Marks M S, Levi B Z, Segars J H, Driggers P H, Hirschfeld S, Nagata T, Appella E, Ozato K
Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 1992 Feb;6(2):219-30. doi: 10.1210/mend.6.2.1569965.
H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.
H-2RIIBP是核激素受体超家族的成员，可与主要组织相容性复合体I类基因的II区增强子结合。基于其与果蝇XR2C/CF1的同源性，H-2RIIBP可能在发育过程中发挥作用。通过使用杆状病毒表达系统，产生了大量重组H-2RIIBP。重组蛋白在昆虫细胞核中积累。然后产生了一系列与重组H-2RIIBP反应的单克隆抗体。利用这些抗体开发了一种DNA-蛋白质免疫沉淀试验，能够区分H-2RIIBP与未感染昆虫细胞中表达的内源性II区结合因子的DNA结合特异性。我们发现H-2RIIBP与雌激素反应元件的结合亲和力与对II区增强子的亲和力相当。H-2RIIBP也能与一些（但不是全部）甲状腺激素反应元件和视黄酸反应元件结合，尽管亲和力较低。在不额外添加配体的情况下就证明了其与这些元件的结合。通过该试验确定的H-2RIIBP结合特异性与通过蛋白质免疫印迹法和凝胶迁移率变动分析评估的特异性一致。此外，甲基化干扰试验表明H-2RIIBP识别保守的激素反应基序GG(T/A)CA。综上所述，这些数据表明H-2RIIBP能够与多种基因的激素反应元件结合。它们提示H-2RIIBP可能发挥多效性功能。
