ACTIVATION-INDUCED SUBCELLULAR REDISTRIBUTION OF G(S-ALPHA)

We have examined the subcellular distribution of alpha(s), the alpha s ubunit of the heterotrimeric G protein G(s), by using immunofluorescen ce microscopy. In transiently transfected HEK293 cells, wild-type alph a(s) localizes to the plasma membrane. However, a mutationally activat ed alpha(s) (alpha(s)R201C) is diffusely distributed throughout the cy toplasm. Similarly, cholera toxin activation of alpha(s) causes it to redistribute from the plasma membrane to cytoplasm in stably transfect ed cells. In HEK293 cells stably transfected with alpha(s) and the bet a(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 a llows alpha(s) to return to the plasma membrane, demonstrating the rev ersibility 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 en docytosis of agonist-activated beta-AR failed to block agonist-stimula ted redistribution of alpha(s). These findings demonstrate that activa ted alpha(s) reversibly undergoes a translocation from the plasma memb rane to cytoplasm and begin to address the relationship between regula ted trafficking of a seven-transmembrane receptor and its cognate G pr otein.