Lundstrom K, Mills A, Allet E, Ceszkowski K, Agudo G, Chollet A, Liljestrom P
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
J Recept Signal Transduct Res. 1995 Jan-Mar;15(1-4):23-32. doi: 10.3109/10799899509045204.
The expression of two G protein-coupled receptors was studied in several cell lines using the Semliki Forest virus expression system. Human neurokinin-2 and dopamine D3 receptor cDNAs were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper RNA into BHK cells resulting in in vivo packaging of high titer SFV-NK-2 and SFV-D3 virus stocks, respectively. Infection of BHK, HOS and CHO cells with the recombinant NK-2 virus resulted in high levels of receptor expression as detected by metabolic labelling with [35S]-methionine. The expression of the NK-2 receptors in the cell membrane was demonstrated by Flow cytometry experiments on infected BHK and CHO cells with fluoresceinyl-NKA as the ligand. Saturation binding assays on membranes prepared from SFV-NK-2 infected CHO cells with [3H] GR100679 showed maximum receptor densities of 6.5 pmol receptor/mg protein. Additionally, the expressed NK-2 receptors were able to stimulate Ca2+ mobilization in CHO cells indicating functional coupling to G proteins. CHO cells infected with SFV-D3 also produced high levels of receptor as evidenced by both [35S]methionine labelling and [3H]spiperone binding.
利用辛德毕斯病毒表达系统，在几种细胞系中研究了两种G蛋白偶联受体的表达情况。将人神经激肽-2和多巴胺D3受体cDNA导入pSFV1载体。体外转录的RNA与pSFV辅助RNA共电穿孔导入BHK细胞，分别在体内包装产生高滴度的SFV-NK-2和SFV-D3病毒株。用重组NK-2病毒感染BHK、HOS和CHO细胞后，通过[35S]-甲硫氨酸代谢标记检测到高水平的受体表达。以荧光素-NKA为配体，对感染的BHK和CHO细胞进行流式细胞术实验，证实了NK-2受体在细胞膜上的表达。用[3H]GR100679对SFV-NK-2感染的CHO细胞制备的膜进行饱和结合试验，结果显示最大受体密度为6.5 pmol受体/mg蛋白。此外，表达的NK-2受体能够刺激CHO细胞中的Ca2+动员，表明其与G蛋白发生了功能偶联。用[35S]甲硫氨酸标记和[3H]螺哌隆结合实验均证明，感染SFV-D3的CHO细胞也产生了高水平的受体。
