Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines
作者:Tejal Choksi、Debbie L Hay、Stephen Legon、David R Poyner、Stefanie Hagner、Stephen R Bloom、David M Smith
DOI:10.1038/sj.bjp.0704761
日期:2002.7
The calcitonin receptor‐like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene‐related peptide (CGRP) and/or adrenomedullin in transfected cells.
There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells.
We confirmed CGRP subtype 1 receptor (CGRP1) pharmacology in SK‐N‐MC neuroblastoma cells. L6 myoblast cells expressed both CGRP1 and adrenomedullin receptors whereas Rat‐2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP2 receptor pharmacology for Col‐29 colonic epithelial cells, which, instead were CGRP1‐like in this study.
L6, SK‐N‐MC and Col‐29 cells expressed mRNA for RAMP1 and RAMP2 but Rat‐2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA.
SK‐N‐MC, Col‐29 and Rat‐2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor.
These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.
British Journal of Pharmacology (2002) 136, 784–792; doi:10.1038/sj.bjp.0704761
降钙素基因相关肽(CGRP)和/或肾上腺髓质素的受体由降钙素受体样受体(CRLR)及其特异性受体活动修饰蛋白(RAMPs)形成。
目前关于CRLR/RAMP复合物能否形成内源性CGRP和肾上腺髓质素受体的证据较少。因此,我们研究了已知表达这些受体的实验细胞株中,CGRP和/或肾上腺髓质素的结合是否与CRLR和RAMPmRNA的表达相关。同时,我们还使用了特异性的人类或大鼠CRLR抗体来检测这些细胞中是否存在CRLR。
我们证实了CGRP亚型1受体(CGRP1)在SK-N-MC神经母细胞瘤细胞中的药理学活性。L6肌母细胞同时表达CGRP1和肾上腺髓质素受体,而Rat-2成纤维细胞仅表达肾上腺髓质素受体。相反,Col-29结肠上皮细胞中未能证实CGRP2受体的药理学活性,而在本研究中这些细胞表现出类似CGRP1的特征。
L6、SK-N-MC和Col-29细胞表达RAMP1和RAMP2mRNA,Rat-2成纤维细胞仅表达RAMP2mRNA。所有细胞株中均未检测到可检测水平的RAMP3mRNA。
SK-N-MC、Col-29和Rat-2成纤维细胞中表达了CRLRmRNA。然而,通过Northern blot分析,一种来源的L6细胞中未检出CRLRmRNA。相反,另一种来源的L6细胞中检测到了CRLRmRNA。所有细胞株均表达了CRLR蛋白。因此,即使在Northern blot分析中未检测到CRLRmRNA,也不能排除该受体的存在。
这些数据有力支持了CRLR与适当的RAMPs共同作为CGRP和肾上腺髓质素的结合位点存在于培养的细胞中。
British Journal of Pharmacology (2002) 136, 784–792; doi:10.1038/sj.bjp.0704761