Figure 5. Functional characterization of Gβγ-mediated PI3Kγ activation.

(A) PI3Kγ membrane recruitment via two Gβγ subunits, Gβγ^HD and Gβγ^CTD. Dashed red line 1 indicates the conformational flexibility of the CTD domain of PI3Kγ upon Gβγ^CTD binding that would allow it to move along the plane of the membrane, as suggested by the State 1 and 2 structures we determined. Dashed red line 2 represents the fact that the p110γ subunit is still not fully engaged with the membrane even when tethered to the membrane (in this example by Gβγ^CTD), as suggested previously¹³. (B) Surface representation of the PI3Kγ·ADP–Gβγ^HD–Gβγ^CTD State 2 structure. The sites targeted by site-directed mutagenesis represent those for Gβγ binding (p110γ-Leu551 and p101-⁷¹¹KRL⁷¹³) and regulation (p110γ-Leu564 and p101-Ile689). (C-F) Dose response curves of PI3Kγ variants (WT, black triangles; p110γ–L551Ap101, yellow circles; p110γp101-CTD-GS and p110γp101-CTD-GSS, green and purple circles; p110γ–L64Sp101, red circles; p110γp101-I689T, blue circles. Specific activity is nmol ADP·μmol enzyme⁻¹·min⁻¹. (G-J) Neutrophil migration imaging and tracking in a zebrafish embryo 3 days post-fertilization. (G) Quantification of neutrophil tracks under control (PBS) or p101 knock down (MO) background. Representative images are shown in Extended Data Fig 11A–B. (H-J) Quantification of neutrophil tracks under p101 knockdown (MO) background of neutrophils without or with expressing human WT p101 (H), mutant human p101-CTD-GS (I), or mutant human p101-I692T (J). Representative images are shown in Extended Data Fig 11C–E. In (G-J), three independent experiments with n>20 are compiled. p values represent overall effects using the Gehan-Breslow-Wilcoxon test.