Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

Abstract

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a hetero-dimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ binding domain, recruiting the hetero-dimer to the membrane and allowing for engagement of a secondary Gβγ binding site in p110γ. Multiple oncogenic mutations mapped to these novel interfaces and enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

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

“…One of the primary differences between p101 and p84 regulatory subunits is their differential ability to be recruited by lipidated Gbg subunits. We have previously identified that this binding occurs at the C-terminal domain of p101, in a region we defined as the Gbg binding domain (GBD) (28,30). The Alphafold2 model of the p110g-p84 structure allowed us to examine differences in this domain.…”

“…Biochemical reconstitution studies have defined two major signaling proteins that mediate PI3Kg activation downstream of cell surface receptors, lipidated Gbg subunits released by activated GPCRs, and GTP loaded lipidated Ras. The presence of p101 and p84 regulatory subunits dramatically alter the activation by each of these stimuli, with in vitro the p110g-p101 complex is activated ~100 fold by Gbg, while p110g-p84 is activated ~5 fold (28)(29)(30)(31)(32). In cells the p110g-p84 complex is poorly recruited to cell membranes by Gbg subunits, with it requiring Ras for membrane localization (32).…”

“…Extensive biophysical experiments on the free p110g catalytic subunit and the p110g-p101 complex have revealed insight into the architecture and regulation of p110g (28,30,(33)(34)(35). The p110g catalytic subunit is composed of an adaptor binding subunit (ABD), a Ras binding domain (RBD) that mediates activation downstream of Ras, a C2 domain, a helical domain, and a bilobal kinase domain.…”

“…The p110g catalytic subunit is composed of an adaptor binding subunit (ABD), a Ras binding domain (RBD) that mediates activation downstream of Ras, a C2 domain, a helical domain, and a bilobal kinase domain. The cryo-EM structure of the p110g-p101 complex revealed that p110g binds to the p101 regulatory subunit through the C2 domain, and the RBD-C2 and C2-helical linkers (30). We previously mapped a putative Gbg binding interface in the helical domain of p110g (28), with an additional binding site in the C-terminal domain of p101 (28,30).…”

“…The cryo-EM structure of the p110g-p101 complex revealed that p110g binds to the p101 regulatory subunit through the C2 domain, and the RBD-C2 and C2-helical linkers (30). We previously mapped a putative Gbg binding interface in the helical domain of p110g (28), with an additional binding site in the C-terminal domain of p101 (28,30). Mutations in Gbg have differential effects on either p110g or p110g-p101 activation (36), but the full molecular details of how Gbg binds to either p110g or p101 is still unclear.…”