biostudies-literatureTatsumi MTakeda Science FoundationJapan Agency for Medical Research and DevelopmentNational Institutes of HealthJapan Science and Technology AgencyNIGMS NIH HHSNIH HHSJapan Society for the Promotion of Science11119https://www.ebi.ac.uk/biostudies/studies/S-EPMC11096383biostudies-literatureUnknown14(1)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 Gα₁₂_/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.Scientific reportsIdentification of Gα₁₂-vs-Gα₁₃-coupling determinants and development of a Gα_12/13-coupled designer GPCR.PMC11096383JP22J10475SC2GM130480P21H04791, JP21H05113, JPJSBP120213501 and JPJSBP120218801JPMJFR215T, JPMJMS2023 and 22714181SC2 GM130480JP22ama121038 and JP22zf0127007Ikuta TCruz CInoue ATatsumi MNakamura GKamakura NAbrol RKuwabara RfalseIdentification 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 Gα₁₂_/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.2024-01-01T00:00:00Z2024 May2026-06-01T11:58:36.424Z2026-04-08T12:09:22.658ZS-EPMC110963833875024710.1038/s41598-024-61506-4