Venepally P, Reddy L G, Sani B P
Kettering-Meyer Laboratories, Southern Research Institute, Birmingham, Alabama 35255, USA.
Biochemistry. 1996 Aug 6;35(31):9974-82. doi: 10.1021/bi9603276.
The involvement of cellular retinoic acid-binding protein I (CRABP I) in the RA signaling was investigated by examining its effects on the interaction of retinoid receptors (RARs and RXRs) with RA-response elements (RAREs) as well as on the RA-induced transcription mediated by retinoid receptors. Analysis of the expression of mouse CRABP I from a cDNA expression plasmid in COS-1 cells revealed that this protein was about 5-fold more abundant in cytosol than in nuclei. The identity and the localization of CRABP I in the cytoplasm as well as the nuclei were also confirmed by the immunoperoxidase staining of the transfected COS-1 cells with CRABP I-specific antibody. When the nuclear extract containing a 10-fold molar excess of CRABP I was incubated with RAR alpha extract in the presence of [3H]RA and resolved on an FPLC size-exclusion column, a 20% decrease in the bound radioactivity in the RAR alpha fraction was accompanied by a proportional increase in the CRABP I fraction. In contrast, the addition of CRABP I did not significantly affect the interaction of RAR alpha or RAR alpha-RXR alpha heterodimers with RAREs. Moreover, the coexpression of CRABP I in CV-1 cells did not markedly inhibit or enhance the transcription activated by RARs and RAR alpha-RXR alpha heterodimers under RA concentrations ranging from 10(-10) to 10(-6) M. These results demonstrate that CRABP I, while it might be important for RA homeostasis, is not directly involved in the retinoid receptor-mediated RA-signaling pathway.
通过研究细胞视黄酸结合蛋白I（CRABP I）对类视黄醇受体（RARs和RXRs）与视黄酸反应元件（RAREs）相互作用的影响以及对类视黄醇受体介导的视黄酸诱导转录的影响，来探究其在视黄酸（RA）信号传导中的作用。对COS-1细胞中来自cDNA表达质粒的小鼠CRABP I表达进行分析发现，该蛋白在细胞质中的丰度比在细胞核中高约5倍。用CRABP I特异性抗体对转染的COS-1细胞进行免疫过氧化物酶染色，也证实了CRABP I在细胞质以及细胞核中的身份和定位。当在存在[3H]视黄酸的情况下，将含有10倍摩尔过量CRABP I的核提取物与RARα提取物一起孵育，并在FPLC尺寸排阻柱上进行分离时，RARα组分中结合放射性降低20%的同时，CRABP I组分中相应增加。相反，添加CRABP I对RARα或RARα-RXRα异二聚体与RAREs的相互作用没有显著影响。此外，在10(-10)至10(-6)M的视黄酸浓度范围内，CRABP I在CV-1细胞中的共表达并未显著抑制或增强RARs和RARα-RXRα异二聚体激活的转录。这些结果表明，CRABP I虽然可能对视黄酸稳态很重要，但并不直接参与类视黄醇受体介导的视黄酸信号通路。
