Figure 6. Relative rates of Gβγ release by Gαi versus Gαs-coupled receptors.
(A) A schematic representation of the experiment that monitors Gβγ release by BRET. Upon agonist stimulation, GPCRs release Gα and Gβγ. The dissociation of Gα-Venus and Gβγ-NLuc results in a decrease of the BRET signal. Released Gβγ-NLuc was chelated by masGRK3ct, a fusion of the C-terminal PH domain of GRK3 and a myristic acid attachment peptide. (B)-(E) Representative time-resolved BRET ratio curves. In (B) and (C), M2Rs released more Gβγ than β2ARs did within the same time period, independent of which Gα-Venus construct was used. In (D) and (E), D2Rs released more Gβγ than β1ARs did within the same time period, independent of which Gα-Venus construct was used. See also Figure 6—figure supplements 1 and 2, and Table 1.
Figure 6—figure supplement 1. M2Rs catalyze release of Gβγ at higher rate compared to β2ARs.
(A) Structural comparison between Gαs and Gαi1. Crystal structures of Gαs (Blue, PDB: 1AZT) and Gαi1 (Red, PDB: 1GG2) were superimposed. The Venus insertion sites are indicated as arrows. The Cα atoms of residues Gαs-113 and 144, and Gαi1-91 and 121 are represented as spheres. (B) (C) Time-resolved BRET ratio curves from HEK-293T cells transiently transfected with different GPCRs and Gα-Venus constructs.
Figure 6—figure supplement 2. D2Rs catalyze release of Gβγ at higher rate compared to β1ARs.
(A) A representative voltage-clamp recording of a HEK-293T cell transiently transfected with GIRK channels and D2Rs. The membrane potential was held at −80 mV. 10 µM Dopamine (Dopa) was applied as indicated. (B) Validation of the function of Venus-inserted Gαs. Time-resolved BRET ratio curves from HEK-293T cells transiently transfected with β1ARs and different Gα-Venus constructs were shown. (C) (D) Time-resolved BRET ratio curves from HEK-293T cells transiently transfected with different GPCRs and Gα-Venus constructs.