Zhou D, Quach K M, Yang C, Lee S Y, Pohajdak B, Chen S
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Mol Endocrinol. 2000 Jul;14(7):986-98. doi: 10.1210/mend.14.7.0480.
PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 278-300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins.
富含脯氨酸的核受体共调节蛋白（PNRC）是在以牛类固醇生成因子1（SF1）为诱饵对人乳腺cDNA表达文库进行酵母双杂交筛选时被鉴定出来的。PNRC的独特之处在于其分子量为35 kDa，明显小于迄今报道的大多数共调节蛋白，并且富含脯氨酸。免疫荧光和蛋白质印迹分析证实了PNRC的核定位。在酵母双杂交试验中，PNRC以不依赖配体的方式与孤儿受体SF1和雌激素相关受体α1（ERRalpha1）相互作用。还发现PNRC以依赖配体的方式与所有测试的核受体的配体结合域相互作用，这些核受体包括雌激素受体（ER）、雄激素受体（AR）、糖皮质激素受体（GR）、孕激素受体（PR）、甲状腺激素受体（TR）、视黄酸受体（RAR）和类视黄醇X受体（RXR）。核受体与PNRC结合需要功能性的AF2结构域。此外，进行了体外谷胱甘肽-S-转移酶下拉试验，以证明PNRC与诸如SF1等核受体之间的直接接触。利用表达PNRC和ER的Hela细胞进行了免疫共沉淀实验，以确认PNRC与核受体在体内以依赖配体的方式相互作用。发现PNRC作为共激活因子发挥作用，增强由SF1、雌激素相关受体1（ERR1）、PR和TR介导的转录激活。通过使用酵母双杂交试验检测PNRC的一系列缺失突变体，发现羧基末端区域的一个23个氨基酸（aa）的序列（aa 278-300）对于与核受体的相互作用至关重要且足够。该区域富含脯氨酸，并包含一个SH3结合基序S-D-P-P-S-P-S。诱变研究结果表明，该基序中的两个保守脯氨酸（P）残基对于PNRC与核受体的相互作用至关重要。在同一酵母双杂交筛选研究中分离出的另一种蛋白质中也发现了确切的23个氨基酸序列。这两种蛋白质属于核受体共调节蛋白的一个新家族。
