Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and...

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H₂ and H₄ receptors (H₂R and H₄R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H₂R and H₄R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H₂R and H₄R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H₂R and H₄R from the mini-G protein recruitment assays using HEK293T cells. Compared to H₂R-mGs expressing cells, histamine responses were weaker (pEC₅₀, E_max) for H₂R-mGsi and -mGsq. By contrast, the H₄R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H₂R to mGs and H₄R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H₂R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H₄R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H₂R function in the brain, as well as of the pharmacological potential of H₄R selective drugs.