Class IB phosphoinositide 3-kinase ␥ (PI3K␥) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3K␥ consists of a catalytic p110␥ and a regulatory p87 PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein ␤␥ (G␤␥)-mediated PI3K␥ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110␥ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3K␥-complemented bone marrow-derived mast cells (BMMCs) from p110␥ -/-mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3K␥ activation. Although p101 strongly interacted with G␤␥, thereby mediating PI3K␥ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing G␤␥-dependent membrane recruitment of p87/p110␥ by direct interaction with p110␥, suggesting the indispensability of Ras for activation of p87/p110␥. Consequently, interference with Ras signaling indeed selectively blocked p87/p110␥, but not p101/ p110␥, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110␥ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3K␥ that could open up new possibilities for therapeutic intervention.confocal life cell imaging ͉ G protein ͉ receptor signaling ͉ mast cells

Background: p87 and p101 represent non-catalytic subunits of class I B PI3K␥. Results: Expression and activity of PI3K␥ is modified differently by p87 and p101 in vitro and in living cells. Conclusion: Non-catalytic subunits of PI3K␥ represent two different regulators in the absence of G␤␥ or Ras. Significance: p87 and p101 determine diversity within class I B PI3K␥ and allow integration in distinct PI3K␥ signaling pathways.

Class IB phosphoinositide 3-kinases (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled-receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody (mAb(A)p110γ) to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ-interface, hydrogen-deuterium exchange coupled to mass spectrometry measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30 times more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

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