Morphine induces terminal μ-opioid receptor desensitization by sustained phosphorylation of serine-375

Abstract

Morphine is a poor inducer of l-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala 2 -MePhe 4 -Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxyterminal residue 375 (Ser 375 ). Ser 375 phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser 375 -phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser 375 .

Agonist-directed Interactions with Specific β-Arrestins Determine μ-Opioid Receptor Trafficking, Ubiquitination, and Dephosphorylation

“…1B). Consistent with previous observations in other heterologous expression systems (3,10), morphine induced significantly less robust phosphorylation of this residue than DAMGO regardless of genotype, thus demonstrating that the agonist-dependent differences in MOR phosphorylation at Ser-375 are preserved in the absence of ␤-arrestins.…”

“…Subsequent studies have demonstrated that morphine's attenuated ability to promote MOR internalization is a consequence of morphine being less effective at promoting MOR phosphorylation and ␤-arrestin recruitment compared with other opioids. In fact, overexpression of GRK2 in cells can augment morphine-induced MOR phosphorylation, ␤-arrestin2 recruitment, and internalization (3,9,10). These data also suggest that in addition to the ligand, MOR regulation and trafficking are also dependent on the complement of intracellular interacting proteins expressed in residence with the receptor.…”

“…Further, MOR phosphorylation at Ser-375 was used to assess the state of dephosphorylation in these studies for several reasons. First, this residue has been shown to be important for MOR internalization and desensitization, and both of these events are known to also involve ␤-arrestins (10,26). Second, morphine-promoted MOR phosphorylation requires this residue (10).…”

“…Several studies have shown that the specific pattern of phosphorylation of these residues depends on the agonist used (10, 24 -26, 28, 29). Importantly, Ser-375 has been correlated with regulatory events in which ␤-arrestins have been implicated, including receptor internalization and desensitization (10,26). Therefore, we assessed MOR phosphorylation at Ser-375 using a phospho-specific antibody.…”

“…Agonist-induced ␤-Arrestin Recruitment-Receptor phosphorylation has been shown to enhance GPCR-␤-arrestin interaction affinities (1,3,9,10,33). To ascertain whether different agonists impart preference for recruiting individual ␤-arrestins to the receptor, ␤arr1/2-KO MEFs were transiently transfected with HA-MOR and either ␤arr1-GFP or ␤arr2-GFP, and ␤-arrestin translocation was determined by confocal microscopy (Fig.…”