Evaluation of the involvement of tyrosine, serine, cysteine and histidine residues in the binding of estrogen, progesterone, and glucocorticoid to their respective receptors

Abstract

The involvement of serine, tyrosine, histidine, and cysteine residues in the binding of hormone to the steroid receptors was investigated using chemical modification techniques. The human estrogen receptor was expressed in a baculovirus vector system in order to produce large quantities of a fully functional receptor. Upon treatment with trypsin, a 28 kDa fragment was generated which retained hormone binding activity. The ability of this fragment to bind to the estrogen responsive element was abolished. This trypsinized fragment was identified on 10-30% glycerol density gradients as a complex which migrated with a sedimentation coefficient of 3-4 S. The native, untreated receptor migrated as a larger 8S oligomeric complex. The tyrosine, serine, histidine, and cysteine residues which were located in this fragment were then evaluated to determine if these amino acids were directly involved in the binding of estradiol to the estrogen receptor. Upon treatment of the human estrogen receptor with the cysteine modification agents p-chloromercuribenzoic acid (PCMB) and N-ethylmaleimide (NEM), the tyrosine modification agents N-acetylimidazole (NAcI), p-nitrobenzenesulfonyl fluoride (NBS), and tetranitromethane (TNM), the serine modification agents phenylmethanesulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP), or the histidine modification agent diethylpyrocarbonate (DFP), specific ({dollar}\sp3{dollar}H) estradiol binding to both the native, untreated receptor and trypsin-treated receptor was inhibited. These results indicated that serine, tyrosine, cysteine, and histidine residues specifically located in the hormone binding domain were involved in the binding of estradiol to the human estrogen receptor. These same chemical agents were utilized to investigate whether a similar binding mechanism existed in the human glucocorticoid and progesterone receptors. Specific binding of ({dollar}\sp3{dollar}H) dexamethasone to the human glucocorticoid receptor was inhibited by NAcI, TNM, PMSF, and DFP, while specific binding of ({dollar}\sp3{dollar}H) R5020 to the progesterone receptor was inhibited by NBS, TNM, PMSF, DFP, PCMB, and NEM. These results suggested that serine, tyrosine, and cysteine all had a similar function in the binding of ligand to the steroid hormone receptors. The involvement of these amino acids in the binding of the estradiol-estrogen receptor complex to DNA was analyzed using DNA-cellulose/DEAE-cellulose column chromatography. While the modification of serine and histidine residues did not effect the binding of the ER complex to DNA, specific binding was inhibited by tyrosine and cysteine modification. The inhibition induced by PCMB and NEM most likely resulted in a modification of the cysteines involved in the zinc finger motif, with a consequential disruption of DNA binding.