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

Abstract

Expression of functionally active mammalian histamine H₁- and H₂-receptors was recently demonstrated in Sf9 cells. Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf9 membranes showed expression of Gα isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf9 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 Gα, and phosphate-labelling of Gβ declined in cell membranes. Some 48h 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, Sf9 cells infected only for 28h allowed studies on histamine-induced G protein coupling. In membranes obtained from H₁-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into G _q/11-like proteins whereas in membranes containing H₂-receptors histamine enhanced GTP azidoanilide-labelling of G _q/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 Sf9 G proteins couple to mammalian histamine receptors and secondly that H₁-receptors activate only G_q/11, whereas H₂-receptors activate G_q/11 and G_s, but neither receptor couples to G_i/o or G₁₂. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components.