Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors
Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors
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DOI：
10.1074/jbc.m112084200
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发表时间：
2002-04-26
影响因子：
4.8
通讯作者：
Firsov, D
中科院分区：
文献类型：
--
作者：
Flahaut, M;Rossier, BC;Firsov, D
受体活性修饰蛋白RAMP 1、RAMP 2和RAMP 3负责限定与降钙素受体样受体（CRLR）的配体的亲和力。也有人提出，受体活性修饰蛋白（RAMP）是CRLR转运到细胞表面所需的分子伴侣。在这里，我们已经研究了CRLR和RAMP在运输CRLR/RAMP异二聚体的质膜通过使用一个高度特异性的结合试验，允许定量检测细胞表面表达的CRLR或RAMP在非洲爪蟾卵母细胞表达系统中的各自的作用。我们证明：（i）异二聚体组装不是CRLR有效细胞表面表达的先决条件，（ii）N-糖基化RAMP 2和RAMP 3在细胞表面表达，并且它们转运至质膜需要N-聚糖，（iii）RAMP 1不是N-糖基化的，并且仅在与CRLR形成异二聚体时转运至质膜，和（iv）在RAMP 1序列中引入N-糖基化位点（D58 N/G60 S、Y 71 N和K103 N/P105 S）使得这些突变体的细胞表面表达水平与CRLR共表达的野生型RAMP 1的水平相似。我们的数据反对RAMP的伴侣功能，并确定了N-糖基化在将这些分子靶向细胞表面中的作用。
Receptor activity modifying proteins RAMP1, RAMP2, and RAMP3 are responsible for defining affinity to ligands of the calcitonin receptor-like receptor (CRLR). It has also been proposed that receptor activity-modifying proteins (RAMP) are molecular chaperones required for CRLR transport to the cell surface. Here, we have studied the respective roles of CRLR and RAMP in transporting CRLR/RAMP heterodimers to the plasma membrane by using a highly specific binding assay that allows quantitative detection of cell surface-expressed CRLR or RAMP in the Xenopus oocytes expression system. We show that: (i) heterodimer assembly is not a prerequisite for efficient cell surface expression of CRLR, (ii) N-glycosylated RAMP2 and RAMP3 are expressed at the cell surface and their transport to the plasma membrane requires N-glycans, (iii) RAMP1 is not N-glycosylated and is transported to the plasma membrane only upon formation of heterodimers with CRLR, and (iv) introduction of N-glycosylation sites in the RAMP1 sequence (D58N/G60S, Y71N, and K103N/P105S) allows cell surface expression of these mutants at levels similar to that of wild-type RAMP1 co-expressed with CRLR. Our data argue against a chaperone function for RAMP and identify the role of N-glycosylation in targeting these molecules to the cell surface.