Agonist Dependency Of The Second Phase Access Of Beta-Arrestin 2 To The Heteromeric Mu-V1b Receptor

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to beta -arrestin 2 and the mu -opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that beta -arrestin 2 closely located near the heteromer mu -V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the mu -V1b heteromer and beta -arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, beta -arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of beta -arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the mu -V1b heteromer to reduce the second-phase access of beta -arrestin 2 and thereby to alleviate tolerance to morphine analgesia.