G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors.
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.
Leopoldt
D
D
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Harteneck
C
C
Nürnberg
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006632:Histamine / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D007313:Insecta; D008322:Mammals; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011968:Receptors, Histamine / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction
D007106:Immune Sera; D008565:Membrane Proteins; D011968:Receptors, Histamine; D011994:Recombinant Proteins; D006632:Histamine; D019204:GTP-Binding Proteins