G-PROTEINS ENDOGENOUSLY EXPRESSED IN SF-9 CELLS - INTERACTIONS WITH MAMMALIAN HISTAMINE-RECEPTORS

Expression of functionally active mammalian histamine H-1- and H-2-rec eptors was recently demonstrated in Sf 9 cells, Either receptor elicit ed phosphoinositide degradation leading to an increased cytoplasmic ca lcium concentration. In the present study we focussed on identifying t he Sf 9 guanine nucleotide-binding proteins (G proteins) involved. Imm unodetection of Sf 9 membranes showed expression of G alpha isoforms b elonging to all four G protein subfamilies. During prolonged baculovir us infection of Sf 9 cells, binding of guanosine 5'-o-(3-thiotriphosph ate) as well as the intensities of G protein immunoreactivity, pertuss is toxin-mediated ADP-ribosylation. GTP azidoanilide labelling of G al pha, and phosphate-labelling of G beta declined in cell membranes. Som e 48 h after infection with mammalian histamine receptor-encoding viru ses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed recep tors. In contrast, Sf 9 cells infected only for 28 h allowed studies o n histamine-induced G protein coupling. In membranes obtained from H-1 -receptor-expressing cells, histamine increased incorporation of GTP a zidoanilide 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-ex pressing cells histamine induced the release of calcium from intracell ular stores. This study shows firstly that Sf 9 G proteins couple to m ammalian 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 nei ther 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 co mponents.