Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling
Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling
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DOI：
10.1074/jbc.m004534200
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发表时间：
2000-09-22
影响因子：
4.8
通讯作者：
Sakata, T
中科院分区：
文献类型：
--
作者：
Kuwasako, K;Shimekake, Y;Sakata, T
表达降钙素受体样受体(CRLR)及其受体活性修饰蛋白(RAMPs)可产生降钙素基因相关肽(CGRP)受体(CRLR/RAMP1)和肾上腺髓质素(AM)受体(CRLR/RAMP2或-3)，利用CRLR和绿色荧光蛋白(CRLR-GFP)的嵌合体研究CRLR和绿色荧光蛋白(CRLR-GFP)在稳定转导的HEK 293细胞中的定位和转运，在RAMP和RAMP共转染的情况下，CRLR-GFP不能对CGRP或AM产生反应，而且在激动剂作用后，质膜上没有发现CRLR-GFP，其定位没有变化。当CRLR-GFP与RAMPS稳定共表达时，CRLR-GFP出现在细胞表面，并且在细胞内cAMP的产生和钙动员中完全活跃，激动剂介导的CRLR-GFP在RAMP1/CGRP或AM、RAMP2/AM和RAMP3/AM中以相似的动力学发生，表明RAMPS对CRLR内化存在配体特异性的调节。这种内化被高渗介质(0.45M蔗糖)和罗丹明标记的转铁蛋白平行定位强烈地抑制，这表明CRLR的内吞作用主要通过依赖于笼蛋白的途径发生。很大一部分CRLR在与配体结合时靶向于溶酶体，内化的CRLR的回收效率不高。在稳定表达CRLR-GFP和Myc-RAMPS的HER 293细胞中，这些罗丹明标记的RAMP在配体存在和不存在的情况下与CRLR-GFP共定位，从而使CRLR与RAMP通过笼蛋白包裹的小泡一起内吞，内化的两个分子都针对降解途径。
Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3), A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without cotransfection of RAMPs, CRLR-GFP failed to generate responses to CGRP or AM without RAMPs, Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization, Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 M sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HER 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands, Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.