Receptor-mediated activation of heterotrimeric G-proteins in living cells.
Receptor-mediated activation of heterotrimeric GTP-binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein-coupled receptors.
Department of Biological Chemistry, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism*; D004735:Energy Transfer; D005453:Fluorescence; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D008024:Ligands; D008164:Luminescent Proteins; D011953:Receptors, Cyclic AMP; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D015124:8-Bromo Cyclic Adenosine Monophosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C012873:2'-deoxy-5'-adenosine monophosphate; D000242:Cyclic AMP; D020962:Heterotrimeric GTP-Binding Proteins