Differential Activity of the G Protein β5γ2 Subunit at Receptors and Effectors

Abstract The G protein β5 subunit differs substantially in amino acid sequence from the other known β subunits suggesting that βγ dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the β5 subunit, recombinant β5γ2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the γ2 subunit (γ2HF). The function of the pure β5γ2HF dimers was examined in three assays: activation of pure phospholipase C-β in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of α subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the β5γ2HF dimer was compared with that of the β1γ2HF dimer, which has demonstrated activity in these assays. The β5γ2HF dimer activated phospholipase C-β with a potency and efficacy similar to that of β1γ2 or β1γ2HF; however, it was markedly less effective than the β1γ2HF or β1γ2 dimer in its ability to activate type II adenylyl cyclase (EC50 of ∼700 nm versus 25 nm). Both the β5γ2HF and the β1γ2HF dimers supported coupling of M1 muscarinic receptors to the Gq α subunit. The ETB receptor coupled effectively to both the Gi and Gq α subunits in the presence of the β1γ2HF dimer. In contrast, the β5γ2HF dimer only supported coupling of the Gq α subunits to the ETB receptor and did not support coupling of the Gi α subunit. These results suggest that the β5γ2HF dimer binds selectively to Gq α subunits and does not activate the same set of effectors as dimers containing the β1subunit. Overall, the data support a specialized role for the β5 subunit in cell signaling.