Abstract Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells by diverse stimuli and can form two distinct complexes, with the p110γ catalytic subunit binding to either p101 or p84 regulatory subunits. These two complexes are differentially activated by G-protein coupled receptors (GPCRs) and Ras, but the molecular details of this activation are still unclear. Using a combination of X-ray crystallography, HDX-MS, EM, molecular modeling, and biochemical assays we reveal molecular differences between the two p110γ-p84 and p110γ-p101 complexes that explain their differential activation. The structure of p110γ-p84 shows a similar assembly to p110γ-p101 at the p110γ interface, however the interface in p110γ-p84 is dynamic and is evolutionarily conserved to be less stable compared to p110γ-p101. The p110γ-p84 complex is only weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation through an interaction with the p110γ helical domain. The interfaces of the p101 GBD with Gβγ, and the p110γ helical domain with Gβγ were determined using computational alphafold2 modeling and HDX-MS. There are distinct differences in the C-terminal domain of p84 and p101, which allows p101 to bind Gβγ subunits, while p84 does not. The two Gβγ interfaces in p110γ and p101 are distinct, revealing how unique mutants of Gβγ cause differential disruption of PI3Kγ complex activation. Overall, our work provides key insight into the molecular basis for how different PI3Kγ complexes are activated.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs