BRET studies using full-length G protein α subunits

The Nanoluc luciferase coding sequence was inserted in the human Gα₁₃ sequence immediately following residue Arg128, with (GlySerGly)₄ linkers on either side. This position has previously been verified as maintaining Gα₁₃ function in Förster resonance energy transfer studies that introduced a fluorophore at this location (31). HEK293 cells genome-edited to lack expression of both Gα₁₂ and Gα₁₃ were transiently transfected 2 d prior to the experiment to coexpress hGPR35 tagged at the C terminus with enhanced yellow fluorescent protein (eYFP) and the indicated Nanoluc-containing Gα₁₃ protein. BRET assays were conducted as described above. Subsequent studies employed this base Nanoluc-containing Gα₁₃ construct in which residues G.H5.22 (Gln) and G.H5.23 (Leu) were altered to the corresponding residues of Gα₁₂ (G.H5.22, Asp) or (G.H5.23, Ile).