Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-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 I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 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 Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.

GPCRome-wide analysis of G-protein-coupling diversity using a computational biology approach.

GPCRs are master regulators of cell signaling by transducing extracellular stimuli into the cell via selective coupling to intracellular G-proteins. Here we present a computational analysis of the structural determinants of G-protein-coupling repertoire of experimental and predicted 3D GPCR-G-protein complexes. Interface contact analysis recapitulates structural hallmarks associated with G-protein-coupling specificity, including TM5, TM6 and ICLs. We employ interface contacts as fingerprints to cluster Gs vs Gi complexes in an unsupervised fashion, suggesting that interface residues contribute to selective coupling. We experimentally confirm on a promiscuous receptor (CCKAR) that mutations of some of these specificity-determining positions bias the coupling selectivity. Interestingly, Gs-GPCR complexes have more conserved interfaces, while Gi/o proteins adopt a wider number of alternative docking poses, as assessed via structural alignments of representative 3D complexes. Binding energy calculations demonstrate that distinct structural properties of the complexes are associated to higher stability of Gs than Gi/o complexes. AlphaFold2 predictions of experimental binary complexes confirm several of these structural features and allow us to augment the structural coverage of poorly characterized complexes such as G12/13.

Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels.

Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding.