Simmons C F, Schwartz A L
Mol Pharmacol. 1984 Nov;26(3):509-19.
The intracellular pathways taken by galactose-terminal glycoproteins were examined following endocytosis by the asialoglycoprotein receptor in monolayers of the human hepatoma cell line, Hep G2. In addition to a pathway leading to lysosomal degradation, single cohort kinetics revealed that up to 28% of surface-bound and internalized 125I-asialoorosomucoid (ASOR) eventually returned undegraded to the extracellular medium over 6 hr in the presence or absence of free ASOR in the exocytosis medium. This reappearance of ligand in the exocytosis medium represented a constant fraction of surface bound and internalized 125I-ASOR, and followed pseudo-first order kinetics with t1/2 = 84 min (long transit pool). Under conditions of enhanced ligand-receptor dissociation (incubation with 100 mM N-acetylgalactosamine (GalNAc), at least 50% of initially internalized 125I-ASOR returned to the cell surface as ligand-receptor complexes, followed by dissociation of free ligand into the exocytosis medium. This rapid transit pool of ligand also displayed pseudo-first order kinetics with t1/2 = 24 min. Exocytosis of 125I-Gal-cytochrome c, a synthesized ligand displaying rapid dissociation from the asialoglycoprotein receptor (ASGP-R), paralleled the kinetics of the rapid transit pool of 125I-ASOR (t1/2 = 28 min). Furthermore, in addition to spontaneous dissociation from ASPG-R following return to the cell surface, studies conducted in saponin-permeabilized monolayers support the return of free intracellular 125I-Gal-cytochrome c to the cell surface during exocytosis. The rapid transit pool of ligand was insensitive to inhibition by 10 mM sodium azide or 0.1 mM primaquine. In contrast, the long transit pool destined for exocytosis was inhibited 50% by 10 mM sodium azide, but insensitive to inhibition by 0.1 mM primaquine. These data suggest that, following internalization by the ASGP-R, a major pathway of ligand movement includes the rapid return of ligand-receptor complexes and/or free ligand to the cell surface. Return of ligand-receptor complexes or free ligand to the cell surface occurs prior to an acidic sorting compartment, can involve multiple cycles of return to the cell surface, and may involve passage through other nonlysosomal intracellular organelles.
在人肝癌细胞系Hep G2的单层细胞中，通过去唾液酸糖蛋白受体进行内吞作用后，研究了半乳糖末端糖蛋白所采取的细胞内途径。除了导致溶酶体降解的途径外，单组动力学显示，在胞吐介质中存在或不存在游离去唾液酸血清类黏蛋白（ASOR）的情况下，高达28%的表面结合并内化的125I-去唾液酸血清类黏蛋白最终在6小时内未被降解地返回细胞外介质。配体在胞吐介质中的这种重新出现代表了表面结合并内化的125I-ASOR的恒定比例，并遵循假一级动力学，半衰期为84分钟（长转运池）。在增强配体-受体解离的条件下（与100 mM N-乙酰半乳糖胺（GalNAc）孵育），至少50%最初内化的125I-ASOR作为配体-受体复合物返回细胞表面，随后游离配体解离到胞吐介质中。这种配体的快速转运池也显示假一级动力学，半衰期为24分钟。125I-半乳糖-细胞色素c（一种从去唾液酸糖蛋白受体（ASGP-R）快速解离的合成配体）的胞吐作用与125I-ASOR快速转运池的动力学平行（半衰期为28分钟）。此外，除了返回细胞表面后从ASPG-R自发解离外，在皂素通透的单层细胞中进行的研究支持游离细胞内125I-半乳糖-细胞色素c在胞吐作用期间返回细胞表面。配体的快速转运池对10 mM叠氮化钠或0.1 mM伯氨喹的抑制不敏感。相反，注定用于胞吐的长转运池被10 mM叠氮化钠抑制50%，但对0.1 mM伯氨喹的抑制不敏感。这些数据表明，在被ASGP-R内化后，配体移动的主要途径包括配体-受体复合物和/或游离配体快速返回细胞表面。配体-受体复合物或游离配体返回细胞表面发生在酸性分选区室之前，可能涉及多次返回细胞表面的循环，并且可能涉及通过其他非溶酶体细胞内细胞器。
