Selkirk J V, Price G W, Nahorski S R, Challiss R A
Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, UK.
Neuropharmacology. 2001 Apr;40(5):645-56. doi: 10.1016/s0028-3908(00)00208-2.
In this study the effects of cell background on the coupling of the type 1alpha metabotropic glutamate (mGlu1alpha) receptor to different G protein sub-populations by recombinant expression of this receptor subtype in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells have been investigated. Receptor-G protein interactions were assessed using [(35)S]GTPgammaS binding and subsequent Galpha subunit-specific immunoprecipitation. In a CHO cell line (CHO-lac-mGlu1alpha), where mGlu1alpha receptor expression is under inducible control, stimulation of membranes with the mGlu receptor agonist quisqualate resulted in an increase in specific [(35)S]GTPgammaS binding to G(q/11)alpha only, whereas in a BHK cell line (BHK-mGlu1alpha) agonist stimulation increased [(35)S]GTPgammaS binding to G(q/11)alpha and also to pertussis toxin (PTx)-sensitive G(i/o) proteins (assessed using G(i1/2)alpha- and G(i3/o)alpha-specific antibodies). These data are consistent with our previous observations of dual, antagonistic G(q/11)/G(i/o) regulation of phospholipase C (PLC) in BHK-mGlu1alpha cells, whereas no evidence was found for a G(i/o) modulation of PLC activity in the CHO-lac-mGlu1alpha cell line. PTx pre-treatment of either cell line had no effect on either the magnitude or the concentration-dependency of agonist-stimulated [(35)S]GTPgammaS-G(q/11)alpha binding, excluding the possibility that receptor-G(i/o) uncoupling can unmask an increase in receptor-G(q/11) interaction. mGlu1alpha receptor expression per se had little effect on Galpha protein expression levels in either CHO or BHK cell lines, with the possible exception of a small, but consistent increase in G(o)alpha expression in BHK-mGlu1alpha cells compared to the vector-transfected control cell line (BHK-570). Semi-quantitative assessment of mGlu1alpha receptor immunoreactivity and [(3)H]quisqualate saturation binding analysis demonstrated a ca 10-fold higher mGlu1alpha receptor content in BHK cells. Whether the higher receptor expression level in BHK-mGlu1alpha cells underlies the additional G(i/o) coupling observed in this cell line, or additional factors contribute to the phenomenon are discussed.
在本研究中，通过在幼仓鼠肾（BHK）细胞和中国仓鼠卵巢（CHO）细胞中重组表达1α型代谢型谷氨酸（mGlu1α）受体亚型，研究了细胞背景对该受体与不同G蛋白亚群偶联的影响。使用[³⁵S]GTPγS结合以及随后的Gα亚基特异性免疫沉淀来评估受体 - G蛋白相互作用。在一种CHO细胞系（CHO - lac - mGlu1α）中，mGlu1α受体表达受诱导控制，用mGlu受体激动剂quisqualate刺激细胞膜仅导致与G（q/11）α的特异性[³⁵S]GTPγS结合增加，而在BHK细胞系（BHK - mGlu1α）中，激动剂刺激增加了与G（q/11）α以及百日咳毒素（PTx）敏感的G（i/o）蛋白的[³⁵S]GTPγS结合（使用G（i1/2）α和G（i3/o）α特异性抗体评估）。这些数据与我们之前在BHK - mGlu1α细胞中对磷脂酶C（PLC）的双重、拮抗性G（q/11）/G（i/o）调节的观察结果一致，而在CHO - lac - mGlu1α细胞系中未发现PLC活性的G（i/o）调节证据。对任一细胞系进行PTx预处理对激动剂刺激的[³⁵S]GTPγS - G（q/11）α结合的幅度或浓度依赖性均无影响，排除了受体 - G（i/o）解偶联可揭示受体 - G（q/11）相互作用增加的可能性。mGlu1α受体表达本身对CHO或BHK细胞系中的Gα蛋白表达水平影响很小，与载体转染的对照细胞系（BHK - 570）相比，BHK - mGlu1α细胞中G（o）α表达可能有小幅但一致的增加除外。对mGlu1α受体免疫反应性的半定量评估和[³H]quisqualate饱和结合分析表明，BHK细胞中的mGlu1α受体含量高约10倍。讨论了BHK - mGlu1α细胞中较高的受体表达水平是否是该细胞系中观察到的额外G（i/o）偶联的基础，或者是否有其他因素导致了这一现象。
