The human H1R gene subcloned into a pEG BacMam vector, was expressed in HEK293S GnTI– (N-acetylglucosaminyl-transferase I–negative) cells using FectoPRO, in FreeStyle™ 293 Expression Medium supplemented with 2% FBS. The cells were supplemented with 10 mM sodium butyrate to boost protein expression 18 h post-transfection and cultivated in suspension at 30 °C for additionnal 48 h.

Histamine exerts critical physiological roles by activating four receptor subtypes, each exhibiting a specific G protein preference. Among these, the histamine H4 receptor (H4R) modulates chemotaxis and interferon production through Gi protein activation, suggesting its therapeutic potential. Despite its physiological significance, the mechanisms underlying H4R signalling and G protein preference across histamine receptors remain poorly understood. Here, we present the cryo-electron microscopy structure of the H4R-Gi complex, revealing unique mechanisms of histamine recognition and receptor activation. We further solved the structures of the histamine H1 receptor (H1R) bound to the non-canonical G proteins Gi and Gs. Through a combination of functional and computational analyses, we identified the intracellular loop 2 as a critical determinant of G protein preference in H1R and H4R. Collectively, our comprehensive study revealed the structural basis for distinct mechanisms of ligand recognition and receptor activation, offering a profound insight into G protein preference across receptor subtypes.