Mouse receptor-activity-modifying proteins 1, -2 and -3: amino acid sequence, expression and function
Mouse receptor-activity-modifying proteins 1, -2 and -3: amino acid sequence, expression and function
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DOI：
10.1016/s0303-7207(00)00212-4
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发表时间：
2000-04-25
影响因子：
4.1
通讯作者：
Born, W
中科院分区：
文献类型：
--
作者：
Husmann, K;Sexton, PM;Born, W
降钙素受体样受体(CRLR)作为肾上腺髓质素(ADM)或降钙素(CT)基因相关肽(CGRP)受体，需要新的受体活性修饰蛋白(RAMP)发挥作用。在这里，代表GenEMBL数据库中表达序列标签(EST)的小鼠的cDNA克隆已经被鉴定。它们编码的蛋白质与人类RAMP1、-2和-3的氨基酸序列同源性分别为70%、68%和54%。对PolA(+)RNA的Northern印迹分析表明，小鼠RAMP1(MRAMP1)编码0.8kb的mRNA，主要存在于胚胎和成人的脑和肺以及成人的骨骼肌中。小鼠RAMP2编码0.8kb和1.2kb的mRNA在所有组织中均被识别，其中以胚胎脑、肺和肠道以及成人心、肺、骨骼肌和脑中的表达水平最高。单个1.2kb的mRAMP3编码转录本主要在胚胎和成人脑中表达。在共表达大鼠CRLR(RCRLR)和mRAMP1的COS-7细胞中，rαCGRP(8-37)、rαCGRP和rβCGRP可抑制[I-125]h-CGRP结合，IC50分别为1.4+/-0.5、4.5+/-0.6和7+/-0.3 nM。R-β-CGRP和r-α-CGRP对CyclicAMP的最大刺激作用分别为10倍，EC50分别为0.65+/-0.67和0.86+/-0.6 nM。在共表达rCRLR和mRAMP2的同一细胞中，RADM和RADM(20-50)取代了[I-125]r-ADM的结合，其IC50分别为1.9+/-0.5和3.4+/-1.4 nM，RADM对cAMP积累的最大刺激倍数为7倍，EC50为0.82+/-0.85 nM。在共表达rCRLR和mRAMP3的细胞中，rαCGRP(8-37)、rβCGRP、rαCGRP、RADM和RADM(20-50)抑制[I-125]H-CGRP结合，IC50在3-22 nM之间。RADM刺激cAMP聚集，EC50为5.1+/-2.7 nM，分别是rαCGRP和rβCGRP的12倍和11倍。总而言之。小鼠RAMPI、-2和-3的氨基酸序列与相应的人类RAMP序列具有很高的同源性。RCRLR与mRAMP1、-2或-3在COS-7细胞中的GO表达显示出明显的CGRP-、ADM-或ADM/CGRP受体。(C)2000由爱思唯尔科学爱尔兰有限公司出版。保留所有权利。
The calcitonin receptor-like receptor (CRLR) requires novel receptor-activity-modifying proteins (RAMPs) for its function as an adrenolnedullin (ADM) or a calcitonin (CT) gene-related peptide (CGRP) receptor. Here, mouse cDNA clones representing expressed sequence tags (ESTs) in the GenEMBL database have been identified. They encode for proteins with 70, 68 and 54% amino acid sequence identity with respect to human RAMP1, -2 and -3. On Northern blot analysis of polyA(+) RNA mouse RAMP1 (mRAMP1) encoding mRNA with an apparent size of 0.8 kb was predominantly observed in embryonic and adult brain and lung and in adult skeletal muscle. Mouse RAMP2 encoding 0.8 and 1.2 kb mRNA were recognized in all tissues analyzed with the highest levels in embryonic brain, lung and gut and in adult heart, lung, skeletal muscle and brain. A single 1.2 kb mRAMP3 encoding transcript was mainly expressed in embryonic and adult brain. In COS-7 cells co-expressing rat CRLR (rCRLR) and mRAMP1, [I-125]h alpha CGRP binding was inhibited by r alpha CGRP(8-37), r alpha CGRP and r beta CGRP with IC50 of 1.4 +/- 0.5, 4.5 +/- 0.6 and 7 +/- 0.3 nM, respectively. CyclicAMP accumulation was maximally stimulated tenfold by r beta CGRP and r alpha CGRP with EC50 of 0.65 +/- 0.67 and 0.86 +/- 0.6 nM. In the same cells co-expressing rCRLR and mRAMP2, binding of [I-125]r-ADM was displaced by rADM and rADM(20-50) with IC50 of 1.9 +/- 0.5 and 3.4 +/- 1.4 nM, respectively, and a maximal sevenfold stimulation of cAMP accumulation was observed with rADM with an EC50 of 0.82 +/- 0.85 nM. In the cells co-expressing rCRLR and mRAMP3, [I-125]h alpha CGRP binding was inhibited by r alpha CGRP(8-37), r beta CGRP, r alpha CGRP, rADM and rADM(20-50) with IC50 between 3 and 22 nM. cAMP accumulation was stimulated by rADM with an EC50 of 5.1 +/- 2.7 nM that was 12-fold and 11-fold lower than that of r alpha CGRP and r beta CGRP. In conclusion. mouse RAMPI, -2 and -3 exhibit high amino acid sequence homology to the corresponding human RAMPs. Go-expression of rCRLR with mRAMP1, -2 or -3 in COS-7 cells revealed distinct CGRP-, ADM- or ADM/CGRP receptors. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.