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

Abstract

Class IB phosphoinositide 3-kinase (PI3K{gamma}) is activated in immune cells by diverse stimuli and can form two distinct complexes, with the p110{gamma} 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 p110{gamma}-p84 and p110{gamma}-p101 complexes that explain their differential activation. The structure of p110{gamma}-p84 shows a similar assembly to p110{gamma}-p101 at the p110{gamma} interface, however the interface in p110{gamma}-p84 is dynamic and is evolutionarily conserved to be less stable compared to p110{gamma}-p101. The p110{gamma}-p84 complex is only weakly recruited to membranes by G{beta}{gamma} subunits alone and requires recruitment by Ras to allow for G{beta}{gamma} activation through an interaction with the p110{gamma} helical domain. The interfaces of the p101 GBD with G{beta}{gamma}, and the p110{gamma} helical domain with G{beta}{gamma} 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 G{beta}{gamma} subunits, while p84 does not. The two G{beta}{gamma} interfaces in p110{gamma} and p101 are distinct, revealing how unique mutants of G{beta}{gamma} cause differential disruption of PI3K{gamma} complex activation. Overall, our work provides key insight into the molecular basis for how different PI3K{gamma} complexes are activated.