DELINEATION OF THE ENDOCYTIC PATHWAY OF SUBSTANCE-P AND ITS 7-TRANSMEMBRANE DOMAIN NK1 RECEPTOR
DELINEATION OF THE ENDOCYTIC PATHWAY OF SUBSTANCE-P AND ITS 7-TRANSMEMBRANE DOMAIN NK1 RECEPTOR
复制标题
DOI：
10.1091/mbc.6.5.509
复制
发表时间：
1995-05-01
影响因子：
3.3
通讯作者：
BUNNETT, NW
中科院分区：
文献类型：
--
作者：
GRADY, EF;GARLAND, AM;BUNNETT, NW
由神经激肽 1 受体 (NK1-R) 介导的神经肽 P 物质 (SP) 的许多作用会导致脱敏和再敏化，这可能与 NK1-R 的内吞作用和再循环有关。我们使用花青 3-SP 和 NK1-R 抗体通过共聚焦显微镜描绘了转染细胞中的内吞途径。 SP和NK1-R被内化到相同的网格蛋白免疫反应性囊泡中，然后分类到不同的区室中。 NK1-R 与早期内体标记共定位，但不与晚期内体或溶酶体标记共定位。我们通过将细胞与细胞外表位的抗体一起孵育来定量细胞表面的 NK1-R。暴露于 SP 后，表面 NK1-R 发生损失并随后恢复。高渗蔗糖和钾消耗（网格蛋白介导的内吞作用的抑制剂）可以防止这种损失。回收率独立于新的蛋白质合成，因为它不受放线菌酮的影响。恢复需要内体酸化，因为它被 H+-ATP 酶抑制剂阻止。通过色谱法检查内化 I-125-Sp 的命运。 SP 在细胞表面和早期内体中是完整的，但在核周囊泡中缓慢降解。我们得出结论，SP 诱导 NK1-R 的网格蛋白依赖性内化。 SP/NK1-R 复合物在酸化内体中解离。 SP 被降解，而 NK1-R 则循环到细胞表面。
Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was' unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H+-ATPase inhibitor. The fate of internalized I-125-Sp was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.