Pratt W B, Jolly D J, Pratt D V, Hollenberg S M, Giguere V, Cadepond F M, Schweizer-Groyer G, Catelli M G, Evans R M, Baulieu E E
Institut National de la Santé et de la Recherche Médicale U33, Université Paris-Sud, Bicêtre, France.
J Biol Chem. 1988 Jan 5;263(1):267-73.
This work was initiated to determine if a specific region of the glucocorticoid receptor determines the formation of the inactive (i.e. non-DNA-binding) 9 S form of the receptor recovered in cytosol preparations. It is known that the murine glucocorticoid receptor of the nti phenotype, which consists of only the carboxyl-terminal 40-kDa peptide containing the DNA-binding and steroid-binding domains separated by a short linker region, is recovered in hypotonic lysates as a 9 S heteromeric complex (Gehring, U., and Arndt, H. (1985) FEBS Lett. 179, 138-142). To further localize the domain required for formation of the 9 S complex, we have determined the sedimentation coefficients of receptors produced in COS-7 cells transfected with several mutants of the human glucocorticoid receptor gene. Deletion of the DNA-binding domain results in a 9 S complex that is somewhat less stable than the wild type receptor during sucrose gradient centrifugation. Deletion of the linker region yields a molybdate-stabilized 9 S complex, but deletion of the entire steroid-binding domain or internal deletion of the amino-terminal two-thirds of this domain yields receptors that are constitutive transcriptional activators and are present in cytosol only in the 4 S form. Taken together, these observations demonstrate that the steroid-binding domain contains the features required for formation of the 9 S heteromeric complex, and they are consistent with the proposal that the steroid-binding domain normally represses receptor function.
开展这项研究是为了确定糖皮质激素受体的特定区域是否决定了在胞质溶胶制剂中回收的受体非活性（即非DNA结合）9S形式的形成。已知nti表型的小鼠糖皮质激素受体仅由羧基末端40 kDa肽组成，该肽包含由短连接区隔开的DNA结合域和类固醇结合域，在低渗裂解物中作为9S异源复合物回收（Gehring, U., and Arndt, H. (1985) FEBS Lett. 179, 138 - 142）。为了进一步定位形成9S复合物所需的结构域，我们测定了用人类糖皮质激素受体基因的几个突变体转染的COS - 7细胞中产生的受体的沉降系数。DNA结合域的缺失导致一种9S复合物，在蔗糖梯度离心过程中其稳定性略低于野生型受体。连接区的缺失产生一种钼酸盐稳定的9S复合物，但整个类固醇结合域的缺失或该结构域氨基末端三分之二的内部缺失产生的受体是组成型转录激活剂，仅以4S形式存在于胞质溶胶中。综上所述，这些观察结果表明类固醇结合域包含形成9S异源复合物所需的特征，并且与类固醇结合域通常抑制受体功能的提议一致。
