G proteins in reverse mode: receptor-mediated GTP release inhibits G protein and effector function.
Active G protein-coupled receptors activate heterotrimeric Galphabetagamma proteins by catalyzing the exchange of GDP by GTP at the Galpha 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 G(o) 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 G(o) proteins by active alpha(2A)-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 Galphabetagamma. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.
Hommers
Leif G
LG
Institute of Pharmacology & Toxicology, University of Würzburg, 97078 Würzburg, Germany.
Klenk
Christoph
C
Dees
Christian
C
Bünemann
Moritz
M
D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000502:physiology*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D018408:Patch-Clamp Techniques; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
C547768:ADRA2A protein, human; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins