Madziva M T, Edwardson J M
Department of Pharmacology, University of Cambridge, UK.
Eur J Pharmacol. 2001 Sep 28;428(1):9-18. doi: 10.1016/s0014-2999(01)01266-3.
The muscarinic M4 receptor for acetylcholine was tagged at its C terminus with green fluorescent protein (GFP) and expressed in NG108-15 cells, which normally express this receptor subtype. The binding affinity of the antagonist N-methylscopolamine was not significantly affected by the presence of the GFP tag, whereas the affinity of the receptor for the agonist carbachol was reduced by four-fold. Stimulation of the tagged receptor resulted in inhibition of adenylyl cyclase. Following agonist stimulation, the tagged receptor was slowly internalized, and became partially co-localized with the endosomal marker Texas Red-transferrin after 30 min. There was little co-localization with the lysosomal marker 1gp120 even after 60 min of internalization. Finally, the tagged receptor, unlike the endogenous receptor, failed to recycle to the plasma membrane on removal of the agonist. We conclude that the GFP-tagged muscarinic M4 receptor does not traffic normally in NG108-15 cells, most likely because of its gross overexpression.
乙酰胆碱的毒蕈碱型M4受体在其C末端用绿色荧光蛋白（GFP）标记，并在通常表达该受体亚型的NG108-15细胞中表达。拮抗剂N-甲基东莨菪碱的结合亲和力不受GFP标签存在的显著影响，而受体对激动剂卡巴胆碱的亲和力降低了四倍。标记受体的刺激导致腺苷酸环化酶的抑制。激动剂刺激后，标记受体缓慢内化，并在30分钟后与内体标记物德克萨斯红转铁蛋白部分共定位。即使内化60分钟后，与溶酶体标记物1gp120的共定位也很少。最后，与内源性受体不同，标记受体在去除激动剂后未能再循环到质膜。我们得出结论，GFP标记的毒蕈碱型M4受体在NG108-15细胞中不能正常运输，很可能是因为其过度表达。
