Components of the G signaling cascade exhibit distinct changes in mobility and membrane domain localization upon β -adrenergic receptor activation.

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the "second wave" of signaling from endosomes. Here, we set out to determine whether G signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin-coated structures (CCSs) or caveolae upon activation by β -adrenergic receptors (β AR). Activated β AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after β AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gα and Gγ exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon β AR stimulation, no redistribution to CCSs, and weak activation-independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated G signaling cascade members and confirm that β AR and arrestin 3 accumulate in CCSs, while Gα , Gγ and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.