Intracellular pocket and ICL2 motifs govern G-protein coupling bias in NK1 receptor (TACR1) and NK2 receptor (TACR2).

The tachykinin receptors NK1 receptor (TACR1) (NK1R) and NK2 receptor (TACR2) (NK2R) are G-protein-coupled receptors (GPCRs) that preferentially bind substance P (SP) and neurokinin A (NKA), respectively. Although these ligands differ in their N-terminal sequences, a conserved C-terminal motif confers partial cross-reactivity. In COS-7 cells, both NK1R/SP and NK2R/NKA activate Gq and Gs signaling pathways to a certain extent. NK1R/SP displays an approximately threefold preference for Gs coupling, whereas NK2R/NKA couples comparably to Gq and Gs, albeit with lower overall potency. In contrast, NK1R/NKA preferentially activates Gq, while NK2R/SP exhibits negligible activity. Receptor activation enables insertion of the C-terminal segment of the Gα α5-helix (so-called "wavy-hook") into the intracellular receptor pocket, involving intracellular loop 2 (ICL2). We investigate the structural basis for differential Gq and Gs coupling by NK1R and NK2R by SP and NKA. Analysis of structures identifies four key differences between NK1R and NK2R located at the base of the intracellular pocket and within ICL2. Reciprocal mutagenesis swapping the motifs between NK1R and NK2R demonstrates that alterations in this region can re-direct G-protein coupling and signaling bias.