Some G protein heterotrimers physically dissociate in living cells

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, G alpha beta gamma heterotrimers physically dissociate after activation, liberating free G alpha subunits and G beta gamma dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Ga subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-G alpha greatly decreased the lateral mobility of intracellular G beta(1 gamma 2)-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-G alpha(oA) lost the ability to restrict G beta(1 gamma 2)-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-G alpha(i3) and CFP-TM-G alpha(s), retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on G beta(1 gamma 2)-YFP mobility induced by immobile CFP-TM-GaoA and CFP-TM-Gai3 but had no effect on the constraint induced by CFP-TM-Gas. These results demonstrate the physical dissociation of heterotrimers containing G alpha(oA) and G alpha(i3) subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., G alpha(s)) may signal without physically dissociating.