title: Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.
abstract: Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.forename: Aliaksei
authors.initials: A
authors.lastname: Shymanets
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.lastname: Prajwal
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.
authors.forename: Oscar
authors.initials: O
authors.lastname: Vadas
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Czupalla
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, U.S.A.
authors.forename: Jaclyn
authors.initials: J
authors.lastname: LoPiccolo
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, U.S.A.
authors.forename: Michael
authors.initials: M
authors.lastname: Brenowitz
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Centre, University of Torino, 10126 Turin, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ghigo
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Centre, University of Torino, 10126 Turin, Italy.
authors.forename: Emilio
authors.initials: E
authors.lastname: Hirsch
authors.affiliation: Mass Spectrometry Group, Leibniz Institute for Molecular Pharmacology, 13125 Berlin, Germany.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.affiliation: *Department of Molecular Cell Biology, Centre for Molecular Biomedicine, Jena University Hospital, 07745 Jena, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wetzker
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Harteneck
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany bernd.nuernberg@uni-tuebingen.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nürnberg
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D058544:Class Ib Phosphatidylinositol 3-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D058544:Class Ib Phosphatidylinositol 3-Kinase
keywords: G-protein; Gβγ; p101; p87; phosphoinositide 3-kinase γ (PI3Kγ); signal transduction