Challenging activity and signaling bias in tachykinin NK1 and NK2 receptors by truncated neuropeptides.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P (SP) and neurokinin A (NKA), respectively. The peptide ligands share a common C-terminal sequence, Phe-X-Gly-Leu-Met-NH, which contributes to their partial cross-reactivity with each other's nonnative receptors. This study examines the impact of truncated tachykinin SP and NKA analogs on signaling activity. SP and NKA were progressively truncated, yielding the shortest versions SP(6-11) and NKA(5-10) with free and acetylated N terminal. A total of 12 SP and 10 NKA analogs were evaluated for activity in bioluminescence resonance energy transfer-based cAMP and IP accumulation assays targeting both NK1R and NK2R, corresponding to G protein and G protein activation, respectively. As previously demonstrated, the first three amino acids are dispensable. When activated by SP analogs, NK1R favors activation of G over G, though this difference diminishes with shorter analogs. In contrast, when NK1R is activated by NKA analogs, the G potency exceeds G potency by nearly an order of magnitude. For NK2R activation by NKA analogs, there are only minor differences between G and G potencies, with a slight preference for higher G potency. The N-terminal charge status plays a key role, leading to significant differences in analog potency. These findings provide valuable insight into how specific receptor-ligand interactions influence downstream G-protein signaling in G protein-coupled receptors, which are highly relevant for therapeutic applications. Finally, the proposed "message-address" model of neuropeptide signaling is assessed for NK1R and NK2R using truncated SP and NKA analogs.