Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine.
Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine.
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对 1, 25(OH)(2)D(3) 假定质膜受体的免疫化学研究。
DOI：
10.1152/ajpendo.2000.278.6.e1104
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
McManus,W
中科院分区：
文献类型：
--
作者：
Nemere,I;Ray,R;McManus,W
针对1，25-二羟基维生素D3[1，25（OH）2D 3; BLM-VDR]的假定基底侧膜（BLM）受体的NH 2-末端产生抗血清。在BLM蛋白的Western分析中，抗体（Ab）099对64.5-kDa条带是单特异性的。64.5 kDa的蛋白质也被标记的亲和配体[14 C] 1，25（OH）2D 3-溴乙酸;标签在过量的未标记的开环类固醇的存在下减少。抗核VDR的单克隆抗体（9A 7）未能在BLM组分中检测到适当的条带。用Ab 099（而不是9A 7）预孵育分离的肠细胞会影响以下两个1，25（OH）2D 3介导的信号转导事件：增加细胞内钙和蛋白激酶C活性。通过Western分析和荧光显微镜观察，Ab 099反应性的亚细胞分布显示BLM中的浓度最高，其次是内质网。暴露于1，25（OH）2D 3为10 s或血管灌注5 min的BLM-VDR抗原的细胞核定位的时间依赖性增加，通过电子显微镜判断，而24，25-二羟维生素D3未能增加细胞核中的抗原标记。用溶剂或1，25（OH）2D 3处理的分离肠细胞制备亚细胞组分，Western印迹的光密度定量证实了细胞核中假定的BLM-VDR的增加。结果表明，该蛋白为1，25（OH）2D 3的一种新的细胞表面结合蛋白。
Antisera were raised against the NH2-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D3[1,25(OH)2D3; BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [14C]1,25(OH)2D3-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)2D3-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)2D3for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24,25-dihydroxyvitamin D3failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)2D3confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)2D3has been identified.