Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors
Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors
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DOI：
10.1074/jbc.m511147200
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发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Kitamura, K
中科院分区：
文献类型：
--
作者：
Kuwasako, K;Cao, YN;Kitamura, K
受体活性修饰蛋白（RAMP）使降钙素受体样受体（CRLR）能够作为降钙素基因相关肽受体（CRLR/RAMP 1）或肾上腺髓质素（AM）受体（CRLR/RAMP 2或-3）发挥功能。在这里，我们研究了人类RAMP 1，-2，3（hRAMP 1，-2，3）的细胞质C-末端尾（C-tails）的功能，通过将它们的C-末端缺失或进行性截短突变体连接到稳定表达hCRLR的HEK-293细胞中。从hRAMP 1中删除C-尾对突变异源二聚体的表面表达、功能或细胞内运输几乎没有影响。相比之下，从hRAMP 2中删除C-尾破坏了hCRLR向细胞表面的转运，导致I-125-hAM结合的显著降低和诱发的cAMP积累。hRAMP 2突变体的转染效率与野生型hRAMP 2相当;此外，免疫细胞化学分析表明，突变体hRAMP 2保持在内质网内。流式细胞仪分析表明，删除C-尾从hRAMP 3显着增强AM诱发的内化的突变异源二聚体，虽然有激动剂亲和力没有变化。通过去除hRAMP 2和-3的6个C-末端氨基酸或将它们的C-尾彼此交换来截短C-尾对hCRLR的表面表达、激动剂亲和力或内化没有影响，这表明hRAMP C-尾内的高度保守的Ser-Lys序列参与两种AM受体的细胞运输。值得注意的是，从hRAMP中删除相应的C-尾对hCRLR的溶酶体分选没有影响。因此，hRAMP 2和-3的各自的C-尾差异地影响hCRLR表面递送和内化。
Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin ( AM) receptor (CRLR/RAMP2 or - 3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, - 2, and - 3 (hRAMP1, - 2, and - 3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in I-125-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wildtype hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and - 3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and - 3 differentially affect hCRLR surface delivery and internalization.