Overall, this work provides insight into the molecular mechanisms mediating differential PI3Kγ activation by Ras and GPCR signaling. Finally, computational modeling, HDX-MS, and mutagenesis were used to define the Gβγ binding interfaces with both the C-terminal domain of p101 and the helical domain of p110γ. The p110γ-p84 complex can only be potently activated and membrane recruited by Gβγ when Ras is present, where the p110γ-p101 complex can be robustly activated by Gβγ subunits alone. Membrane reconstitution experiments using HDX-MS and single-molecule total internal reflection fluorescence (TIRF) microscopy to study membrane recruitment of p110γ-p84 and p110γ-p101 by lipidated Gβγ and Ras shows that p110γ-p84 requires Ras for membrane localization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments revealed that the p110γ-p84 is dynamic relative to the p110γ-p101 complex. To decipher the molecular mechanism for why p101 and p84 subunits differentially regulate p110γ activation, we determined the structure of the p110γ-p84 complex using a combined X-ray crystallography, EM, and computational modeling approach. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation.
The p110γ-p84 complex is dynamic compared with p110γ-p101. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras.
0 kommentar(er)
