Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling

Abstract

Opioid tolerance is well described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the μ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor's cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor's cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.

Chronic morphine induces adaptations in opioid receptor signaling in a thalamo-cortico-striatal circuit that are projection-dependent, sex-specific and regulated by mu opioid receptor phosphorylation

“…Instead, chronic morphine treatment did not induce any facilitation in 10 S/T-A mice in this study, suggesting alternative mechanisms are responsible. In studies which have observed presynaptic tolerance, the effect was attributed to a downregulation in the number of functional receptors due to phosphorylation and internalization (Jullié et al, 2022; Jullié et al, 2020). This mechanism is in line with our findings, as tolerance did not develop at MThal-ACC terminals in 10 S/T-A mice.…”

“…Within the presynaptic compartment, multiple adaptations to chronic opioid exposure have been observed; tolerance in some instances (Atwood et al, 2014; Fyfe et al, 2010; Matsui et al, 2014), while enhanced opioid efficacy, or facilitation, in others (Chieng & Williams, 1998; Hack et al, 2003; Ingram et al, 1998; Pennock et al, 2012). MOR phosphorylation also regulates presynaptic cellular tolerance in cultured striatal neurons (Jullié et al, 2022; Jullié et al, 2020), however, the role of phosphorylation in mediating presynaptic facilitation and tolerance in intact brain circuits is not established. Complicating the matter, studies investigating these differences have been done across species and brain regions making it difficult to generalize how a particular cell type or synapse will adapt to repeated opioid exposure.…”

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers

“…Ultimately, extensive future work is needed to directly probe the β-arr–mediated internalization, trafficking, and signaling properties of mGluRs in the synaptic context where little is known about GPCR regulation. Notably, two recent studies of opioid receptors have shown that GRKs and β-arrs can drive rapid endocytosis and long-term down-regulation of receptor function in presynaptic boutons ( 74 , 75 ). However, little is known about lysosomal targeting and potential proteolysis of axonal/presynaptic GPCRs, motivating future work on mGluR8.…”

Subcellular location defines GPCR signal transduction

“…Transgenic HeLa cells stably expressing N-terminally signal sequence (ss) SEP-tagged DOR were generated by cloning a “CAG promoter, ssSEP-DOR, RGK promoter, PuroR” cassette into a piggyBac transposon plasmid (Addgene #84239), cotransfection of the plasmid with piggyBac transposase, followed by flow cytometry-assisted sorting of GFP-positive cells and puromycin selection. The following published plasmids were used for transient expression: ssfDOR (mouse) ( 7 ), ssfMOR (mouse) ( 7 ), ssfβ2AR (mouse) in pcDNA3.1 ( 5 ), ssfMOR (mouse)-EGFP ( 7 ), ssSEP-(GFP variant Superecliptic pHluorin) DOR (mouse) ( 69 ), β-arrestin2 (bos taurus)-mCherry ( 5 ), GRK2 (bos taurus)-EGFP in pCAGGS-SE ( 70 ), LgBiT-β-arrestin2 (human) in pNBe3 ( 28 ), ManII-BFP2 ( 64 ), and GalT-DsRed2 ( 6 ). N-terminally tagged mRuby2-mGo, mRuby2-mGsi, and mRuby2-mGs ( 33 ) were from Nevin Lambert.…”