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.

A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus

“…The high affinity and GTP-sensitive binding sites are thought to reflect receptors coupled to G proteins, whereas the low affinity sites represent uncoupled receptors. In Sf9 cells, various G protein subtypes are expressed in different amounts depending on the class of G pro- tein (12,46). The small number of the high affinity binding sites observed in BV expressing BLT1 alone may represent a receptor population coupled to endogenous G proteins.…”

“…5c, Table I). Because G␤ subunits have been reported to be detected in Sf9 cells (46), the endogenous G␤␥ subunits of Sf9 cells could well be recruited to the membrane fraction co-expressing receptor and the G␣ subunit and could reconstitute the heterotrimer with exogenous G␣ i1 . The BV released from the co-infected cells also exhibited high affinity for ligand binding comparable with the binding affinity observed in mammalian expression systems (29) (Fig.…”

Acylation of Gα13 is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation

“…[28]. Antibodies AS343 raised against the C-terminal domain of GK 13 (amino acids 367^377, LHDNLKQLMLQ) were used for immunoprecipitation [14,29]. Antibodies were kindly provided from the laboratory of G. Schultz (Berlin, Germany).…”

Characteristics of the Ca2+‐dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells

“…Wojcikiewicz et al, 1994;Arias-MontanÄ o et al, 1994) complicates the interpretation of the Ca 2+ -dependence of the inhibitory action of histamine. However, there is no evidence that H 1 -receptors can couple to G i (Leopoldt et al, 1997) and no indication of PKC-mediated inhibition of cyclic AMP accumulation, such as has been reported for the type VI cyclase (Lai et al, 1997), since the broad-spectrum PKC inhibitor K-252A had no eect on the inhibitory eect of histamine. Further, the inhibitory action of thapsigargin provides strong evidence for a Ca 2+ -dependent inhibition of cyclic AMP accumulation in U373 MG cells.…”