Activation-induced subcellular redistribution of G(s alpha)

We have examined the subcellular distribution of alpha(s), the alpha subunit of the heterotrimeric G protein G(s), 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(s)R201C) 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.