Receptor activity-modifying protein (RAMP) isoform-specific regulation of adrenomedullin receptor trafficking by NHERF-1
Receptor activity-modifying protein (RAMP) isoform-specific regulation of adrenomedullin receptor trafficking by NHERF-1
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DOI：
10.1074/jbc.m501751200
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发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Parameswaran, N
中科院分区：
文献类型：
--
作者：
Bomberger, JM;Spielman, WS;Parameswaran, N
受体活性修饰蛋白（RAMPs 1-3）是单一的跨膜辅助蛋白，对各种g蛋白偶联受体的质膜表达和受体表型至关重要。肾上腺髓质素（AM）是一种血管舒张配体的功能性受体，由RAMP2或RAMP3和降钙素受体样受体（CRLR）组成。现在已知RAMP3蛋白-蛋白相互作用调节AM2受体的再循环。本研究的主要目的是确定RAMP3的其他相互作用伙伴，并确定它们在CRLR-RAMP3贩运中的作用。g蛋白偶联受体的转运受到Na+/H+交换调节因子-1 （NHERF-1）的调控，NHERF-1是一种含有两个串联PSD-95/Discs-large/ZO-1同源性（PDZ）结构域的衔接蛋白。在表达AM2受体的HEK 293T细胞中，该复合物经历激动剂诱导的脱敏和内化。然而，在NHERF-1的存在下，尽管AM受体（CRLR/RAMP3）经历了脱敏，但受体复合物的内化被阻断。叠加实验和突变分析表明，RAMP3和NHERF-1通过NHERF-1上的PDZ型结构域相互作用。当CRLR与RAMP1或RAMP2共表达时，CRLR- ramp复合物的内化不受NHERF-1的影响。NHERF-1上ezrin/radixin/moesin （ERM）结构域的突变表明，NHERF-1通过将复合物粘附在肌动蛋白细胞骨架上，抑制了CRLR/RAMP3复合物的内化。当在内源性表达CRLR-RAMP3复合物和NHERF-1的人近端小管细胞原代培养中进行检测时，CRLR-RAMP复合物在激动剂刺激下脱敏，但不能内化。通过RNA干扰技术敲除RAMP3或NHERF-1，可使激动剂诱导的CRLR-RAMP复合体内化。这些使用内源性和过表达细胞模型的结果表明，NHERF-1和RAMP3在AM受体内化中的新功能，并提示受体运输的其他调节机制。
Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.