Santini F, Keen J H
Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Cell Biol. 1996 Mar;132(6):1025-36. doi: 10.1083/jcb.132.6.1025.
The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we studied the relationship between activation of IgE-bound high affinity Fc receptors (FCepsilonRI) and coated pit formation. Because these receptors are rapidly internalized via clathrin-coated pits only when cross-linked by the binding of multivalent antigens, we were able to separate activation from internalization by using an immobilized antigen. The FCepsilonRIs, initially uniformly distributed over the cell surface. relocalized and aggregated on the antigen-exposed membrane. The process was specific for the antigen, and temperature- and time-dependent. This stimulation initiated a cascade of cellular responses typical of FCepsilonRI signaling including membrane ruffling, cytoskeletal rearrangements, and, in the presence of Ca2+, exocytosis. Despite these responses, no change in coated pit disposition or in the distribution of clathrin and assembly protein AP2 was detected, as monitored by immunoblotting and by quantitative (vertical sectioning) confocal microscopy analysis of immunofluorescently stained cells. Specifically, there was no decrease in the density of clathrin-coated pits in regions of the cell membrane not in contact with the antigen, and there was no apparent increase in clathrin-coated pits in proximity to stimulated FCepsilonRI receptors as would have been expected if the receptors were inducing formation of new pits by active recruitment. These results indicate that de novo formation of clathrin-coated pits is not a prerequisite for rapid internalization or a direct response to stimulation of FCepsilonRI receptors. Therefore, increases in coated pits reported to occur in response to activation of some signaling receptors must be consequences of the signal transduction processes, rather than strictly serving the purpose of the internalization of the receptors.
曾经作为内吞作用靶点的受体出现在被膜小窝中的基本机制是一个重要但尚未解决的问题。具体而言，被激活的受体是仅仅在遇到预先存在的被膜小窝时被捕获，随后迅速被内化？还是受体本身通过主动募集，聚集可溶性包被和胞质成分，并启动新的被膜小窝的快速组装，进而介导其内化？为了探究这个问题，我们研究了与IgE结合的高亲和力Fc受体（FCεRI）的激活与被膜小窝形成之间的关系。由于这些受体仅在被多价抗原交联时才通过网格蛋白包被的小窝迅速内化，我们能够通过使用固定化抗原来将激活与内化分开。FCεRI最初均匀分布在细胞表面，在抗原暴露的膜上重新定位并聚集。该过程对抗原具有特异性，并且依赖于温度和时间。这种刺激引发了一系列典型的FCεRI信号传导的细胞反应，包括膜褶皱、细胞骨架重排，以及在Ca2+存在下的胞吐作用。尽管有这些反应，但通过免疫印迹以及对免疫荧光染色细胞的定量（垂直切片）共聚焦显微镜分析监测发现，被膜小窝的分布或网格蛋白和组装蛋白AP2的分布没有变化。具体而言，在细胞膜未与抗原接触的区域，网格蛋白包被的小窝密度没有降低；并且在受刺激的FCεRI受体附近，网格蛋白包被的小窝也没有明显增加，而如果受体通过主动募集诱导新小窝形成，情况应该是相反的。这些结果表明，网格蛋白包被小窝的从头形成不是FCεRI受体快速内化的先决条件，也不是对其刺激的直接反应。因此，据报道响应某些信号受体激活而出现的被膜小窝增加必定是信号转导过程的结果，而不仅仅是为了受体的内化。
