Activation-induced subcellular redistribution of Gs alpha.
We have examined the subcellular distribution of alpha s, the alpha subunit of the heterotrimeric G protein Gs, by using immunofluorescence microscopy. In transiently transfected HEK293 cells, wild-type alpha s localizes to the plasma membrane. However, a mutationally activated alpha s (alpha sR201C) is diffusely distributed throughout the cytoplasm. Similarly, cholera toxin activation of alpha s causes it to redistribute from the plasma membrane to cytoplasm in stably transfected cells. In HEK293 cells stably transfected with alpha s and the beta 2-adrenergic receptor (beta-AR), stimulation of the beta-AR by the agonist isoproterenol also causes a translocation of alpha s from the plasma membrane to cytoplasm. Replacing the agonist with antagonist allows alpha s to return to the plasma membrane, demonstrating the reversibility of alpha s translocation. Receptor-activated alpha s does not colocalize with internalized beta-AR at endosomes. Incubation of cells in hypertonic sucrose to inhibit clathrin-coated pit-mediated endocytosis of agonist-activated beta-AR failed to block agonist-stimulated redistribution of alpha s. These findings demonstrate that activated alpha s reversibly undergoes a translocation from the plasma membrane to cytoplasm and begin to address the relationship between regulated trafficking of a seven-transmembrane receptor and its cognate G protein.
Wedegaertner
P B
PB
Department of Cellular and Molecular Pharmacology, University of California at San Francisco, USA.
Bourne
H R
HR
von Zastrow
M
M
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D001693:Biological Transport, Active; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol