DeMarzo A M, Oñate S A, Nordeen S K, Edwards D P
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
Biochemistry. 1992 Nov 3;31(43):10491-501. doi: 10.1021/bi00158a012.
We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.
我们之前利用免疫共沉淀分析法报道，来自T47D人乳腺癌细胞的人孕酮受体B型（PR-B）在溶液中可与A型受体（PR-A）形成二聚体，且二聚化程度与受体对特定DNA序列的结合活性相关[DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72 - 76]。这表明溶液中的二聚化是受体激活过程中的一个中间步骤。本研究检测了孕酮拮抗剂RU486对孕酮受体（PR）溶液中二聚化的影响。通过免疫共沉淀分析确定，RU486的结合并未损害受体的二聚化；相反，与孕激素激动剂R5020相比，该拮抗剂促进了更有效的溶液二聚化。这种增强的受体二聚化与与RU486结合的转化受体更高的DNA结合活性相关。与R5020相比，RU486先前已被证明会在人PR中产生另外两种改变。溶液中的PR-RU486复合物在含盐蔗糖密度梯度上的沉降速率（6 S）比PR-R5020复合物（4 S）更快，并且在RU486存在的情况下，PR-DNA复合物在凝胶迁移分析中的电泳迁移率更快。我们目前表明，6 S的PR-RU486复合物是受体单体，而非二聚体。在杆状病毒载体昆虫细胞中分别表达的重组PR-A和PR-B也观察到了RU486促进的沉降速率增加和凝胶迁移分析中迁移率增加。这些结果表明，RU486在溶液中的PR单体以及与DNA结合的二聚体中均诱导了独特的构象变化。我们还研究了RU486诱导的PR构象变化是否会阻止与RU486结合的PR多肽与另一个与R5020结合的PR多肽形成异二聚体。为评估此情况，将在全细胞中分别与R5020或RU486结合的PR-A和PR-B在体外混合。PR-A-RU486能够与PR-B-R5020形成二聚体，溶液中形成的异二聚体以及与特定DNA结合的异二聚体均证明了这一点。在体外形成异二聚体的能力增加了RU486在体内的拮抗作用可能部分通过与激动剂结合的一个受体亚基和与RU486结合的另一个受体亚基之间的异二聚化以显性负性方式发挥作用的可能性。
