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

Abstract

The G protein ␤ 5 subunit differs substantially in amino acid sequence from the other known ␤ subunits suggesting that ␤␥ dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the ␤ 5 subunit, recombinant ␤ 5 ␥ 2 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 ␥ 2 subunit (␥ 2HF ). The function of the pure ␤ 5 ␥ 2HF dimers was examined in three assays: activation of pure phospholipase C-␤ in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of ␣ subunits to the endothelin B (ET B ) and M 1 muscarinic receptors. In each case, the efficacy of the ␤ 5 ␥ 2HF dimer was compared with that of the ␤ 1 ␥ 2HF dimer, which has demonstrated activity in these assays. The ␤ 5 ␥ 2HF dimer activated phospholipase C-␤ with a potency and efficacy similar to that of ␤ 1 ␥ 2 or ␤ 1 ␥ 2HF ; however, it was markedly less effective than the ␤ 1 ␥ 2HF or ␤ 1 ␥ 2 dimer in its ability to activate type II adenylyl cyclase (EC 50 of ϳ700 nM versus 25 nM). Both the ␤ 5 ␥ 2HF and the ␤ 1 ␥ 2HF dimers supported coupling of M 1 muscarinic receptors to the G q ␣ subunit. The ET B receptor coupled effectively to both the G i and G q ␣ subunits in the presence of the ␤ 1 ␥ 2HF dimer. In contrast, the ␤ 5 ␥ 2HF dimer only supported coupling of the G q ␣ subunits to the ET B receptor and did not support coupling of the G i ␣ subunit. These results suggest that the ␤ 5 ␥ 2HF dimer binds selectively to G q ␣ subunits and does not activate the same set of effectors as dimers containing the ␤ 1 subunit. Overall, the data support a specialized role for the ␤ 5 subunit in cell signaling.

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

“…The combinations of β 5 γ-dimers found in our screen are in line with other studies (29,47). Especially the β 5 γ 2 -dimer is a well characterized combination (8,(50)(51)(52), which is reported to be unstable in certain detergents (53). Our finding that the identified β 5 γ-dimers interact moderately with the α i1 -subunit and, to a lesser extent, with the α sL -subunit has also been reported in previous studies (8,(50)(51)(52).…”

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

“…As each ␣ subunit was purified from a column of immobilized ␤␥ dimers after activation with AlCl 3 and NaF, which induces a conformational change, the proteins are properly folded, active molecules. Moreover, each preparation of ␣ subunit coupled to the appropriate recombinant receptors (29,31,36); the G q ␣ preparation was able to markedly activate PLC␤ (Fig. 2B), and the preparations of G s ␣ have been demonstrated to activate adenyl cyclase (31).…”

“…The purified ␣ subunits were concentrated to a volume of 100 -200 l, separated into aliquots, and stored at Ϫ80°C. Each preparation of G protein ␣ subunit used in this study was functional based on its ability to couple to recombinant receptors and ␤␥ subunits when reconstituted into Sf9 cell membranes (29,31,36). SDS gels documenting the purity of the G␣ subunits prepared by this procedure are published elsewhere (29,31).…”

“…The procedures used for purification of the ␣ subunits of G i , G q , G s , and G o were very similar; our methods for purification of G s and G q have been described in detail (29,31). Briefly, the desired G␣ subunit was overexpressed in Sf9 insect cells, with ␤ 1 and ␥ 2 subunits engineered to have hexahistidine and FLAG tags at their N termini (33).…”

“…Protein was concentrated using an Amicon Ultra 30 concentrator and exchanged twice using Ni 2ϩ -NTA base buffer containing 0.1% Chaps. The ␤ 5 ␥ 2 HF dimer was purified as described (29). Verification of the proper post-translational processing of the ␥ subunit in the dimer was accomplished using matrix-assisted laser desorption ionization mass spectrometry as described (38).…”