Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Cell Biochem. 2010 Jul 1;110(4):926-34. doi: 10.1002/jcb.22606.
Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25-dihydroxyvitamin D(3) (1,25D(3)) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand-dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos-1 cells transfected with human VDR and histidine-tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D(3) inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D(3) to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand-binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand-dependent heterodimerization and nuclear localization protect the VDR from these modifications.
其他人以及我们之前曾表明维生素 D 受体 (VDR) 可被 26S 蛋白酶体降解，而 1,25-二羟基维生素 D(3) (1,25D(3)) 的治疗可抑制这种降解。在本研究中，我们发现，在成骨细胞中，但不在肠上皮细胞中，VDR 易受 26S 蛋白酶体的降解。成骨细胞中 VDR 降解的亚细胞部位是细胞质，而配体依赖性保护 VDR 免受 26S 蛋白酶体降解的部位是染色质。这些核定位与 VDR 免受 26S 蛋白酶体降解之间的直接关系使我们假设未占据的细胞质 VDR 是多泛素化的底物，这使 VDR 成为 26S 蛋白酶体降解的目标，而核定位具有保护 VDR 免受多泛素化和降解的能力。为了验证这些假设，我们使用了转染了人 VDR 和组氨酸标记的泛素表达载体的 Cos-1 细胞。我们发现未占据的 VDR 被多泛素化，而 1,25D(3) 抑制了这种修饰。VDR 核定位信号中的突变（R49W/R50G 和 K53Q/R54G/K55E）或 VDR 与视黄酸 X 受体的二聚化界面中的突变（M383G/Q385A）消除了 1,25D(3) 保护 VDR 免受多泛素化的能力，尽管这些突变对 VDR 的配体结合活性没有影响。因此，我们得出结论，在某些细胞环境中，未占据的细胞质 VDR 易受多泛素化和蛋白酶体降解的影响，而配体依赖性异二聚化和核定位可保护 VDR 免受这些修饰。
