G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION

• Published in: The Journal of biological chemistry 2010-03, Vol.285 (11), p.8227-8233
• Main Authors: Hommers, Leif G, Klenk, Christoph, Dees, Christian, Bünemann, Moritz
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Fluorescence Resonance Energy Transfer; G Protein-Coupled Inwardly-Rectifying Potassium Channels - physiology; GTP-Binding Protein alpha Subunits - genetics; GTP-Binding Protein alpha Subunits - metabolism; GTP-Binding Protein beta Subunits - genetics; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - genetics; GTP-Binding Protein gamma Subunits - metabolism; GTP-Binding Proteins - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Triphosphate - metabolism; Guanosine Triphosphate - pharmacology; Heterotrimeric GTP-Binding Proteins - genetics; Heterotrimeric GTP-Binding Proteins - metabolism; Humans; Kidney - cytology; Neurobiology; Patch-Clamp Techniques; Receptors, Adrenergic, alpha-2 - genetics; Receptors, Adrenergic, alpha-2 - physiology; Receptors, G-Protein-Coupled - metabolism; Signal Transduction; Signal Transduction - physiology; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.015388

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and Go protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of Go proteins by active α₂A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.