title: Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).
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.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Oscar
authors.initials: O
authors.lastname: Vadas
authors.forename: Hashem A
authors.initials: HA
authors.lastname: Dbouk
authors.forename: Aliaksei
authors.initials: A
authors.lastname: Shymanets
authors.forename: Olga
authors.initials: O
authors.lastname: Perisic
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.forename: Widian F
authors.initials: WF
authors.lastname: Abi Saab
authors.forename: Bassem D
authors.initials: BD
authors.lastname: Khalil
authors.forename: Christian
authors.initials: C
authors.lastname: Harteneck
authors.forename: Anne R
authors.initials: AR
authors.lastname: Bresnick
authors.forename: Bernd
authors.initials: B
authors.lastname: Nürnberg
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Backer
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
mesh_terms: D000818:Animals; D002633:Chemotaxis; D058544:Class Ib Phosphatidylinositol 3-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D018631:ras Proteins / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C523442:PIK3R3 protein, human; D018631:ras Proteins
keywords: HDX-MS; PIK3CG; PIK3R5; PIP3; oncogene