Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P-2 to PtdIns-3,4,5-P-3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P-3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class I-B p110gamma/p101 PI3Kgamma is activated by Gbetagamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells Gbetagamma acts as a membrane anchor or an allosteric activator of PI3Kgamma, and which role its noncatalytic p101 subunit plays in its activation by Gbetagamma. Using GFP-tagged PI3Kgamma subunits expressed in HEK cells, we show that Gbetagamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3Kgamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110gamma displayed basal enzymatic activity, but was further stimulated by Gbetagamma, even in the absence of p101. Therefore, we conclude that in vivo, Gbetagamma activates PI3Kgamma by a mechanism assigning specific roles for both PI3Kgamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gbetagamma with p110gamma contributes to activation of PI3Kgamma.