Grady E F, Slice L W, Brant W O, Walsh J H, Payan D G, Bunnett N W
Department of Surgery, University of California, San Francisco 94143.
J Biol Chem. 1995 Mar 3;270(9):4603-11. doi: 10.1074/jbc.270.9.4603.
Endocytosis of the gastrin releasing peptide receptor (GRP-R) may regulate cellular responses to GRP. We observed endocytosis in transfected epithelial cells by confocal microscopy using cyanine 3-GRP (cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies. At 4 degrees C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 degrees C, ligand and receptor were internalized into early endosomes with fluorescein isothiocyanate-transferrin. After 10 min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min cy3-GRP was in large, central vesicles, while GRP-R was at the cell surface. We quantified surface GRP-R using an antibody to an extracellular epitope and an 125I-labeled secondary antibody. After exposure to GRP, there was a loss and subsequent recovery of surface GRP-R. Recovery was unaffected by cycloheximide, and thus independent of new protein synthesis, but was attenuated by acidotropic agents, and therefore required endosomal acidification. Internalization of 125I-GRP, assessed using an acid wash, was maximal after 10-20 min, and was clathrin-mediated since it was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP and its receptor are rapidly internalized into early endosomes and then dissociate in an acidified compartment. GRP is probably degraded whereas the GRP-R recycles.
胃泌素释放肽受体（GRP-R）的内吞作用可能调节细胞对胃泌素释放肽（GRP）的反应。我们使用菁3-GRP（菁3.18标记的胃泌素释放肽）和GRP-R抗体，通过共聚焦显微镜观察了转染上皮细胞中的内吞作用。在4℃时，菁3-GRP和GRP-R局限于质膜。在37℃孵育5分钟后，配体和受体与异硫氰酸荧光素-转铁蛋白一起内化到早期内体中。10分钟后，菁3-GRP和GRP-R存在于核周小泡中，60分钟时，菁3-GRP存在于大的中央小泡中，而GRP-R位于细胞表面。我们使用针对细胞外表位的抗体和125I标记的二抗对表面GRP-R进行定量。暴露于GRP后，表面GRP-R先减少随后恢复。恢复不受放线菌酮影响，因此与新蛋白质合成无关，但受亲酸性试剂减弱，因此需要内体酸化。用酸洗评估的125I-GRP内吞作用在10 - 20分钟后达到最大值，且是网格蛋白介导的，因为它受到高渗蔗糖和苯砷氧化物的抑制。因此，GRP及其受体迅速内化到早期内体中，然后在酸化区室中解离。GRP可能被降解，而GRP-R则循环利用。
