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

Abstract

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-NH2, which contributes to their partial cross-reactivity with each other's non-native 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 BRET-based cAMP and IP3 accumulation assays targeting both NK1R and NK2R, corresponding to Gs protein and Gq protein activation, respectively. As previously demonstrated, the first three amino acids are dispensable. When activated by SP analogs, NK1R favors activation of Gs over Gq, though this difference diminishes with shorter analogs. In contrast, when NK1R is activated by NKA analogs, the Gq potency exceeds Gs potency by nearly an order of magnitude. For NK2R activation by NKA analogs, there are only minor differences between Gq and Gs potencies, with a slight preference for higher Gq 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 GPCRs, 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.