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. 2013 Nov 19;110(47):18862-7.
doi: 10.1073/pnas.1304801110. Epub 2013 Nov 4.

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs)

Oscar Vadas  1 , Hashem A Dbouk, Aliaksei Shymanets, Olga Perisic, John E Burke, Widian F Abi Saab, Bassem D Khalil, Christian Harteneck, Anne R Bresnick, Bernd Nürnberg, Jonathan M Backer, Roger L Williams
Affiliations

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs)

Oscar Vadas et al. Proc Natl Acad Sci U S A. .

Abstract

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

Keywords: HDX-MS; PIK3CG; PIK3R5; PIP3; oncogene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mapping regulatory interactions of p110γ with p101, with membranes and with Gβγ using HDX-MS. (A) Mapping of the HDX changes in p110γ induced by binding p101. Peptides with significant changes are colored on the ribbon diagram of the p110γ structure (Protein Data Bank 1E8X) according to the color scheme shown (red and orange for increased HDX, cyan and blue for decreased HDX). (Lower) A linear plot highlighting changes in HDX between the two states (y axis) as a function of the central residue number for each peptic peptide (x axis). The schematic drawing on the Right illustrates the two states that were compared in the HDX-MS analysis. (B) Mapping of the HDX changes in p110γ induced by binding of p110γ/p101 to lipid membranes (illustrated as in A). (C) Mapping of the HDX changes in p110γ caused by interaction of prenylated Gβγ with the membrane-bound p110γ/p101 complex (illustrated as in A). The pink star indicates the position of residues mutated for in vitro and cellular characterization.
Fig. 2.
Fig. 2.
GPCR-mediated activities of p110γ/p101 in vitro and in cells. (A) Effect of the p110γ -552RK/DD mutation on in vitro activation by Gβγ of either the free p110γ catalytic subunit (Upper) or the p110γ/p101 complex (Lower). The error bars illustrate SDs of three independent replicates. (B) Representative images of HEK293T cells stably expressing the fMLP receptor and transfected with the described PI3Kγ constructs. PIP3 formation in the plasma membrane was detected by translocation of the transfected GFP-Grp1PH domain. Basal and fMLP-stimulated (1–2 min) states are shown. (C) Quantification of the cellular activity assessed by GFP-Grp1PH translocation. (D) Activation of p110γ/p101 signaling by LPA in cells, as detected by pAkt Western blot. (E) Quantification of LPA-PI3Kγ–mediated Akt activation. The graph shows pAkt/Akt ratios normalized to unstimulated WT-p110γ/p101. The graph shows mean ± SD of at least three independent experiments. P values calculated by two-tailed t test. (F) Transformation of NIH 3T3 cells measured by colony formation in soft agar resulting from transfection with the indicated constructs. (Upper) Western blot showing p110γ expression, using an anti-myc antibody. Graph is as in E. (G) Chemotaxis of HEK293E cells expressing WT or mutant p110γ toward media with or without LPA. (Upper) Western blot showing expression levels of p110γ as detected by anti-myc antibody. Graph is as in E with two replicates.
Fig. 3.
Fig. 3.
Effect of p101 mutations on cellular activity and transformation. (A) Schematic representation of the two states compared by HDX-MS to identify changes in p101 induced by prenylated Gβγ on membranes. In this experiment, p101 was always in complex with p110γ. (B) Linear plot of changes in HDX rates in p101 upon binding prenylated Gβγ as a function of central residue number. White indicates regions for which no peptides were detected in the HDX-MS experiment. (C) Schematic representation of p101 tetra-alanine mutants that were generated for biochemical characterization. Above the blocks illustrating positions of mutations is a bar colored according to the HDX results (blue represents a decrease in HDX, gray no change, and white regions not covered by peptides in the HDX-MS experiment). (D) Representative images of HEK293T cells transfected with the described PI3Kγ constructs. PIP3 production is indicated by translocation of the GFP-Grp1PH domain construct to the plasma membrane (as for Fig. 2B). (E) A quantitation of Grp1PH translocation for all of the p101 tetra-alanine mutants. (F) In vitro lipid kinase activity as a function of Gβγ concentration for two selected p101 mutants in complexes with wild-type or 552DD-mutated p110γ. Error bars show SD for at least three replicates. (G) Transformation of NIH 3T3 cells measured by colony formation in soft agar resulting from transfection with the indicated constructs.
Fig. 4.
Fig. 4.
Schematic representation of changes in HDX rate for p110γ and p101 observed when transiting through different activation states. Blue coloring indicates reduction in exchange, and increases are shown in red. Yellow highlights structural elements that differ between the PI3Kγ states.
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