Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3K␣, -␤, and -␦ isoforms coupled to receptor tyrosine kinases and a PI3K␥ isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K␥ catalytic subunit (p110␥) and G␤␥ complexes. When phosphatidylinositol was used as a substrate, G␤␥ stimulated p110␥ lipid kinase activity more than 60-fold (EC 50 , ϳ20 nM). Stimulation was inhibited by G␣ o -GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110␥ did not affect G␤␥-mediated enzymatic stimulation, whereas incubation of G␤␥ with a synthetic peptide resembling a predicted G␤␥ effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110␥. G␤␥ complexes bound to N-as well as C-terminal deletion mutants of p110␥. Correspondingly, these enzymatically inactive N-and C-terminal mutants inhibited G␤␥-mediated activation of wild type p110␥. Our data suggest that (i) p110␥ directly interacts with G␤␥, (ii) the pleckstrin homology domain is not the only region important for G␤␥-mediated activation of the lipid kinase, and (iii) G␤␥ binds to at least two contact sites of p110␥, one of which is close to or within the catalytic core of the enzyme.

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.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5‐trisphosphate (InsP3)‐mediated Ca2+ release and store‐dependent ‘capacitative’ Ca2+ entry through Ca(2+)‐permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store‐operated channels. The related trp‐like (trpl) gene product also forms a Ca(2+)‐permeable cation channel, but is not activated by store depletion. Co‐expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33‐fold in comparison with co‐expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside‐out patches. We conclude that trpl is regulated by G11 proteins in a membrane‐confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store‐operated mechanisms but also by directly stimulating cation channels.

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In this study, G␤ specificity in the regulation of G␤␥-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase C␤ (PLC␤) isozymes was examined. Recombinant mammalian G␤ 1-3 ␥ 2 complexes purified from Sf9 membranes stimulated PI3K␥ lipid kinase activity with similar potency (10 -30 nM) and efficacy, whereas transducin G␤␥ was less potent. Functionally active G␤ 5 ␥ 2 dimers were purified from Sf9 cell membranes following coexpression of G␤ 5 and G␥ 2-His . This preparation as well as G␤ 1 ␥ 2-His supported pertussis toxin-mediated ADP-ribosylation of G␣ i1 . G␤ 1 ␥ 2-His stimulated PI3K␥ lipid and protein kinase activities at nanomolar concentrations, whereas G␤ 5 ␥ 2-His had no effect. Accordingly, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , significantly stimulated the lipid kinase activity of PI3K␤ in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLC␤ 2 and PLC␤ 1 . However, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , activated PLC␤ 3 . Experimental evidence suggests that the mechanism of G␤ 5 -dependent effector selectivity may differ between PI3K and PLC␤. In conclusion, these data indicate that G␤ subunits are able to discriminate among effectors independently of G␣ due to selective protein-protein interaction.