A lipidated peptide derived from the C-terminal tail of the vasopressin 2 receptor shows promise as a new $β$-arrestin inhibitor
$β$-arrestins play pivotal roles in seven transmembrane receptor (7TMR) signalling and trafficking. To study their functional role in the regulation of specific receptor systems, current research relies mainly on genetic tools, as few pharmacological options are available. To address this issue, we designed and synthesised a novel lipidated phosphomimetic peptide inhibitor targeting $β$-arrestins, called ARIP, which was developed based on the C-terminal tail (A343-S371) of the vasopressin V2 receptor. As the V2R sequence has been shown to bind $β$-arrestins with high affinity and stability, we added an N-terminal palmitate residue to allow membrane tethering and subsequent cell entry. Here, using BRET2-based biosensors, we demonstrated the ability of ARIP to inhibit agonist-induced $β$-arrestin recruitment on a series of 7TMRs belonging to class A (low stable associations with arrestins) or class B (high stability), with efficiencies that dependent on receptor type. In addition, we showed that ARIP was unable to recruit $β$-arrestins to the cell membrane by itself, and that it did not interfere with canonical G protein signalling. Molecular modelling studies also revealed that ARIP binds $β$-arrestins in the same way as V2Rpp, the phosphorylated peptide derived from the V2R C-terminal domain, and that replacing the p-Ser and p-Thr residues of V2Rpp with Glu residues does not alter the inhibitory activity of ARIP on $β$-arrestin recruitment. Importantly, ARIP exerted an opioid-sparing effect in vivo, as intrathecal injection of ARIP potentiated the analgesic effect of morphine in the tail-flick nociceptive model, a behavioural response consistent with $β$-arrestin genetic inhibition. ARIP therefore represents a promising pharmacological tool for investigating the fine-tuning roles of $β$-arrestins in 7TMR-driven pathophysiological processes.