The mu opioid receptor (mOR) is the primary target for opioid analgesics that are widely used and abused. Opioid receptors are removed from the cell surface after activation, causing down regulation of responses. Receptor recycling to the surface, which allows recovery of cellular responses, determines acute tolerance to opioids, and might have long‐term effects on signaling. Whether and how mOR recycling is acutely regulated by homologous signals is not clear. Here we used total internal reflection fluorescence microscopy (TIR‐FM) to directly visualize and quantify individual receptor recycling events, to test whether mOR recycling is regulated by opioid signaling downstream of the receptor itself. Attenuation of continued signaling, by removing the agonist, reduced the frequency of recycling events, indicating that opioid signaling increases the rate of mOR recycling. This homologous regulation was reduced in cells treated with the G protein inhibitor pertussis toxin, but not a Protein Kinase A inhibitor. Further, inhibition of G beta‐gamma (Gbg), Phospholipase C, or Protein Kinase C (PKC) abolished the decrease in recycling caused by agonist removal, while activation of Gbg or PKC caused an increase in recycling even in the absence of agonist. This suggests that mOR recycling is regulated by PKC activation through Gbg. Importantly, the homologous regulation was abolished by mutating the serine 363 residue on mOR, a PKC phosphorylation site. Our results support a model where Gbg activation of PLC/PKC and phosphorylation of mOR site S363 is required for the homeostatic regulation of mOR recycling. This represents a novel mechanism for homologous regulation of receptor recycling, which could provide control points for regulation by different opioid drugs. Support or Funding Information Supported by the NIH Cellular and Molecular Biology Training Grant T32‐GM007315 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides an as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that two related peptides, Dynorphin A and Dynorphin B, bind and activate KOR to similar extents in mammalian neuroendocrine cells and rat striatal neurons, but localize KOR to distinct intracellular compartments and drive different post-endocytic fates of the receptor. Strikingly, localization of KOR to the degradative pathway by Dynorphin A induces sustained KOR signaling from these compartments. Our results suggest that seemingly redundant endogenous peptides can fine-tune signaling by regulating the spatiotemporal profile of KOR signaling.

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Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that, although highly related Dynorphins bind and activate KOR to similar extents on the cell surface, they localize KOR to distinct subcellular compartments, dictate different post-endocytic fates of the receptor, and differentially induce KOR signaling from the degradative pathway. Our results show that seemingly redundant endogenous opioid peptides that are often co-released can in fact fine-tune signaling by differentially regulating the subcellular spatial profile of GPCR localization and signaling.