Identification of Gα₁₂-vs-Gα₁₃-coupling determinants and development of a Gα_12/13-coupled designer GPCR

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα₁₂ and Gα₁₃ belong to the G₁₂ subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα₁₂ or Gα₁₃, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα₁₂-coupled designer receptor exclusively activated by designer drugs (DREADD; G₁₂D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα₁₂-vs-Gα₁₃ selectivity. Residue-swapping experiments showed that G₁₂D distinguishes differences between Gα₁₂ and Gα₁₃ in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378^G.H5.23 in Gα₁₂ interacts with N103^2.39, S169^3.53 and Y176^34.53 in G₁₂D, while H364^G.H5.09 in Gα₁₂ interact with Q264^5.71 in G₁₂D. Screening of mutations at these positions in G₁₂D identified G₁₂D mutants that enhanced coupling with Gα₁₂ and to an even greater extent with Gα₁₃. Combined mutations, most notably the dual Y176^34.53H and Q264^5.71R mutant, further enhanced Gα_12/13 coupling, thereby serving as a potential Gα_12/13-DREADD. Such novel Gα_12/13-DREADD may be useful in future efforts to develop drugs that target Gα_12/13 signaling as well as to identify their therapeutic indications.