Sunn K L, Cock T A, Crofts L A, Eisman J A, Gardiner E M
Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, 2010 Australia.
Mol Endocrinol. 2001 Sep;15(9):1599-609. doi: 10.1210/mend.15.9.0693.
The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.
核激素受体N端区域在转录调控中的重要性日益受到认可。由于可变VDR基因转录本提示可能存在N端延长的受体，我们对其天然存在情况、反式激活能力及亚细胞定位进行了研究。通过使用异构体特异性和非选择性抗VDR抗体进行蛋白质印迹分析，在人肾组织以及成骨细胞（MG63）、肠细胞（Int - 407、DLD - 1和COLO 206F）和肾上皮细胞（786）人细胞系中，除了先前已识别的48 kDa VDRA形式外，还检测到一种新的54 kDa VDRB1蛋白。VDRB1的表达水平约为VDRA的三分之一。当用维生素D反应性启动子瞬时转染到内源性VDR低的细胞系中时，异构体特异性VDRB1表达构建体产生的配体依赖性反式激活低于VDRA。绿色荧光蛋白标记的VDR异构体的细胞内定位模式不同。在没有1,25 - 二羟基维生素D3的情况下，VDRB1表现为离散的核内斑点，而VDRA产生弥漫性核荧光。经1,25 - 二羟基维生素D3处理后，两种VDR异构体均表现出相似的弥漫性核信号。在没有1,25 - 二羟基维生素D3的情况下，VDRB1斑点与SC - 35斑点和早幼粒细胞白血病核体的一个子集部分共定位。这些数据首次证明了VDRB1的存在，VDRB1是一种新的N端变体人VDR，在人组织和细胞系中的表达水平与VDRA相当。其特征是反式激活活性降低以及配体反应性斑点状核内定位。VDRB1的核内区室化和功能活性改变可能介导了这种受体异构体的特殊生理作用。
