Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits.
Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits.
复制标题
大鼠肝细胞半乳糖/N-乙酰半乳糖胺特异性受体的亲和标记：优先标记其中一个亚基。
DOI：
10.1021/bi00394a005
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Lee,YC
中科院分区：
文献类型：
--
作者：
Lee,RT;Lee,YC
约翰霍普金斯大学生物学系和麦科勒姆-普拉特研究所，马里兰州巴尔的摩，邮编21218接收日期：1987年2月2日;修订后的《曼尼普尔文》接收日期：1987年4月24日摘要：半乳糖/乙酰半乳糖胺特异性受体大鼠肝细胞的脱唾液酸糖蛋白受体（也称为脱唾液酸糖蛋白受体）由三个亚基组成，其中之一[43千道尔顿（kDa）]的丰度（高达总蛋白的70%）高于两个次要种类（52和60 kDa）。当肝细胞膜上的受体用125 I标记的高亲和性试剂[在半乳糖残基上含有芳基叠氮基的三触角糖肽; Lee，R. T.，和李，Y。C.（1986）Biochemistry 25，6835-6841]，标记主要（51-80%）发生在次要条带之一（52 kDa）上。同样，亲和力结合，7V-乙酰半乳糖胺修饰的乳过氧化物酶放射性碘标记的相同的52 kDa的带优先。与此相反，光亲和标记和乳过氧化物酶催化碘化的纯化，洗涤剂溶解的受体导致的分布的thalamel是可比的考马斯亮蓝染色模式的三个条带，即，43 kDa的带是主要的带标记。这些和其他实验结果表明，优先标记的次要带和低效的标记的主要带在肝细胞膜上的这些亚基的膜上的特定的拓扑结构的安排。我们推测，在天然的，膜boundstate的受体，52-kDa的次要带是拓扑突出，而主要的（43 kDa）带被部分掩盖。这种部分掩蔽可能是由于膜上受体亚单位的紧密堆积而形成晶格[哈代，M. R.，汤森河R.，Parkhurst，S. M.，& Lee，Y. C.（1985）Biochemistry 24，22-28]。在哺乳动物肝细胞表面存在识别半乳糖（Gal）1或GalNAc的跨膜受体[称为脱唾液酸糖蛋白（ASGP）受体]。尽管该受体的确切生理功能还不清楚，但受体及其配体的细胞内运动是受体系统的较好研究之一[综述参见Ashwell和Harford（1982）和Breitfeld et al.（1985）]。根据目前的概念，该受体系统的主要操作模式如下：去唾液酸糖蛋白（配体）最初被肝细胞表面上的受体结合，受体-配体复合物通过包被的小凹被内吞到囊泡中，受体和配体在到达溶酶体之前的一个阶段彼此解离，然后受体穿梭回到细胞表面，而配体则进入溶酶体被降解。该受体也以比表面受体丰富2-4倍的量存在于大鼠肝细胞的内膜中（Breitfeld等人，1985年）。内部受体似乎在生物化学和免疫化学上与表面受体相似（Pricer & Ashwell，1976），但对于表面和内部受体之间的功能关系存在冲突的观点（Ashwell & Har-ford，1982）。
Department of Biology and McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218 Received February 2, 1987; Revised Manuscript Received April 24, 1987 abstract: The galactose/W-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C.(1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, 7V-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptorresulted in a distribution of thelabel that is comparable to the Coomassie blue stainingpattern of the three bands; ie, the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulatethat in the native, membrane-boundstate of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C.(1985) Biochemistry 24, 22-28]. ere exists on the surface of mammalian hepatocytes a transmembrane receptor that recognizes galactose (Gal) 1 or GalNAc [known as asialoglycoprotein (ASGP) receptor]. Though exact physiological function of this receptor is not clearly understood, intracellular movement of the receptor and its ligands is one of the better studied of the receptor systems [for a review, see Ashwell and Harford (1982) and Breitfeld et al.(1985)]. Accordingto the current concept, the major mode of operation of this receptor system is as follows: An asialoglycoprotein (ligand) is initially bound by the receptors on the hepatocyte surface, the receptor-ligand complex is endocytosed in vesicles via coated pits, the receptor and the ligand dissociate from each other at one of the stages before reaching lysosome, and the receptor then shuttles back to the cell surface while the ligand proceeds to lysosome to be degraded. The receptor is also present in the internal membranes of rat hepatocytes in a quantity that is 2-4 times more abundant than the surface receptor (Breitfeld et al., 1985). Internal receptors appear to be biochemically and immunochemically similar to the surface receptors (Pricer & Ashwell, 1976), but conflicting views exist for the functional relationship between the surface and internal receptors (Ashwell & Har-ford, 1982).