No abstract

G protein-coupled receptor desensitization and trafficking are important regulators of opioid receptor signaling that can dictate overall drug responsiveness in vivo. Furthermore, different -opioid receptor (OR) ligands can lead to varying degrees of receptor regulation, presumably because of distinct structural conformations conferred by agonist binding. For example, morphine binding produces a OR with low affinity for ␤-arrestin proteins and limited receptor internalization, whereas enkephalin analogs promote robust trafficking of both ␤-arrestins and the receptors. Here, we evaluate OR trafficking in response to activation by a novel -selective agonist derived from the naturally occurring plant product, salvinorin A. It is interesting that this compound, termed herkinorin, does not promote the recruitment of ␤-arrestin-2 to the OR and does not lead to receptor internalization. Moreover, whereas G protein-coupled receptor kinase overexpression can promote morphine-induced ␤-arrestin interactions and OR internalization, such manipulations do not promote herkinorin-induced trafficking. Studies in mice have shown that ␤-arrestin-2 plays an important role in the development of morphine-induced tolerance, constipation, and respiratory depression. Therefore, drugs that can activate the receptor without recruiting the arrestins may be a promising step in the development of opiate analgesics that distinguish between agonist activity and receptor regulation and may ultimately lead to therapeutics designed to provide pain relief without the adverse side effects normally associated with the opiate narcotics.

The neoclerodane diterpene salvinorin A, found in the leaves of Salvia divinorum, is a potent κ-opioid receptor agonist, making it an attractive scaffold for development into a treatment for substance abuse. Although several successful semisynthetic studies have been performed to elucidate structure–activity relationships, the lack of analogues with substitutions to the furan ring of salvinorin A has prevented a thorough understanding of its role in binding to the κ-opioid receptor. Herein we report the synthesis of several salvinorin A derivatives with modified furan rings. Evaluation of these compounds in a functional assay indicated that sterically less demanding substitutions are preferred, suggesting the furan ring is bound in a congested portion of the binding pocket. The most potent of the analogues successfully reduced drug-seeking behavior in an animal model of drug-relapse without producing the sedation observed with other κ-opioid agonists.

Morphine and other opiates mediate their effects through activation of the -opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, ␤-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of ␤-arrestin, ␤-arrestin1 and ␤-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting ␤-arrestin2 specifically. In this study, we examine the different contributions of ␤-arrestin1-and ␤-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both ␤-arrestins. Here we show that morphine only recruits ␤-arrestin2, whereas the MOR-selective enkephalin [D-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]enkephalin (DAMGO), recruits either ␤-arrestin. We show that ␤-arrestins are required for receptor internalization and that only ␤-arrestin2 can rescue morphine-induced MOR internalization, whereas either ␤-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, ␤-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of ␤-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and ␤-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.Morphine and other opiates are among the most clinically useful analgesics, and their actions are mediated largely through activation of -opioid receptors (MORs).3 As a G protein-coupled receptor (GPCR), the MOR is subject to regulation paradigms that include phosphorylation by GPCR kinases (GRKs) and subsequent interactions with ␤-arrestins (␤-arrestin1, also known as arrestin2, and ␤-arrestin2, also known as arrestin3). ␤-Arrestins can then initiate receptor internalization, which in turn can promote both receptor down-regulation and resensitization (1-3). ␤-Arrestins can also facilitate these regulatory events by scaffolding ubiquitination machinery, such as E3 ligases, to GPCRs, as has been shown for the ␤ 2 adrenergic receptor (␤ 2 AR), V2 vasopressin receptor, and the chemokine receptor (CXCR4) (4 -6), however this has not been demonstrated for the MOR. MOR regulation has been shown to be contingent upon the particular agonist acting at the receptor as morphine promotes different regulatory events than other opioid ligands, including the D-enkephalin analog, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO), fentanyl, methadone, and etorphine, although all of these ligands are full agonists at the MOR with respect to G protein coupling (7). The difference between agonists was first recognized when Arden et al. (8) observed that although DAMGO promotes robust internalizatio...

Background: 6Ј-Guanidinonaltrindole (6Ј-GNTI) activates G protein coupling to -opioid receptors (KOR) without ␤-arrestin2 recruitment in transfected cells. Results: In striatal neurons, 6Ј-GNTI activates Akt but not ERK1/2; U69,593 activates both kinases. Conclusion: In neurons, U69,593-induced activation of ERK1/2 is ␤-arrestin2-dependent, whereas activation of Akt is G protein-mediated. Significance: Identification of KOR signaling pathways in endogenous systems will inform the development of KOR-directed medications.