The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP₃ production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101^VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

PI3Kγ 亚型被髓样细胞中的 Gi 偶联 GPCR 激活，但 PI3Kγ 的两个内源性复合物 p101 / p110γ 和 p84 / p110γ 通过 Gβγ 接受直接调节或通过 RAS 进行间接调节的程度以及这些输入的充足性有争议或不清楚。我们生成了具有点突变的小鼠，这些突变阻止了 Gβγ 与 p110γ (RK552DD) 或 p101 (VVKR777AAAA) 的结合，并研究了这些突变在原发性中性粒细胞和中性粒细胞炎症小鼠模型中的影响。Gβγ 与 p110γ 结合的​​丧失大大降低了各种 GPCR 激动剂对中性粒细胞中 p101 / p110γ 和 p84 / p110γ 的激活。Gβγ 与 p101 结合的丧失导致更多不同的影响，这取决于激动剂和细胞反应，在 PIP _{3 中}看到的减少最大初级中性粒细胞响应 LTB4 和 MIP-2 以及在巯基乙酸盐诱导的腹膜炎或 MIP2 诱导的耳袋炎症期间中性粒细胞的迁移中产生。我们还观察到 p101 ^VVKR777AAAA中性粒细胞对f 的p84 依赖性 ROS 反应增强。MLP 和 C5a，表明 p101 / p110γ 和 p84 / p110γ 之间可能存在 GPCR 激活下游 Gβγ 亚基的竞争。在缺乏 p101 和 p84 调节亚基的小鼠中，GPCR 没有激活中性粒细胞中的 p110γ，表明 RAS 与 p110γ 的结合不足以支持这种细胞类型中的 GPCR 激活。这些发现定义了 Gβγ 亚基在激活两种内源性 PI3Kγ 复合物方面的直接作用，并表明调节性 PI3Kγ 亚基偏向于不同 GPCR 的激活。