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 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.

• Authors: D Leopoldt, C Harteneck, B Nürnberg
• Journal: Naunyn-Schmiedeberg's archives of pharmacology (Naunyn Schmiedebergs Arch Pharmacol) Vol. 356 Issue 2 Pg. 216-24 (Aug 1997) ISSN: 0028-1298 [Print] Germany
• PMID: 9272728 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Immune Sera
• Membrane Proteins
• Receptors, Histamine
• Recombinant Proteins
• Histamine
• GTP-Binding Proteins

Topics

• Amino Acid Sequence
• Animals
• Baculoviridae (metabolism)
• Cell Line
• GTP-Binding Proteins (chemistry, immunology, metabolism)
• Histamine (metabolism)
• Immune Sera (immunology)
• Insecta
• Mammals
• Membrane Proteins (metabolism)
• Molecular Sequence Data
• Receptors, Histamine (biosynthesis, genetics, metabolism)
• Recombinant Proteins (biosynthesis)
• Signal Transduction