Figure 1.

Identification of the Gα protein involved in β₁AR signalling. (a) Schematic representation of the Gα-Rluc/Gγ-GFP biosensor used to identify the Gα proteins involved in β₁AR signalling. (b–k) HA-β₁AR HEK 293 stable cell lines were transfected with a constant amount of Gα-Rluc (BRET donor) and untagged Gβ₁, along with increasing amounts of Gγ₁-GFP construct (BRET acceptor). Cells were stimulated (red curves) or not (black curves) with 1 μM isoproterenol and BRET values collected. NetBRET values were calculated by subtracting the background BRET signal detected in cells expressing the Rluc-fused constructs alone (donor-Rluc) from the BRET values obtained in cells expressing the energy donor and acceptor (donor-Rluc and acceptor-GFP). BRET titration curves were generated for 10 different Gα subunits: Gα_s (b), Gα_q (c), Gα₁₂ (d), Gα₁₃ (e), Gα_i1 (f), Gα_i2 (g), Gα_i3 (h), Gα_z (i), Gα_oA (j) and Gα_oB (k). Values represent mean ± SEM of 3 independent experiments performed in triplicate. Responses for Gα_s, Gα_i2, Gα_z and Gα₁₂ were further analyzed in subsequent sections.