title: Dissociated GαGTP and Gβγ protein subunits are the major activated form of heterotrimeric Gi/o proteins.
abstract: Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.
authors.affiliation: From the Faculty of Science, University of South Bohemia, Branisovska 31a, 37005 Ceske Budejovice, Czech Republic and.
authors.forename: Alexey
authors.initials: A
authors.lastname: Bondar
authors.forename: Josef
authors.initials: J
authors.lastname: Lazar
mesh_terms: D004789:Enzyme Activation / Q000502:physiology; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans
chemical_list: D019922:GAP-43 Protein; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Adrenergic Receptor; Cell Signaling; Heterotrimeric G Proteins; Membrane Proteins; Microscopic Imaging; Plasma Membrane; Potassium Channels; Two-photon Polarization Microscopy