Braiterman L T, Chance S C, Porter W R, Lee Y C, Townsend R R, Hubbard A L
Department of Cell Biology/Anatomy, Johns Hopkins Medical School, Baltimore, Maryland 21205.
J Biol Chem. 1989 Jan 25;264(3):1682-8.
Mammalian hepatic asialoglycoprotein receptors (ASGP-R) are composed of two unique, but closely related polypeptides, which in the rat are designated rat hepatic lectins 1 and 2/3 (RHL 1, RHL 2/3). Despite numerous studies, the composition of a functional ASGP-R has remained unclear. We examined this question in rat hepatoma tissue culture (HTC) cells (which lack endogenous ASGP-R) that were co-transfected with cDNAs for both RHL 1 and RHL 2/3. The original population was cloned, but derivatives were unstable. We therefore used fluorescence-activated cell sorting to separate a subpopulation of cells (positive) that specifically endocytosed fluoresceinated asialoorosomucoid (ASOR) from one that did not (negative). We then used indirect immunofluorescence with polypeptide-specific ASGP-R antibodies, immunoanalysis, and binding and uptake studies with two Gal ligands (ASOR and NAc-galactosylated poly-L-lysine (Gal-Lys] to further define the ASGP-R status in these two populations. As reported by others, we found that expression of both RHL 1 and RHL 2/3 in the positive cells resulted in binding, uptake and degradation of ASOR, the most commonly used ASGP-R ligand. The negative cells expressed only RHL 1 and neither bound nor processed ASOR. However, the presence of RHL 1 was sufficient for specific high affinity binding and processing of the synthetic ligand, Gal-Lys, by negative cells. These results show that RHL 1 can function as an ASGP-R, given a highly galactosylated ligand, and that RHL 2/3 must play an important role in the organization of native ASGP-R in the membrane.
哺乳动物肝脏去唾液酸糖蛋白受体（ASGP-R）由两种独特但密切相关的多肽组成，在大鼠中它们被命名为大鼠肝脏凝集素1和2/3（RHL 1、RHL 2/3）。尽管进行了大量研究，但功能性ASGP-R的组成仍不清楚。我们在大鼠肝癌组织培养（HTC）细胞（缺乏内源性ASGP-R）中研究了这个问题，这些细胞同时被转染了RHL 1和RHL 2/3的cDNA。最初的细胞群体被克隆，但衍生物不稳定。因此，我们使用荧光激活细胞分选技术，从一个不（阴性）特异性内吞荧光标记的去唾液酸血清类黏蛋白（ASOR）的细胞群体中分离出一个亚群（阳性）细胞。然后，我们使用针对多肽特异性ASGP-R抗体的间接免疫荧光、免疫分析以及与两种半乳糖配体（ASOR和N-乙酰半乳糖基化聚-L-赖氨酸（Gal-Lys））的结合和摄取研究，以进一步确定这两个群体中ASGP-R的状态。正如其他人所报道的，我们发现阳性细胞中RHL 1和RHL 2/3的表达导致了ASOR（最常用的ASGP-R配体）的结合、摄取和降解。阴性细胞仅表达RHL 1，既不结合也不处理ASOR。然而，RHL 1的存在足以使阴性细胞对合成配体Gal-Lys进行特异性高亲和力结合和处理。这些结果表明，在有高度糖基化配体的情况下，RHL 1可以作为ASGP-R发挥作用，并且RHL 2/3在膜中天然ASGP-R的组织中必须发挥重要作用。
