G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors
G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors
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DOI：
10.1007/pl00005044
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发表时间：
1997-08-01
影响因子：
3.6
通讯作者：
Nurnberg, B
中科院分区：
文献类型：
--
作者：
Leopoldt, D;Harteneck, C;Nurnberg, B
最近在Sf 9细胞中证实了功能活性的哺乳动物组胺H-1-和H-2-受体的表达。在本研究中，我们集中在确定的Sf 9鸟嘌呤核苷酸结合蛋白（G蛋白）参与。Sf 9膜的免疫检测显示属于所有四个G蛋白亚家族的G α亚型的表达。在Sf 9细胞的长时间杆状病毒感染期间，鸟苷5 '-o-（3-硫代三磷酸）的结合以及G蛋白免疫反应性的强度，百日咳毒素介导的ADP-核糖基化。GTP叠氮酰苯胺标记的G α，和磷酸盐标记的G β细胞膜下降。一些48小时后感染哺乳动物组胺受体编码病毒几乎没有功能耦合的配体激活的受体昆虫G蛋白的观察，尽管高水平的表达受体。相比之下，Sf 9细胞感染仅28小时允许组胺诱导的G蛋白偶联的研究。在从H-1受体表达细胞获得的膜中，组胺增加GTP叠氮酰苯胺掺入G（q/11）样蛋白，而在含有H-2受体的膜中，组胺增强G（q/11）样和G（s）样蛋白的GTP叠氮酰苯胺标记。在Fura负载的H-1和H-2受体表达细胞中，组胺诱导细胞内钙库的钙释放。本研究首先表明，Sf 9 G蛋白与哺乳动物组胺受体偶联，其次，H-1受体仅激活G（q/11），而H-2受体激活G（q/11）和G（s），但不与G（1/0）或G（12）偶联。最后，杆状病毒感染后的时间对于研究重组表达和内源性信号转导组分之间的功能偶联是至关重要的。
Expression of functionally active mammalian histamine H-1- and H-2-receptors was recently demonstrated in Sf 9 cells, Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf 9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf 9 membranes showed expression of G alpha isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf 9 cells, binding of guanosine 5'-o-(3-thiotriphosphate) as well as the intensities of G protein immunoreactivity, pertussis toxin-mediated ADP-ribosylation. GTP azidoanilide labelling of G alpha, and phosphate-labelling of G beta declined in cell membranes. Some 48 h after infection with mammalian histamine receptor-encoding viruses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed receptors. In contrast, Sf 9 cells infected only for 28 h allowed studies on histamine-induced G protein coupling. In membranes obtained from H-1-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into G(q/11)-like proteins whereas in membranes containing H-2-receptors histamine enhanced GTP azidoanilide-labelling of G(q/11)-like and G(s)-like proteins. In fura-loaded H-1- and H-2-receptor-expressing cells histamine induced the release of calcium from intracellular stores. This study shows firstly that Sf 9 G proteins couple to mammalian histamine receptors and secondly that H-1-receptors activate only G(q/11), whereas H-2-receptors activate G(q/11) and G(s), but neither receptor couples to G(1/0) or G(12). Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components.