Abstract
To determine if activation of the glucocorticoid receptor involves covalent charge modification of the steroid-binding protein, unactivated and activated IM-9 cell glucocorticoid receptors were examined by high resolution two-dimensional gel electrophoresis. As previously reported (Smith, A. C., and Harmon, J. M. (1985) Biochemistry 24, 4946-4951), two-dimensional electrophoresis of immunopurified, [3H]dexamethasone mesylate-labeled, steroid-binding protein from unactivated receptors resolves two 92-kDa isoforms (pI congruent to 5.7 and 6.0-6.5). After activation, the apparent pI of neither isoform was altered, indicating that there had been no covalent charge modification of the steroid-binding protein. Thus, the physicochemical changes observed after activation of the steroid receptor cannot be explained by dephosphorylation or other models which involve covalent charge modification of the steroid-binding protein. This conclusion was consistent with the observation that treatment of immunopurified, affinity-labeled receptors with calf intestine alkaline phosphatase did not alter the apparent pI values or distribution of the steroid-binding protein isoforms. However, chromatography of activated steroid-receptor complexes on DNA-cellulose revealed that only the more basic of the two steroid-binding protein isoforms bound to DNA. Therefore, the charge heterogeneity of the steroid-binding protein may be important in regulating the ability of the steroid-binding protein to interact with DNA.
Highlights
IM-9 cell glucocorticoid receptorswere examined by the apparenstizeof steroid-recep~cromplexes when analyzed high resolution two-dimensional gel electrophoresis, by gel filtration and sucrose gradient sedimentation [12,13,14,15]
The relationship between the change in surface chargereflected by the decreased affinity of activated steroid-receptor complexes for DEAE-cellulose andthe decrease insize of the activated complex remains unclear
Our results suggest that there is no such tion (1 ml) was applied to a 3.0-ml column of DEAE-cellulose modification,implying that thesurfacecharge alterations equilibrated in buffer B
Summary
Vol 261, No 28, issue of October 5,pp. 13285-13292,1986 Printed in 11.S.A. Analysis of Glucocorticoid ReceptorActivation by High Resolution Two-dimensional Electrophoresis ofAffinity-labeled Receptor*. M. (1985) Biochemistry 24, 4946-4951), two-dimen- kDa mammalian heat shock protein may be associated with tsmftafhrhoipooreaepnmmttahassratltu(eheesPlnnreoeIortanceictePdrt=mi-Ihovbpae5aoisdhn.tfy7eodlndbraianeeetrniesegtei-ndhscloaepe6nfcbrprio.emot0ivolts-emeaor6disfl.nue,o5rs.nrne)tme.tsowTrocpaohAhlusviaufdrertsais-eg,fblriteteiwtendharmod,ceelitSdo9nipdHv,2gihia-pnlfkdtyirdciDseooaiixctnctaeaaio,oit-icntisnhnhoege-omf ipectachuxocaepHtisliseivtomedsawdtotiihrneloeyvancabeterd(hp,1atet2hwotte-eeruh1-rsn7iolct)aemaa.ctbnhteiierlvbiizacheeteceddtoderinsrefofsociomregmcpueitrarooaitfrcetitdco(h1onbe6nyw,figh1cgli7ouuc)nhrc,adoidtitcitioisobons~anoosiscfcibkotahenitrdeeeeonscwuuenpnpportootocno--r changes observedafter activationof the steroid recep- activate the occupied form of the receptor, such treatment tor cannot bexplainedby dephosphorylation or otherdoes not convert the unoccupied form of the receptor to a models whichinvolve covalent charge modification of DNA-binding protein [18,19]. Activation of steroid-receptor complexes results in the same decreased affinity for DEAE-containing resins [28] are consistent with this hypothesis. This hypothesis is consistent with the increased pl of activated steroidreceptorcomplexes [29,30,31,32]. Fractions were assayed for radioof the steroid-binding proteinbinds to DNA
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