G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors

被引:85
作者
Leopoldt, D [1 ]
Harteneck, C [1 ]
Nurnberg, B [1 ]
机构
[1] FREE UNIV BERLIN, INST PHARMAKOL, D-14195 BERLIN, GERMANY
关键词
G proteins; histamine receptors; signal transduction; Sf9; cells;
D O I
10.1007/PL00005044
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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.
引用
收藏
页码:216 / 224
页数:9
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