Abstract
In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta 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 PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.
Highlights
G-protein-coupled receptors are part of a major signal recognition system in mammalian cells that perceives a vast variety of external physical and chemical entities [1]
Different phosphoinositide 3-kinases (PI3Ks) have been identified as essential elements in the activation of mitogenic signaling cascades by G-proteincoupled receptors [37,38,39]
Since we and others [23, 24, 49] have found that G␥ stimulates PI3K activity by direct interaction with PI3K and PI3K␥, we examined the coupling of different G␥ isoforms to these lipid kinases by employing purified preparations (Fig. 1)
Summary
G-protein-coupled receptors are part of a major signal recognition system in mammalian cells that perceives a vast variety of external physical and chemical entities [1]. Recombinant mammalian G1–3␥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. G1␥2-His stimulated PI3K␥ lipid and protein kinase activities at nanomolar concentrations, whereas G5␥2-His had no effect.
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