Lamaze C, Baba T, Redelmeier T E, Schmid S L
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Mol Biol Cell. 1993 Jul;4(7):715-27. doi: 10.1091/mbc.4.7.715.
The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.
使用穿孔的人A431细胞比较了表皮生长因子（EGF）和转铁蛋白受体介导的内吞作用的生化需求。形态学研究表明，辣根过氧化物酶（HRP）偶联的EGF和金标记的抗转铁蛋白（Tfn）受体抗体在体外内吞过程中共定位。两种配体被隔离到深陷的被膜小窝中都需要ATP水解和胞质因子，并且受到GTPγS的抑制，这表明机制上存在相似性。重要的是，还检测到EGF和Tfn隔离在生化需求上的几个差异。这些差异包括对可溶性AP（网格蛋白组装蛋白）复合物的不同需求、不同的胞质需求以及对酪氨酸激酶抑制剂染料木黄酮的不同敏感性。EGF和Tfn隔离之间检测到的生化差异很可能反映了将EGF受体（R）招募到被膜小窝中的特定需求。该分析提供了一种新方法，可用于确定这些生化差异的分子基础，并阐明配体诱导EGF-R招募到被膜小窝中所涉及的机制。
