STUDIES ON RETINOID-BINDING PROTEINS

由于其疏水性，类维甲酸与 体内的蛋白质。这些蛋白质参与了细胞的运输 维生素从它在肝脏中的储存位置到维生素A--需要 周围组织。此外，细胞对蛋白质的摄取- 相关维生素与维甲酸的细胞内转运 都是由蛋白质推动的。目前的建议旨在 阐明维生素转运的离散步骤 A到它的终极地点(S)的行动。 维生素A通过与视黄醇的结合从肝脏中动员出来 蛋白质(RBP)。将尝试开发一种体外培养技术 允许详细检查视黄醇含量的系统 通过内质网的膜转移 并被新生的RBP收购。此外，稳定的转染体 制造RBP的细胞系，在正常和维生素A中生长- 缺乏培养介质，将被用来解开为什么视黄醇- 缺乏RBP的分子不会离开内质网。 基于已知的RBP的三维结构和 前白蛋白，将尝试绘制相互作用部位的地图 在这两种蛋白质之间，相继使用抗肽 抗体、定点突变和计算机图形学 模特儿。与细胞表面受体相互作用的RBP位点 将通过类似的方法来确定。 大多数细胞含有两种细胞内维甲酸结合蛋白， CRBP和CRABP。将尝试获得cdna克隆。 并阐明了CRABP基因的结构。这个 CRBP在维生素A细胞间转运中的可能作用 肝脏的脂肪储存细胞和肝细胞之间将 走近了。在分离的脂肪储存细胞中CRBP-表达将是 检测和分析CRBP和RBP mRNAs在体内的分布 正常和维生素A缺乏的大鼠组织将使用在 原位杂交技术。 CRBP和CRABP将其配体交付给AS的可能性 然而，尚未发现的细胞内结构将通过 探索抗独特型抗体的使用。这一方法将 有人试图设计出一种体外系统 允许从细胞内结合蛋白转移类维甲酸 亚细胞结构。

Due to their hydrophobic nature retinoids are associated with proteins in vivo. Such proteins are engaged in the transport of the vitamin from its storage site in the liver to vitamin A-requiring peripheral tissues. Moreover, the uptake by cells of the protein- associated vitamin and the intracellular transport of the retinoids are facilitated by proteins. The current proposal is aimed at elucidating the discrete steps involved in the transport of vitamin A to its ultimate site(s) of action. Vitamin A is mobilized from the liver by the retinol-binding protein (RBP). Attempts will be made to develop an in vitro system that allows detailed examination of how retinol is transferred across the membrane of the endoplasmic reticulum and is acquired by the nascent RBP. Moreover, stably transfected cell lines manufacturing RBP, grown in normal and vitamin A- deficient culture media, will be employed to unravel why retinol- deficient RBP molecules do not exit the endoplasmic reticulum. Based on the known three-dimensional structures of RBP and prealbumin, attempts will be made to map the sites of interaction between the two proteins using, in succession, antipeptide antibodies, site-directed mutagenesis and computer graphics modeling. The RBP-site interacting with a cell surface receptor will be identified by a similar approach. Most cells contain two intracellular-retinoid-binding proteins, CRBP and CRABP. Attempts will be made to obtain cDNA clones for CRABP and elucidate the structure of the CRABP gene. The possible role of CRBP in the intercellular transfer of vitamin A between fat storing cells of the liver and hepatocytes will be approached. CRBP-expression in isolated fat storing cells will be examined and the distribution of CRBP and RBP mRNAs in normal and vitamin A-deficient rat tissues will be studied using in situ hybridization techniques. The possibility the CRBP and CRABP deliver their ligands to as yet undiscovered intracellular structures will be examined by exploring the use of anti-idiotypic antibodies. This approach will be complimented by attempts to devise in vitro systems that allow transfer of retinoids from the intracellular binding proteins to subcellular structures.