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

• Published in: Naunyn-Schmiedeberg's archives of pharmacology 1997-08, Vol.356 (2), p.216-224
• Main Authors: Leopoldt, D, Harteneck, C, Nürnberg, B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Baculoviridae - metabolism; Cell Line; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - immunology; GTP-Binding Proteins - metabolism; Histamine - metabolism; Immune Sera - immunology; Insecta; Mammals; Membrane Proteins - metabolism; Molecular Sequence Data; Receptors, Histamine - biosynthesis; Receptors, Histamine - genetics; Receptors, Histamine - metabolism; Recombinant Proteins - biosynthesis; Signal Transduction
• ISSN: 0028-1298
• DOI: 10.1007/PL00005044

Expression of functionally active mammalian histamine H1- and H2-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 H1-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into Gq/11-like proteins whereas in membranes containing H2-receptors histamine enhanced GTP azidoanilide-labelling of Gq/11-like and G(S)-like proteins. In fura-loaded H1- and H2-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 H1-receptors activate only Gq/11, whereas H2-receptors activate Gq/11 and G(S), but neither receptor couples to Gi/o or G12. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components.