sign in sign up
<<
Home , Glucocorticoid, Glucocorticoid receptor, Progesterone receptor

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 1123-1127, February 1989 Biochemistry Binding of accelerates the kinetics of and binding to DNA (hormone receptors/mouse mammary tumor virus/gel retardation/methylation interference) MICHAEL SCHAUER, GEORGES CHALEPAKIS, ToIVO WILLMANN, AND MIGUEL BEATO* Institut fur Molekularbiologie und Tumorforschung, Philipps-Universitat, Emil-Mannkopff-Strasse 2, D-3550 Marburg, Federal Republic of Germany Communicated by Elwood V. Jensen, October 17, 1988

ABSTRACT hormone receptors induce by ation by dimethyl sulfate in vivo only after hormone admin- virtue of their interaction with DNA regulatory sequences. The istration, whereas no protection is detected prior to hormone hormonal response of a particular in vivo correlates with treatment (5). binding of the hormone to the receptor and supposedly reflects To understand the precise role of the hormone in the the degree of occupancy of the corresponding DNA regulatory interaction of the receptor with DNA we have analyzed the sequences. However, in vitro the steroid-free glucocorticoid and ability of the hormone-free to bind to progesterone receptors bind specifically to the regulatory the HRE in the long terminal repeat (LTR) of mouse sequences of mouse mammary tumor virus, thus raising mammary tumor virus (MMTV) in vitro. Contrary to the questions on the role of the hormone in DNA binding in vivo. prediction of the prevalent model, the hormone-free gluco- By using monoclonal , gel retardation assays, and corticoid receptor in crude binds to the HRE of filter binding techniques we show here that binding of a MMTV, yielding a DNase I protection pattern indistinguish- functional steroid to either the glucocorticoid or the proges- able from that of the hormone-bound receptor (6). In the terone receptors influences the kinetics of the -DNA presence and the absence ofthe hormone a thermal activation interaction in vitro. In the presence of hormone the on rate of step is required for DNA binding to occur and activation is receptor binding to DNA fragments with or without regulatory inhibited by molybdate (6). Thus, binding of the sequences is accelerated 2- to 5-fold, and the off rate is hormone to the receptor is not an absolute requirement for accelerated 10- to 20-fold. The receptors complexed to an exposure of the DNA-binding domain of the protein. Similar antihormone bind to DNA with kinetics intermediate between findings have been reported for the purified progesterone those ofthe steroid-free and the hormone-bound protein. Thus, receptor (7). ligand binding accelerates the kinetics of receptor binding to In an attempt to solve the apparent discrepancy between DNA and this could partly account for the behavior of the these results and those reported in vivo (5), we have studied observed in vivo. the influence ofhormone binding on the kinetic parameters of the DNA-binding reaction. Here we show that, although the The cis elements that mediate gene induction by steroid glucocorticoid and the progesterone receptors recognize are hormone-dependent enhancers (1) and have selectively the HRE of MMTV in the absence of hormone, been designated hormone-responsive or -regulatory elements binding ofthe corresponding hormone accelerates the on and (HREs). The trans-acting factors that interact with the HREs offrates ofthe receptorbinding to DNA. On the basis ofthese are the receptors, a family of that findings we proposed a model that could account for the has been widely studied due to their ability to specifically behavior of the hormone receptors observed in vivo. bind the corresponding hormone. The domain ofthe receptor that binds the steroid hormone is located in a large region of MATERIALS AND METHODS the C-terminal half of the protein and is separated from the DNA-binding domain by a hinge region (2). Exactly how the Plasmids and Receptors. The following DNA fragments DNA-binding domain and the steroid-binding domain of the were used in the binding studies. (i) A 600-base-pair (bp) receptor interact is not known. BamHI-EcoRI fragment from p13-13 (8) containing the four The magnitude of the hormone response observed in vivo binding sites previously reported for the glucocorticoid and correlates with the degree of saturation of the corresponding the progesterone receptors (9, 10). (ii) A 450-bp fragment receptor (see, for instance, ref. 3). According to the prevail- containing vector sequences from p13-13. (iii) A 370-bp ing view, binding of the hormone to the receptor is followed fragment containing 10 linked copies of an oligonucleotide by a conformational change of the protein leading to the corresponding to the distal binding site of the exposition of the preformed DNA-binding domain that is MMTV-HRE (9). This oligonucleotide subcloned into the Sal masked in the hormone-free receptor (1). Whether this I site ofpTK-CAT-3 (15) gives rise to the plasmid pMMTV-1. masking is accomplished intramolecularly by the steroid- cytosol was freshly prepared from male rats adre- binding domain or intermolecularly by interaction with other nalectomized 48 hr before the preparation as described (11). factors, such as the 90-kDa , is a matter of The cytosol was passed through a column of calf thymus debate (4). In cell fractionation studies one consequence of DNA cellulose at 0°C to remove DNA-binding proteins (6) hormone administration is the association of the otherwise and incubated with 0.1 ,uM [3H] acetonide cytosolic receptor with the nuclear fraction, suggesting a tighter interaction with DNA in chromatin. In agreement with Abbreviations: TA, , 9a-fluoro-11/3,17a,21- this model, the HRE of the tyrosine amino transferase gene tetrahydroxypregnane-1,4-diene-3,20-dione cyclic 16,17-acetal with in rat liver has been shown to be protected against methyl- acetone; Org 2058, 16a-ethyl-21-hydroxy-19-norpregn-4-ene- 3,20-dione; RU 486, 17f3-hydroxy-llp-(4-dimethylaminophenyl- 1)-17a-(prop-1-ynyl)estra-4,9-dien-3-one; HRE, hormone-respon- The publication costs of this article were defrayed in part by page charge sive or -regulatory element; MMTV, mouse mammary tumor virus; payment. This article must therefore be hereby marked "advertisement" LTR, long terminal repeat. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1123 1124 Biochemistry: Schauer et al. Proc. Natl. Acad. Sci. USA 86 (1989) [9a-fluoro-1 1,X,17a,21-tetrahydroxypregnane-1 ,4-diene-3, allow a precise quantitative evaluation of the kinetic results, 20-dione cyclic 16,17-acetal with acetone (TA); specific in particular of the initial rate of association. For this reason radioactivity, 5 Ci/mmol; 1 Ci = 37 GBq] or without hormone we decided to use a more rapid nitrocellulose filter binding at 250C for 30 min. This cytosol has been shown to be free of assay (10). This method yields highly reproducible results. endogenous (6). The receptor content was When hepatic cytosol is passed through a calf thymus determined by the dextran-coated charcoal technique (12). DNA-cellulose column at 40C before the beginning of the Uterine cytosol was prepared as described (13) from fresh rabbit uteri obtained from animals injected three times with A ON-IRATELTR Z ON-RATLector 3 OFF-RATE/.LTR 17,8- (150 Ag per injection 2, 4, and 6 days before (600 bp) (456 bp) (66 bp) removal of the uteri). The cytosol was passed at 0C first a through a phosphocellulose column and then through 35 a88 DNA-cellulose column. The flow-through ofthe columns was 'TA i -Hormone incubated for 60 min at 0C with 0.1 AM 3H-labeled 16a- 30 36 ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione ([3H]Org 25 2 'TA 66 2058) or without hormone, and the receptor content was 28 28- determined (12). The level ofendogenous progesterone ofthe 4- treated cytosol was determined using a radioimmunoassay kit (Baxter Merz & Dade, Dudingen, Switzerland) and found to 151 15 I0 I be <0.08 ng/ml. Assay. DNA binding of the glucocor- 4 2 4 8 2 4 Immunoprecipitation 8 2 9 8 6 8 16 12 ticoid receptor was detected using the monoclonal GR49/4 as described (6, 14). 3 Nitrocellulose Filter Binding Assay. For the filter binding LTR IHRE Vector assays, incubations were performed at 25°C (11). The off-rate measurements were performed using 500 ng of the unlabeled 48 ; a/ IIU 48 48 LTR fragment as competitor and were corrected for the 'TA as the decay of receptor-DNA complexes described for 38 'TA 38 38... immunoprecipitation assay. 'TA Shift Analysis. The plasmid pMMTV-1 was Gel Mobility 28 - -Hormone 28j- digested with HindIII and BamHI and labeled on both 5' ends, and the 57-bp fragment containing the entire MMTV -HH; 18' 18 region was isolated from a NuSieve (FMC) agarose gel. A typical reaction mixture contained 0.05 ng (5000-10,000 cpm) 44 of end-labeled DNA fragment, 1 ng of cytosolic progesterone a0 4 60 0 2 4 G 10 0 2 -a receptor, which was added last, and 100 ng of poly(dA-dT) in 3 a reaction volume of 25 ul. A 4% polyacrylamide gel (40:1), 1 LTRKRE Vector containing 0.3 x Tris-borate/EDTA buffer, was preelectro- phoresed for 1 hr at 11 V/cm. Following binding, the samples ynylestra-4,-dien--one 4 ,-Hormone were loaded onto the gel and electrophoresis was continued for 45 min. For competition binding reactions, the specific DNA08agments88sdeerind.Vaue ae heavrae-Hormone lt18(.0'\ 68o. B. competitor DNA (500-1000 ng) was added after 30 min of U46 incubation, and the samples were loaded onto the gel at the \ RU406 times indicated in the legend to Fig. 3B. Methylation Interference Experiments. The end-labeled Frshliae yoo toomadeaec48~~~~~~~U8iedrtpsethoga with178-hdoy20- we2028-86dmthlmnphnl1)1c-(rp DNA fragment (see above) was partially methylated (16) and *TA subsequently added to a preparative binding reaction. After acivain 02°C30ash ieic fbnigt 'tifeetS-aeeTA electrophoresis, the separated DNA fragments were elec- d 10283848i0600th 01828r384856fi 18a2838a485 wa6 troblotted overnight onto Whatmann DE81 paper. Radioac- tive bands were localized by autoradiography, and the DNA FIG. 1. Kinetics of glucocorticoid receptor binding to DNA. was eluted from the excised paper and analyzed on a 15% Fresh liver cytosol from adrenalectomized rats, passed through a acrylamide/8 M gel (16). column ofcalfthymus DNA-celluloseb'C,at was incubated with TA, with 17f3-hydroxy-11f3-(4-dimethylaminophenyl-l)-17a-(prop-1- ynyl)estra-4,9-dien-3-one (RU 486), or without hormone. After heat RESULTS activation (25vC, 30am the kinetics ofbinding to different 5'-labeled in DNA fragments was determined. Values are the average of triplicate DNA-Binding Kinetics of the Glucocorticoid Receptor determinations, and the variation of individual measurements was Liver Cytosol. The kinetics of the binding reaction of rat liver <20%. (A) Detection of complexes with monoclonal receptor anti- glucocorticoid receptor to various DNA fragments contain- bodies. Different times after addition of the DNA, the complex of ing or lacking the HRE of MMTV is depicted in Fig. 1. Initial receptor and DNA was measured by incubation with the mouse experiments (Fig. L4) were performed with crude hepatic monoclonal antibody GR49/4 and precipitation with anti-mouse IgG cytosol and monoclonal receptor antibodies as described (6). linked to Pansorbin (6). Thus, for the on-rate measurements (nos. 1 The on and off rates are both accelerated by binding of the and 2), the values at time 0 are already high, because association synthetic glucocorticoid analog TA. The effect on the off rate takes place during immunoprecipitation. For the off-rate measure- is more dramatic, thus explaining the higher DNA binding ments (no. 3), a 100-fold excess of nonradioactive LTR fragment was observed at equilibrium in the absence of the ligand (6). added at time 0. The amount of DNA bound at this point was taken as 100%.o The values are corrected for the decay of the complexes Similar enhancement of the on rate is observed with frag- observed in the absence of nonradioactive competitor DNA. (B) ments containing 10 copies of a synthetic HRE (data not Measurement of on rate with filter binding assay and different DNA shown) and with nonspecific vector DNA fragments (Fig. LA, fragments. Each volume represents the average of three determina- compare 1 and 2). tions that differed by <20%o. (C) Measurement ofthe offrate with the The time needed for immunoisolation ofthe receptor-DNA filter binding assay. The values are the average of duplicate deter- complexes limits the flexibility ofthis technique and does not minations. Individual measurements differed by <15%. Biochemistry: Schauer et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1125 experiments, the extent of DNA binding observed with the 50 .- FIG. 2. Influence of pH on the filter technique is comparable to that determined by immu- binding of glucocorticoid receptor noprecipitation (Table 1). In addition, if the glucocorticoid +TA to DNA. Incubation of cytosol receptor is removed from cytosol by adsorption to a dexa- glucocorticoid receptor, free of -agarose matrix, little DNA binding in the super- hormone or preincubated with 0.1 /LM TA or 0.1 ,uM RU 486, with a natant can be detected by either the immunological assay or 600-bp radioactive LTR fragment the filter assay (Table 1). Thus, we conclude that under these 301w / \ was performed at 250C for 10 min conditions the nitrocellulose filter binding assay reflects as described in Materials and binding of the glucocorticoid receptor to DNA. Methods, with the only difference /RU486' that the final of the A more precise measurement of the reaction rates with the 20. = pH binding filter assay shows that binding of the steroid accelerates the sreaction was adjusted as indicated on rate 4- to 5-fold and the off rate 20-fold (Fig. 1 B and C). -Hormone in the abscissa. The percentage of These effects were observed with two different fragments total DNA bound was determined 10 , I in triplicate by the filter binding containing either the natural LTR HRE (600 bp) or 10 copies 6 6.5 7 7.5 8 technique and the average is of a synthetic 37-bp HRE (370 bp) as well as with a pH shown on the ordinate. nonspecific vector fragment (450 bp). In the presence and in the absence of steroid the on rate was similar for the different uterine binds to the HRE of we fragments (2-fold difference), whereas dissociation from the cytosol selectively MMTV, used the band retardation assay combined with methylation fragment containing the synthetic HRE was slower by a interference factor of 4.5 than from the vector fragment. The effect of experiments (19). For this purpose we used a hormone does not result from a change in the proportion of 57-bp synthetic oligonucleotide containing a perfect copy of receptor molecules able to interact with DNA. At saturating the promoter distal binding site of MMTV from -187 to concentration of DNA >85% of the receptor molecules were -157. A complex, Pr-i, migrating more slowly than free bound to DNA, regardless of whether the hormone was DNA, f, is detected in the presence of cytosol, with or present (data not shown). The rate parameters observed in without hormone, and this complex shows the same mobility the presence of the antihormone RU 486 were intermediate. as observed with partially purified The on rate was very close to that found with the steroid-free (Fig. 3A). receptor, whereas the off rate was closer to that found in the The specificity of the interaction is demonstrated by the presence of TA (Fig. 1). results of methylation interference experiments. As shown in The effects of the hormone ligand are observed at different Fig. 4, the slowly migrating complexes observed in the temperatures (not shown) but depend more strongly on the presence of cytosol show very weak methylation at the pH ofthe incubation mixture (Fig. 2). Similar pH dependence guanine -174 in the strand and at the guanines -180 was found with all DNA fragments tested (not shown). Within and -171 in the antisense strand, indicating that methylation the range studied, the extent of DNA binding observed after at these positions interferes with progesterone receptor 10 min of incubation at 25TC is influenced by pH only in the binding. These are the positions that have been identified as presence of TA, with optimal binding occurring at pH 7.0. sites of intimate contact between the HRE and purified Below pH 6.5 or above 7.5 there is little difference between progesterone or glucocorticoid receptors (ref. 20 and G.C., DNA binding in the absence of hormone or in the presence unpublished results). Thus, these results strongly suggest of either RU 486 or TA. Since binding of TA to the receptor that the complexes observed in the band retardation assay in is optimal at pH 7.5 rather than 7.0 (18), it is improbable that the presence of uterine cytosol reflect specific binding of the the pH effect on DNA binding reflects simply the influence progesterone receptor. The methylation interference pattern of pH on affinity of the receptor for the steroid ligand. It is is not changed by addition ofthe , suggesting that free conceivable that protonation of a histidine residue of the and occupied progesterone receptor recognized the DNA receptor participates in hormone-induced conformational double helix in analogous ways. changes responsible for the observed acceleration of the These results clearly show that the band retardation assay DNA-binding parameters. can be used with crude cytosol to measure the kinetics of DNA-Binding Kinetics of the Progesterone Receptor in dissociation of the progesterone receptor from the LTR Uterine Cytosol. To test whether the progesterone receptor in fragment. As with the glucocorticoid receptor, we find with uterine cytosol that binding of a synthetic agonist, Org 2058, Table 1. Comparison of immunoprecipitation and filter accelerates the rate of the receptor dissociation from the binding assays complex with DNA (Fig. 3B). The half-life of the receptor- % DNA binding DNA complexes is decreased by a factor of20 in the presence Cytosol TA MMTV HRE Vector of the agonist (Fig. 3C). receptor, ng Method (0.1 ,uM) (600 bp) (450 bp) To determine more precisely the kinetic parameters of the DNA binding reaction with the HRE of MMTV we performed 200 Immp. - 35.6 5.1 nitrocellulose filter binding assays using rabbit uterine cyto- 200 Immp. + 21.0 5.0 sol without hormone or after incubation with the synthetic 200 Filter - 40.9 8.2 progestin Org 2058 As with liver addition 200 Filter + (Fig. 5). cytosol, of 21.2 5.5 the hormone analogue accelerates the rate of association of the 11* Filter - 4.1 4.0 to LTR and 11* + receptor vector fragments (Fig. 5A). This effect is Filter 3.5 3.9 less pronounced than for the glucocorticoid receptor, but this Immp., immunoprecipitation. Filter, filter binding. Values repre- may be due to the faster kinetics observed with the uterine sent the average of two duplicate determinations. Variation among cytosol. The effect ofthe ligand on the rate ofdissociation ofthe the duplicates was <15%. Incubation was at 250C for 30 min and progesterone from immunoprecipitation or filtration through nitrocellulose was as receptor DNA is more evident (Fig. SB). In described in the text. particular, with the LTR fragment, the very slow dissociation *The cytosol was passed through a column of rate observed in the absence of hormone is dramatically immobilized to agarose (17). This procedure resulted in removal of accelerated by binding ofOrg 2058. Binding of the antihormone 95% of the [3H]TA-binding activity as determined by the charcoal- RU 486 has little effect on the dissociation rate, which is very dextran technique (12). similar to that of free receptor. 1126 Biochemistry: Schauer et al. Proc. Natl. Acad. Sci. USA 86 (1989) A

Pr-1 FIG. 4. Methylation interference ex- periment with progesterone receptor. The MTV-57 DNA fragment was labeled 1 * _ -180 we w ar on both 5' ends with [y-32P]ATP and T4 W-, _ -174 polynucleotide kinase and premodified 1 2 3 4 5 6 7 189 1 134 by treatment with dimethyl sulfate (16). After incubation with or without cytosol, B the samples were applied onto a poly- Time Competitor w - -171 acrylamide gel (see Fig. 3), and free or 0T (min) 0 OrgrgDNA complexed DNA was isolated. After 0 + + cleavage with piperidine, the samples WIm 2 + + were analyzed in a 20% polyacrylamide/ 2 WI~ urea sequencing gel (16). Lanes 1 and 3, 3 Sp a 8 + + pattern of cleavage with free DNA; lanes 4 12 + + 2 and 4, pattern of cleavage in the re- 5 30 + + tarded bands obtained with uterine cyto- .I ~~~ 9i 0 + sol either free of hormone or incubated 6 - with Org 2058, respectively; lane 5, 7 2 - + cleavage pattern of DNA treated with 8 a 20 - + dimethyl sulfoxide in the absence of cy- 9 45 - + . _- - * tosol. The numbers refer to the transcrip- M. 1 2 3 4 5 tion start point in the MMTV LTR. 1 0 I 90 - + 1 1 9 30 + - - I DISCUSSION 1 2 90 - 1 3 r~~~~ Free DNA The data reported in this paper confirm previous reports showing that the glucocorticoid and the progesterone recep- tors are able to bind to the specific sequences called HREs in C the absence of the corresponding hormone ligand (10, 11). 102 That the observed binding with uterine cytosol is due to the progesterone receptor is suggested by the electrophoretic -Hormone mobility of the receptor-DNA complexes in acrylamide gels la c and by the observed pattern of methylation interference. The 0 level of endogenous progesterone in the uterine cytosol used 4c 101 for binding assays is almost undetectable, and therefore we z 0 conclude that the majority of the progesterone receptor +Org2058 molecules in our preparations are free of hormone. Thus, in the absence of ligand the progesterone receptor binds to the HRE of MMTV. 100 Binding of a synthetic agonist to either the glucocorticoid 0 20 40 60 80 100 or the progesterone receptors accelerates the kinetics of the Time (min) interaction with DNA in vitro. The rate of association of FIG. 3. Band retardation assay with uterine cytosol. (A) Com- receptor and DNA is highly dependent on the starting parison of band retardation by partially purified progesterone recep- concentrations of both macromolecules. Under the condi- tor and uterine cytosol. Cytosolic progesterone receptor was incu- tions used in these studies, this rate is fast in the absence of bated with Org 2058 (10 nM) or without hormone for 1 hr at 00C. hormone, but even faster in the presence of an agonist ligand. Progesterone receptor was partially purified as described in the text. In the case of the glucocorticoid receptor, the increase in the Different amounts of cytosol, with or without hormone, or purified association rate is about 4-fold, whereas it is 2-fold for the progesterone receptor were incubated with 0.05 ng of MTV-57 DNA of the RU 486 to (57-bp HindIII-BamHI fragment from pMMTV-1) for 20 min at room progesterone receptor. Binding antagonist temperature and then loaded onto a polyacrylamide gel, which was the glucocorticoid receptor does not alter the rate of associ- run and processed. Shown is an autoradiogram after overnight ation as compared to the hormone-free protein. exposure. Lanes 1 and 2, free DNA without receptor; lanes 3, 4, 5, A more significant effect ofhormone binding is observed in and 6, 0.125, 0.5, 1.25, and 2.0 ng, respectively, of cytosol receptor studies of the dissociation rate of preformed receptor-DNA incubated with hormone; lanes 7, 8, 9, and 10, 0.125, 0.5, 1.25, and complexes. This reaction follows an apparent first-order 2.0 ng, respectively, of cytosol receptor without hormone; lanes 11, kinetics and probably reflects the intrinsic stability of the 12, 13, and 14, 4, 8, 12, and 16.5 ng, respectively, of purified complexes since the experimental results are markedly in- hormone-receptor complexes. See text for discussion of Pr-1 and f. fluenced by the nature of the DNA fragment used. For the dissociation from a (B) Kinetics of progesterone receptor synthetic and the progesterone receptors, binding of the oligonucleotide. Cytosolic progesterone receptor was incubated with glucocorticoid Org 2058 or without hormone for 1 hr at OC. Amounts of cytosol active hormone analogues enhances the dissociation rate by containing 1 ng of receptor were incubated with 0.05 ng of MTV-57 an order of magnitude. These results are obtained with HRE DNA for 30 min at room temperature (see above). At time 0, 500 ng containing fragments as well as with vector DNA. Binding of of specific competitor DNA was added, incubation was continued at the antihormone RU 486 to either the glucocorticoid or the room temperature, and samples were loaded onto the gel after different time intervals. Lanes 1-5, cytosol incubated with Org 2058; lanes 6-10, cytosol without hormone; lanes 11 and 12, receptor- (C) Quantitative evaluation of the gel shown in B. The bands DNA complexes without added competitor DNA; lane 13, free DNA corresponding to free and bound DNA were quantitated by using a without receptor. The migration of the free DNA and the protein- video microdensitometer, and the values are expressed semilogarith- DNA complexes apparently decreases with increasing incubation mically as % bound DNA as a function of time after addition of times, since the samples were loaded onto the gel at later time points competitor DNA. The values are corrected for the spontaneous decay and thus were electrophoresed for progressively shorter time. of protein-DNA complexes in the absence of competitor DNA. Biochemistry: Schauer et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1127 A HRE observed in vivo (5). Attempts to detect glucocorticoid receptor molecules vivo have LTR (688 bp) Vector bp) bound to the HRE of MMTV in 58 (458 been unsuccessful (22), probably reflecting a relatively short residence time of the hormone-receptor complex on the 48 483 HRE. Recently it has been reported that binding of hormone *Org20g8 but not of an antihormone facilitates the dissociation of the receptor from a complex with the 90-kDa heat shock protein, which seems to be a component of the nonactivated receptor form (4). Our results cannot be influenced by this type of o interaction, since we performed our experiments with ther- / 0 8-Hormone mally activated receptor already free of the 90-kDa protein. We do not know whether the various effects of the ligand (dissociation from the 90-kDa protein, acceleration of DNA / / 2 4 6 a 8 2 _4 6 8 binding kinetics, and activation) are directly re- Time (min) Time (min) lated, but they respond in a similar way to binding of the antihormone RU 486. This ligand is less effective in promoting the dissociation ofthe receptor from the 90-kDa protein, results 18-/LIR (68 bp) 184W46Vector/o (458 bp) in less dramatic changes ofthe DNA-binding kinetics, and does not lead to transcriptional activation. Thus, it is conceivable that -Hormone ~ ~ -oron the three phenomena reflect different aspects of the same 88%.~~~~~~~~~~ ligand-induced conformational change of the receptor. The intimate relationship between these phenomena awaits a better understanding of the receptor structure. 68 11iJR486 68 Independently of the physiological significance of these \AJW486 particular results, we consider it important to draw the attention 48 48 of researchers interested in DNA-binding regulatory proteins to OrS8\Org28' -6- the quantitative and kinetic aspects of the reaction. Conven- ~ ~ ~ 0rg85 tional methods, such as DNase I footprinting, only provide 20 28 A I i information on the 1 28 30 48 8 18 28 38 483 specificity of binding and may only be fully Time (mini) Time (mini) interpretable when complemented by kinetic studies. We thank P. Formstecher (Lille) for the dexamethasone-agarose FIG. 5. Kinetics of association (A) and dissociation (B) of matrix, Hannes Westphal for the monoclonal receptor antibodies, progesterone receptor and DNA. Fresh uterine cytosol, passed and Bernhard Gross for the preparations of receptor. This work was through columns of phosphocellulose and DNA cellulose, was supported by grants from the Deutsche Forschungsgemeinschaft and incubated with Org 2058, with RU 486, or without hormone for 1 hr the Fonds der Chemischen Industrie. at 0OC. After salt activation (0.5 M NaCl, 60 min) the kinetics of 1. Yamamoto, K. R. (1985) Annu. Rev. Genet. 19, 209-252. binding to different 5'-labeled DNA fragments was determined in 2. Evans, R. M. (1988) Science 240, assay. reaction 889-895. duplicate by the filter binding For each association 3. Beato, M., Kalimi, M. & Feigelson, P. (1972) Biochem. (A) 3-5 ng of cytosol progesterone receptor and 5-10 ng of labeled Biophys. ,ul Res. Commun. 47, 1467-1472. DNA fragment were incubated in 60 (final volume) containing 10 4. Groyer, A., Schweizer-Groyer, G., Capedon, F., Mariller, M. & mM Tris HCl (pH 7.0), 0.5 mM Na2EDTA, 100 mM NaCl, 10% Baulieu, E. E. (1987) Nature (London) 328, 624-626. , 1 mM dithiothreitol, and 200 ng of sheared calf thymus 5. Becker, P. B., Gloss, B., Schmid, W., Strahle, U. & Schutz, G. DNA at0OC for different times. For the off-rate measurements (B), (1986) Nature (London) 324, 686-688. after incubation at 250C for 30 min, a 100- to 200-fold excess of 6. Willmann, T. & Beato, M. (1986) Nature (London) 324, 688-691. nonradioactive LTR fragment was added at time 0, and samples were 7. Bailly, A., LePage, C., Rauch, M. & Milgrom, M. (1986) EMBO J. filtered after different time intervals. 5, 3235-3241. 8. Hynes, N. E., van Ooyen, A., Kennedy, N., Herrlich, P., Ponta, H. progesterone receptor resulted in a dissociation rate inter- & Groner, B. (1983) Proc. Natl. Acad. Sci. USA 80, 3637-3641. mediate between that observed with the free receptors and 9. Scheidereit, C., Geisse, S., Westphal, H. M. & Beato, M. (1983) that found with the hormone-bound proteins. Nature (London) 304, 749-752. of the 10. Chalepakis,G., Arnemann, J., Slater, E., Bruller, H. J., Gross, B. How could this kinetic effect induced by binding & Beato, M. (1988) Cell 53, 371-382. ligand be related to the observed behavior of the glucocor- 11. Geisse, S., Scheidereit, C., Westphal, H. M., Hynes, N. E., ticoid receptor in vivo? It is probable that specific DNA Groner, B. & Beato, M. (1982) EMBO J. 1, 1613-1619. binding of regulatory proteins is accomplished by a scanning 12. Beato, M. & Feigelson, P. (1972) J. Biol. Chem. 247, 7890-7896. trial and error mechanism involving multiple association and 13. Westphal, H. M., Fleischmann, G. & Beato, M. (1981) Eur. J. Biochem. 119, dissociation steps (21). If such a mechanism applies to the 101-106. 14. Westphal, H. M., Moldenhauer, G. & Beato, M. (1982) EMBOJ. 1, hormone receptor in vivo, binding of the hormone would 1467-1471. accelerate the kinetics of DNA searching by more than one 15. Cato, A. C. B., Miksicek, R., Schutz, G., Arnemann, J. & Beato, order of magnitude (4-fold increase of the on rate and 20-fold M. (1986) EMBO J. 5, 2237-2240. increase of the off rate). This effect could be sufficient to 16. Maxam, A. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560. overcome a kinetic barrier encountered by the free receptor 17. Idziorek, T., Formstecher, P., Danze, P. M., Sableniere, B., single Lustenberger, P., Dumur, V. & Dautrevaux, M. (1985) Eur. J. and to allow the hormone-receptor complex to localize Biochem. 153, 65-74. regulatory sequences within the eukaryotic genome. The 18. Koblinsky, M., Beato, M., Kalimi, M. & Feigelson, P. (1972) J. kinetic values observed with the antihormone, RU 486, are Biol. Chem. 247, 7897-7904. close to those found with the steroid-free receptor and could 19. Fried, M. & Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505- partly explain the lack of functional activity of RU 486. 6525. 20. to our kinetic measurements the difference in Scheidereit, C. & Beato, M. (1984) Proc. Natl. Acad. Sci. USA 81, According 3029-3033. complex for nonspe- affinity of the glucocorticoid-receptor 21. Berg, 0. G., Winter, R. B. & von Hippel, P. H. (1981) Biochemistry cific and specific DNA is only about 10-fold in vitro. We do 20, 629-648. not know whether this difference is sufficient to account for 22. Cordingley, M. G., Riegel, A. T. & Hager,G. L. (1987) Cell 48, the accumulation of glucocorticoid receptor molecules on the 261-280.