Research ArticleGPCR SIGNALING

Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils

See allHide authors and affiliations

Science Signaling  03 Nov 2020:
Vol. 13, Issue 656, eaaz4003
DOI: 10.1126/scisignal.aaz4003

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

How to activate PI3Kγ in neutrophils

A critical effector of GPCRs in neutrophils is PI3Kγ, but how the two PI3Kγ complexes are activated by Gβγ has been controversial, likely due to a reliance on in vitro assays using cultured cells. Rynkiewicz et al. generated mice with knock-in mutations in the Gβγ-binding sites in the PI3Kγ catalytic subunit p110γ or in the regulatory subunit p101. Loss of Gβγ binding to p110γ had greater effects on signaling in response to different GPCR agonists than loss of Gβγ binding to p101. Although the migration of isolated neutrophils was affected only by loss of Gβγ binding to p110γ, neutrophil migration in mouse models of neutrophilic inflammation was strongly impaired by defective Gβγ binding to either p110γ or p101. These results refine our understanding of how PI3Kγ is activated in neutrophils and emphasize the importance of investigating signaling pathways in physiologically relevant contexts.

Abstract

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 p101VVKR777AAAA 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.

View Full Text

Stay Connected to Science Signaling