radioactivity. The recovery of labeled metabolites was monitored and sucrose gradients as previously described (11). Unless stated otherwas usually 50 to 60% of initial label added. Radioactivity was deter- wise, all gradients contained TKED buffer. Ovalbumin and bovine mined in a toluene scintillation fluid containing 7.5 g of 2,5-diphen- serum albumin were used for the estimation of sedimentation coeffiyloxazole and 0.34 g of 1,4-bis-[2-(4-methyl-5-phenyloxazolyl)]ben- cients. In the experiment described in Fig. 3, the 25°C labeling step zene/liter. was replaced with a 2-h incubation at 0°C since incubation a t 25°C Preparation of Kidney Cell Cytosol-The cells from a confluent has been shown to reduce the ability of the intestinal 1,25-(OH)zD,7 culture were detached from flasks with 10 mM EDTA in phosphatereceptor to enter intoaggregates (26). buffered saline. Cells were rinsedwith phosphate-buffered saline and Molecular Weight Determination by Gel Filtration-A column (2 collected by centrifugation at 50 g for 5 min. One volume of packed X 90 cm) of Sephacryl S-200 (Pharmacia FineChemicals) was packed cells was resuspended in 2 volumes of 50 mM Tris-HCI, 300 mM KCI, by gravity to a final bed volume of 250 ml. The columnwas eluted at 1.5 mM EDTA, and 5.0 mM dithiothreitol, pH 7.4 (TKED buffer). a flow rate of 45 ml/h using a peristaltic pump. The 2-ml fractions Cells were sonicated using a model 350 sonicator (Heat SystemsInc., were collected using an LKB Ultrorac type 7000 fraction collector. N. Y.) at a setting of 5 in the pulse mode for bursts while the cells The void volume, as determined by the elution of blue dextran, was were kepton ice. Cytosolwas obtained by centrifugation of the 95 ml. Standardization of the column was accomplished using 10 mg homogenate at 5,000 X g for 90 min in a Beckman L5-50 ultracentri- each of cytochrome c. pepsin, myoglobin, ovalbumin, bovine serum

Receptor for la,25-Dihydroxyvitamin D3 in Chick Kidney Cells

10162

albumin, and human y-globulin in 1 ml of TKED buffer. A plot of log molecular weight uersus KO (where KO= V, - Vo/VT- Vo; V,, elution volume, VO,void volume, VT,total bed volume) yielded a straight line which was used for the estimation of the molecular weight of unknowns. Miscellaneous Measurements-Radioactivity determinations were carriedout with a Beckrnan LS-1OOC spectrometer,and quench correction was monitored by the use of automatic external standardization. Protein concentration of kidney cytosols was determined by the method of Bradford (27) using crystalline bovine serum albumin as a standard. RESULTS

Metabolism of 25-OH-[26,27-3H]D3 by Cultured Kidney Cells-Fig. 1 represents a high pressure liquid chromatographic separation of metabolites producedfrom25-OH[26,27-'HH]D3 by a primary culture of chick kidney cells. The figure demonstrates that under control conditions (i.e. no 1,252 6 IO 14 18 23 26 30 34 38 (0H)zDa addition prior to assay) the major metabolite of 25OH-[26,27-3H]D3 formedis 1,25-(OH)2[26,27-3H]D3. When 10 FRACTION NO. ( = I m l ] nM 1,25-(OH)zD3 was added to theculture media 24 h prior to FIG. 1. High pressure liquid chromatographic separation of the assay of %-OH-& metabolism, la-hydroxylase activity metabolites. High pressure liquid chromatographic separation of was substantially decreased while 24-hydroxylaseactivity was metabolites of 25-(OH)-[26,27-'H]Daproduced by primary cultures of stimulated. In thisexperiment, control cells generated approx- chick kidney cells. 0, metabolite profile of cells not exposed to 1.25metabolites formed by cells after 1,25-(OH)zD3(10nM) imately 46.5 pmol of 1,25-(OH)zD3/flaskin 30 min. Incubation (0H)zD:J;0, with 1,25-(OH)& (10nM) prior to assay resulted in a de- addition 24 h prior to assay. Incubation of 25-OH-[26,27-3H]D3with all cultures was for 30 min at 37°C. The arrows note the retention to 14.3 pmol/flask/30 min. time of nonradioactive 25-OH-D3, 24,25-(OH)&, and 1,25-(OH)zD3 creased production of 1,25-(OH)zD3 In contrast, 24,25-(OH)JI3production was found to increase which were monitored by absorption at 254 nM. to 36.1 pmol/flask/ in cells exposedto 1,25-(OH)2D3 from 14.1 30 min. In three experiments, 1,25-(OH)& addition was found a f S.E.) decrease in 1,25-(OH)zD3 to cause a 64 f 2% - 9 production and 145 k 18% (8 f S.E.) increase in 24,25(OH)ZD~ production. These results demonstrate that 1,25(OH)zD3acts on cultured kidney cells by altering 25-OH-Ds metabolism. The type of kidney cell that propagates using our culture techniques is unknown. It has previously been shown that culture media in which L-valine has been replaced with Dvaline will select for kidney tubule epithelial cells since these cells are known to contain the enzyme D-amino acid oxidase (28). We found that the use of media containing D-Vdine (DVal modified Eagle's media, GIBCO) had no adverse effect on cell growth rate, cell population at confluency, or la-hydrox45 0" ylase activity when compared with cells grownin either Way40 mouth's media or normal modified Eagle's media. In three u '. 35 3 experiments, all cultures reached confluencyin 72 h at a 30 $ population density of 10 f 3 x 10" & S.E.) cells/flask and were capable of producing 51 f 7 pmol of 1,25-(OH)zD3/flask/ 25 !?A 30 min. No significant differenceswere noted for these param20 = 5? eters using the different culture media. These data suggest I5 2 that our kidney cell cultures contain amino acid ogidase, lo which wouldbe consistent with their being principally epitheA lial in nature. ' Y 5 Identification of a 1,25-(OH,hD3Specific Binding Macromolecule in Cultured Kidney Cell Cytosol-When cytosol FRACTION FRACTION prepared from a confluent culture of chick kidney cells was FIG. 2. Sucrose density gradient analysis of 1,25labeled with 1,25-(OH)2[26,27-3H]D:tand analyzed by sucrose and 25-OH-[26,27-3H]Ds binding by chick density gradient ultracentrifugation in TKED buffer, the gra- (OH)~[26,27-~H]Ds kidney cytosol preparations. Cytosol was prepared as described in dient profiie shown in Fig.2A was obtained. A single binding text. Approximately 1.0 mgof cytosol protein was incubated with peak sedimenting in the 3.7 S region was seen. When labeled vitamin D;3 analogs and binding was analyzed on 4 to 20% sucrose in the presence of excess nonradioactive 1,25-(0H)zDa,this 3.7 gradients. Data were normalized for all gradients to 1 mg/ml of cytosolic protein. A, binding of 2.0 nM 1,25-(OH)2[26,27-'HH]D:1 S peak was totally displaced. (M and ) competition with 200 nM nonradioactive 1,25-(OH)zD:~ In contrast, cytosol prepared frommincedchickkidney in cytosol prepared from cultured kidney cells. B, binding tissue contains at least 1,25-(OH)zD3 binding species (Fig. 2C). (W) of 2.0 n~ 25-OH-[26,27-'HH]D3(A-A) by cultured cell cytosol. C, The macromolecule that sediments at 3.7 S was foundto have binding of 2.0 nM 1,25-(OH)2[26,27-'H]D3 (M and )competition a small portion of bound 1,25-(OH)2[26,27-'H]D:>displaced by with 200 nM nonradioactive 1,25-(OH)& (M by) cytosol prea 100-fold excessof unlabeled 1,25-(OH)zD,+ However, excess pared from minced chick kidney. 0,binding of 2.0 nM 25-OH-[26,27by minced kidney cytosol. 1,25-(OH)zD3also displaced 1,25-(OH)z[26,27-3H]D3 binding "JH]D:t(A-A)

(x

(x

c

f

i

Receptor la,25-Dihydroxyvitamin for from a 6.0 S binder which is shown in Fig. 2 0 to have a greater binding capacity for 25-OH-[26,27-3H]D3.This 6.0 S binder has previously been demonstrated to be the result of serum contaminant in cytosol preparations (29). A 10- to 15-fold decrease in 6.0 S 25-OH-D3 binding protein concentration in cytosol from cultured cells, relative to cytosol from kidney tissue is also shown (Fig. 2, B and D). The multiplicity of 1,25(OH)D3binding sites in cytosolfrom minced tissue convinced us that adequate characterization of the 3.7 S binding macromolecule would not be possible using this preparation. In contrast, cytosol from cultured cells contains a specific 1,25(OH)2D3 binding3.7 S macromolecule which accounts for all saturable binding. Thus, characterization of 1,25-(OH)?Ds binding in this cytosol can beasyumed to be to the 3.7 S binder. All further studies, therefore, used cytosol from cultured chick kidney cells. Fig. 3A shows that when cytosol labeled with 1,25(OH)2[26,27-'H]D3 isanalyzed onsucrose gradients containing reduced potassium chloride concentrations, the 3.7 S binding component is gradually lost with concomitant increases in the amounts of a component with a higher sedimentation coeffcient. Aggregated species sedimented as a broad peak (4 to 5.5 S ) in 60 mMKC1 and as a 6 S species in the absence of salt. Large amounts of aggregated material were also seen in the gradient pellet. Molecular Weight Determination-When 1,25-(OH)2[3H]D3-labeled cytosolwas chromatographed on a Sephacryl

DSin Chick Kidney Cells

10163

S-200 gel permeation column (equilibrated in TKED buffer), the labeled component eluted as a single peakwith an apparent M, = 72,000 (data notshown). Determinationof Binding Constants-Preliminary experiments with cytosol from cultured cells indicated that binding of 1,25-(OH)2[26,27-3H]D3 reached equilibrium by 40 min and remained stable for an additional 60 min (figure not shown). We determined thenature of the label after the binding reaction reached equilibrium. When 2 n~ 1,25-(OH)2[26,27'HID3 was incubated with 2 mg of cytosolic protein at 25°C for 1 h, greater than 95% of the extracted radioactivity chromatographed as standard 1,25-(OH)?D3using the high pressure liquid chromatography system previously described. Furthermore, the bound label was found to be greater than 96% 1,25-(OH)2[26,27-'H]D3.These data indicate that under the

40

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Bound ( p moles/mq protein)

FIG. 4. Saturation analysis of 1,25-(OH),[26,27-'WD3 binding to cytosol from cultured chick kidney cells. Cytosol (0.25

I

IO

20

FRACTION

30

PELLET

NO.

FIG. 3. Sedimentation properties of the chick kidney receptor in the presence of decreasing concentrations of KCl. A, 200 protein) were labeled with 2 nM 1.25p1 of cytosol (1.1 mgof (OH)~[26,27-~H]Dz and analyzed on 4 to 20% sucrose gradients con0.06 M KC1 (M), taining 0.3 M KC1 (M), or in the absence of KC1 (H B, ) cytosol . was labeled with 2 rn 1,25-(OH)2[26,273H]D3alone (0)or in the presence of 20 nM unlabeled 1,25-(OH)& ( O " 0 ) and was analyzed on 4 to 20% sucrose gradients in the presence of 0.3 M KCl. C, cytosol was labeled as in B but was analyzed on a 4 to 20% sucrose gradient in the absence of KC1.

mg) was incubated with 1,25-(OH)~[26,27-~H]D3 (0.01 to 1.0 nM) in a total volume of 0.5 ml at 25OC for 1 h. Specific and saturable binding was assessed by including parallel samples with 100 n~ unlabeled 1,25-(OH)~D3. Specific binding was calculated and free hormone was separated from bound by dextran-coated charcoal as described under "Materials and Methods." A, saturation plot of total (O),specific (0). and nonspecific (A) binding. B, Scatchard analysis of saturable and specific binding. The equilibrium dissociation constant (KO) for specific1,25-(OH)J26,27-3H]D3 binding was determined from the slope of the plotted regression line to be 4.36 X 10"' M and the concentration of binding sites was estimated from the abscissa intersect to be 50.6 fmol/mg of protein. Each point represents the mean of triplicate determinations, the standard error of which was less than 5%.

10164

Receptor la,25-Dihydroxyvitamin for

D3 in Chick Kidney Cells

-

binding conditions used, it can be assumed that little metabolism of the label takes place. tFig. 4.4 shows that specific 1,25-(OH)~Ds binding is satura28 0 om , ble while nonspecific and total binding arenotsaturable. , e ' " 40"-0 Analysis of the saturablebinding by the methodof Scatchard 0 2 4 7 is shownin Fig. 4B. A straight line indicating a single binding site is demonstrated. The slope of the line revealsahigh affinity binding site with an equilibrium dissociation constant (KO)of 4.36 X lo-" M. The intersect of the line with abscissa indicates a maximum binding capacity (N,J of 50.6 fmol/mg y, of protein. In three experiments with cytosol from different u '-. 4 - I I 1 1 cultures, the mean (+ S.E.) KIJwas 5.6 k 2.0 X 10."' M and I 1 1 I 70 BO 90 themean (k S.E.) N,,, was 56.8 k 5.5 fmol/mg of protein. IO 20 30 %E ?GIN? Another technique for evaluation of binding-affinity constants is by estimation of association and dissociation rate constants. Analysis of the rate of association of 1,25-(OH)2D:l with the kidney cytosol at 25"C, shown in Fig. SA, demonstrates that theprocess is relatively rapid. Evaluation of the association constant rate in is shown Fig. 5B. The slope of the regression line ( r = 0.95) which is equal to the second order association rate constant was calculated to be 9.2 X lo7 M" min". Binding of 1,25-(OH)2Dsis also shown in Fig. 5A to be a slowly reversible process. The addition of 500-fold excess unlabeled hormone to thebinding reaction after equilibrium was established results in approximately 'h displacement of v) In bound label within 60 min. The dissociation followed pseudo 'u. L fist order kinetics as indicated by the linear plot ( r = 0.93) of \ u 5 -32.4- 20In (1,25-(0H),[26,27-"H]D3 bound) uersus time. The dissocia1 1 1 1 I I , o; o; io io lo lo tionrateconstantas calculatedfrom the slope of the regres2 4 6 8 1 0 1 2 sion line in Fig. 5C was 5.0 X lo-" min.". The data permits TIME(MIN) kinetic determination of the equilibrium binding constant by FIG. 5. Kinetic analysis of 1,25-(OH)~[26,27-~H]D% binding to chick cytosol. Cytosol (0,25 mg,0,5 ml) was incubated with theratio of the dissociation rateconstanttothe association 0.5 nM 1,25-(OH)2["H]D.jintheabsenceor presence o f 100-fold excess rate 'Onstant (i.e. K' = kd/ka)' A Of 5'42 x 'O"' is nonradioactive 1,25-(OH)2D3.Time points were taken a t various times calculated, a value that is quite similar to the equilibrium by pipetting 0.5-ml aliquots of cytosol into 0.25 ml of dextran-coated dissociation constant derived by Scatchard analysis. charcoal at 0-4°C as described under "Materials and Methods." A, a Specificity of Binding to Chick Kidney Cytosol-To assess plot of saturable 1,25-(OH)2['HID.%binding versus time of incubation the specificity of the kidney cytosol binder for vitamin D:] (0)demonstrates binding equlibrium is established within 20 min and analogs, relevant compounds were incubated with cytosol in 32-

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e

L.

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maintained for an additional 70 min. Addition of 100 nM unlabeled 1,25-(OH)2D:3tothe binding reactionafter 30 min (0)is shown to displace bound label by approximately 35% within 60 min. B, the association of 1,25-(OH)~['H]D.I saturable binding analyzed as a second order binding process revealedan association rate constanto f 9.2 X 10' M- min" as calculated fromthe slope of thelinear regression line. The correlation coefficient for the data was r = 0.95. C, the dissociation of 1,25-(OH)2[:'H]D:lbinding was analyzed by plotting the bound 1,25-(OH)2["H1D,as a function of time after addition of excess

the presence Of 0'5 nM The data shown in Fig. 6 defines control binding, approximately 40 fmol 1125-(0H)Z[26,27-"H]Ds'

_______

_ _ _ _ _ _ , . ~ "

unlabeled 1,25-(0H),D:1. The dissociation rateconstantforthis pseudo first orderreaction was calculatedfrom the slopeof the regression line to be 5.0 X lo-:' min '. Analysis of data revealed a correlation coefficient of r = 0.93.

FIG.6. Competition by vitaminD3 analogs for 1,25-(0H)~[~H]D~ binding sites in chick kidneycell cytosol. Cytosol was incubated with 0.5 nM 1,25(OH)2["H]Daand several concentrations of unlabeled vitamin Da analogs for 1 h at 25°C. The control value of specific 1,25-(OH)2[aH]D:lbinding was definedas 100%and represented approximately 40 fmol/mg of protein. Each point was the mean of pentuplicate determinations (&.E.).

COMPETITOR

CONCENTRATION ( log Molar 1

Receptor for la,25-DihydroxyvitaminD3 in Chick Kidney Cells

10165

binding of relevant ligands with highaftinity andlow capacity. A correlation of receptor Ku value with the concentration of circulating hormone has been used to assess the relevance of binding macromolecules.We presently demonstrateda kidney 1,25-(OH)zDs binderwith a KD that approximatescirculating 1,25-(OH)*D3levels.' Reports have characterized chick intestinal receptors with affinity for 1,25-(OH)2D3 that is an order of magnitude less than we find for the renalbinder (2,3). It is difficult to detemine if the higher affinity value obtained in the kidney indicates site specific differences in 1,25-(OH),Dn DISCUSSION receptor molecules. Technical variations in 1,25-(OH)2D3reThe use of cell isolation and culture techniques facilitated ceptor studies may significantly alter dissociation constant estimation. A higher specific activity 1,25-(OH)2-[26,27-3H]D:, the characterization of the renal 1,25-(OH)& receptor. In (160 Ci/mmol) (as used in this study) has beenshown to yield contrast to cytosol from cultured cells, cytosol from kidney tissue fragments possessed two 1,25-(OH)~D3 binding macro- an apparent K Dvalue of 7.1 X 10"' M for the chick intestinal molecules. One of the binding proteins had a 3.5 to 3.7 S receptor (24). This value represents aseveral-foldhigher sedimentation coefficient and the second was in the 6.0 S affinity than previously reported with lower specific activity (2, 3). Anotherfactor which could influence 1,25-(OH)2D:~ region. All 1,25-(OH)& receptor-like proteins characterized to date have been found to have 3.0 to 3.7 sedimentation affhity constant evaluationwould be the presence of the 6.0 coeffkients (2-5, 11-13). While it is easy to recognize such a S binding protein. Thus, in an earlier study, cytosol prepared binder incytosol from cultured cells, the sucrose gradient from minced chick kidney tissue revealed a KI, of 1.2 X analysis of binding macromolecules from kidney minces re- M (12). This cytosol was shown to be grossly contaminated vealed only slight saturable 1,25-(0H)~D:3 binding to the3.7 S with the lower affinity, higher capacity 6.0 S binder. It seems I we found (5.5 X components. This may duplicate and explain earlier reports reasonable that the higher affinity K ~ value in which a renal 1,25-(OH)2D3receptor was not observed (10, 10"' M) in chick kidneycells may reflect the reduced amounts 11). The 6.0 S binder isclearly shown to havea preference for of 6.0 S contamination in ourcytosol preparation. An anaysis of the binding specificity of the 3.7 S macro25-OH-[26,27-aH]D:iover 1,25-OH[26,27-3H]D3.This binding protein has been reported to result from the interaction of a molecule reveals that of the vitamin Daanalogs tested, 1,25serum 4.0 S 25-OH-D:r transport proteinwith acytosolic factor (OH)2D3 is bound by the receptor with the greatest affinity. (30). It has been suggested that this interaction may occur Both 1-hydroxylation and 25-hydroxylation of vitamin Ds are inside the cell (31). Other evidence from our laboratory indi- shown tobe critical forreceptor binding. The 24,25-dihydroxcates that the6.0 S binder is a serum contaminant of cytosol ylated vitamin D3 analog was the least potent hydroxylated prepared in the presence of blood (29). Our data supports the vitamin DRcompound tested. Vitamin Ds itself was found to latter conclusion in that we found little 6.0 S binder in cytosol be void of receptor binding. The relativepotency of the agents from washed, isolated,and culturedcells. Cytosol from washed tested for receptor binding parallels their reported potency in mincedkidney, on the other hand, was found to possess stimulatingintestinal calcium transportand bonecalcium appreciable 6.0 S binder. Our overall conclusion from sucrose mobilization (34). Stumpf et al. have demonstrated intracellular localization gradient analysis(Fig. 2) is thatcytosol from the kidney tissue minces possesses low saturable binding to heterogeneousbind- of 1,25-(OH)?D3 in rat distal tubules (9). Recently, Colston ing macromolecules while cytosol from cultured kidney cells and Feldman showed that isolated rat renal tubules contain a 3.2 S cytosolic 1,25-(OH)zD:ireceptor-like protein (13).We will yield information specific to the 3.7 S binder. Like other steroid hormone receptors, the chick intestinal presently report that chick kidney cells which propagate in 1,25-(OH)zD3receptor has recently been shown to aggregate culture possessa 1,25-(OH)2Ds receptor. The data suggest in buffers of low ionic strength (26). This process appears to that the kidney cells we are studying are derived from renal be reversible and can be explained, at least in part, by the tubules. Ourfinding that thecells grow equally well in a media nonspecific interaction of the receptorwith other cytoplasmic (D-Vdine MEM, GIBCO) that selectsfor tubule-epithelial cells are shown to components. Similar types of aggregates have been reported cells supports this contention. The cultured respond to 1,25-(OH)2D3 (10 nM) addition by decreasing 1,25in chick bone cytosol (32), and in cytosol from rat bone (5) and intestine(33). In the present study, we have shown similar (OH)2D3production and increasing 24,25-(OH)~D3 production from 25-OH-D3. This has previously been demonstrated in aggregation in chick kidney cell cytosol. Scatchard anaysis reveals that the 1,25-(OH)2Da receptor- vivo and with similar cell culture systems (6, 7, 14, 15). The alteration 25of like protein fromkidney cell cytosol has an apparentdissocia- mechanism of 1,25-(OH)2Da-mediated tion binding constant ( K n ) of 5.6 X lo-'' M and is present at (OH)zD:, metabolism is not simply a product feedback inhia concentration of approximately 57 fmol of receptor/mg of bition, but has been shown to require a period of time and is protein. A similarKn valuewas obtained by kineticevaluation blocked by transcriptional inhibitors (7, 14, 15). Although there are inherent flaws in correlating data from of the association ( k , = 9.2 X 10' M" min") and dissociation (kd = 5.0 X lo-" min") rate constants and subsequentcalcu- other studies on 1,25-(OH)~D.3receptors present in various lation of the K D from the ratio of kd/k,. In a recent study of target organs, a remarkable similarity seems toexist. All 1,25a 10-fold difference be- (OH)2D3receptors are 3.0 to 3.7 S cytosolic macromolecules. the intestinal 1,25-(OH)~D3 receptor, to 10"' M) for 1,25tween the K D values estimated by equilibrium and kinetic Theyhave a highaffinity (KT,= methods was noted. The authors suggested that this finding (OH)2Ds and are present in low concentrations (50 to 300 may be a result of contamination of the intestinal cytosol by fmol/mg of protein). Analog specificity studies for various 25-OH-D:3 6.0 S binder. Our finding of little or no disparity 1,25-(OH)2D3receptors closely resemble the one presented for between kinetic and equilibrium Kr) values in cytosol rela- the kidney 3.7 S binder. The molecular weight and aggregatively free of the 6.0 S 25-OH-Ds binding protein supports this tional properties of 1,25-(OH)2D,~ receptor-like molecules are conclusion. ' A. Hamstra and H. F. DeLuca, unpublished results. An important characteristic of a receptor molecule is the

of 1,25-(OH)2Da/mgof protein, asbeing that which occurs in the absence of competing analog. The relativeaffinity of analogs for receptor binding was determined by the concentration of analog required to decrease 1,25-(OH),-[26,27-"HID3 binding by 50%. From Fig. 6, the relative potency of the various vitamin Da analogs tested can calculated. be Assuming 1,25-(OH)2Dato be 1, the molar potency ratios of the compounds tested are shown to be as follows: 25-OH-D:j = 470; lcu-OH-D:{= 880; 24,25-(OH)2D3= 1765; vitamin D3 > 11,000.

10166

Receptor for la,25-Dihydroxyvitamin 0 3 in Chick Kidney Cells

Commun. 89,56-63 also similar (26, 32). If identical receptors for 1,25-(OH)zD3 mediate each target site response to hormone, site specific 13. Colston, K. W., and Feldman, D. (1979) J. Clin.Endocrinol. Metab. 49,798-800 modulation of 1,25-(OH)2D3activities would be due to factors 14. Henry, H. L. (1979) J.Biol. Chem. 254, 2722-2729 other thanthe hormone-receptor interaction.This might fur- 15. Spanos, E., Brown, D. J., and MacIntyre, I. (1979) FEBS Lett. ther suggestthat site specific receptoragonists or antagonists 105,31-34 for 1,25-(OH)2D3may be difficultto design. 16. Juan, D., and DeLuca, H. F. (1977) Endocrinology 1, 1184-1193 These results suggest that the effects of 1,25-(OH)zD3 on 17. Chandler, J. S., Pike, J. W., and Haussler, M. R. (1979) Biochem. Biophys. Res. Commun. 90,1057-1063 renalvitamin D3 metabolismmayinvolvechangesingene J., Holick, M., Suda, T., Tanaka, Y., and DeLuca, H. F. expression, presumably through a receptor-mediated process. 18. Omdahl, (1971) Biochemistry 10,2935-2940 The use of cell culture techniques should prove beneficial for 19. Napoli, J. L., Fivizzani, M. A., Hamstra, A. J., Schnoes, H. K., studying the relationshipbetween the characterized1,25and DeLuca, H. F. (1979) Anal. Biochem. 96,481-488 (OH)2D3receptor-like proteinand control of renal vitaminDa 20. Frolik, C. A., and DeLuca, H. F. (1973) J. Clin. Invest. 52, 543548 metabolism. 21. Jones, G., and DeLuca, H. F. (1975) J.Lipid Res. 16,448-453 22. Bligh, E. G., and Dyer, W.J . (1959) Can. J . Biochem. Physiol. REFERENCES 1. DeLuca, H. F. (1978) Annu. Reu. Biochem. 45,631-666 2. Tsai, H. C., and Norman, A. W. (1973) J. Biol. Chem. 248,59675975 3. Brumbaueh. P. F.. and Haussler. M. R. (1974) J.Biol. Chem. 249, 1258-1262 4. Kream. B. E.. Jose. M.. Yamada., S.., and DeLuca. H. F. (1977) Science 197, l086-10& 5. Chen, T. L., Hirst, M. A., and Feldman, D. (1979) J.Biol. Chem. 254,7491-7494 6. Tanaka, Y., Lorenc, R. S., and DeLuca, H.F. (1975) Arch. Biochem. Biophys. 171, 521-526 7. Colston, K. W., Evans, I. M. A,, Spelsberg, T. C., and MacIntyre, I. (1977) Biochem. J.164,83-89 8. Chen, T. C., and DeLuca, H. F. (1973) Arch. Biochem. Biophys. 156,321-327 9. Stumpf, W. F., Sar, M., Reid, F. A., Tanaka, Y., and DeLuca, H. F. (1979) Science 206. 1188-1190 10. Brumbaugh, P. F., Hughes, M. R., and Haussler, M. R. (1975) Proc. Natl. Acad. Sci. U. S. A . 72,4871-4875 11. Kream, B. E., Yamada, S., Schnoes, H. K., and DeLuca, H. F. (1977) J . Biol. Chem. 252,4501-4505 12. Christakos, S., and Norman, A. W. (1979) Biochem. Biophys. Res.

37,911-917 23. Tanaka, Y., and DeLuca, H. F. (1980) Methods Enzymol. 67F, 370-385 and DeLuca, H. F. (197 9) Arch. Biochem. Biophys. 24. Mellon, W. S., 197,90-95 25. Scatchard, G . (1949) Ann. N. Y.Acad. Sci. 51,660-672 26. Franceschi, R. T., and DeLuca, H. F. (1979) J. Biol. Chem. 254, 11629-11635 27. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 28. Scott, G., and Migeon, B. R. (1975) Cell 5,ll-17 29. Krearn, B. E., DeLuca, H. F., Moriarity, D. M., Kendrick, N. C., and Ghazarian, J. G . (1979) Arch. Biochem. Biophys. 192,318323 and DeMoor, P. (1977) J. B i d . 30. Van Baelen, H.,Bouillon,R., Chem. 252,2215-2518 31. Cooke, N. E., Walgate, J., and Haddad, J . G . (1979) Clin. Res. 27, 44A 32. Mellon, W. S., and DeLuca, H. F. (1980) J. Biol. Chem. 255, 4081-4086 33. Feldman, D.,McCain, T. A,, Hirst, M. A,, Chen, T. L., and Colston, K. W. (1980) J.Biol. Chem. 254, 10378-10382 34. DeLuca, H. F., Paaren, H. E., and Schnoes, H. K. (1979) Topics in Current Chemistry, Vol. 83,pp. 1-65, Springer-Verlag,Berlin