A PTEN-Regulated Checkpoint Controls Surface Delivery of δ Opioid Receptors

Abstract

The ␦ opioid receptor (␦R) is a promising alternate target for pain management because ␦R agonists show decreased abuse potential compared with current opioid analgesics that target the opioid receptor. A critical limitation in developing ␦R as an analgesic target, however, is that ␦R agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of ␦R in sensory neurons contributes to this low ␦R agonist efficacy in vivo by limiting surface ␦R expression. Using direct visualization of ␦R trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains ␦R in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases ␦R from this checkpoint and stimulates delivery of exogenous and endogenous ␦R to the neuronal surface both in vitro and in vivo. PTEN inhibition in vivo increases the percentage of TG neurons expressing ␦R on the surface and allows efficient ␦R-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the ␦R, explain the low efficacy of ␦R agonists in vivo, and provide evidence that active ␦R relocation is a viable strategy to increase ␦R antinociception.

β-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling

“…These observations may resolve the paradox that while CXCR4 overexpression is associated with metastatic potential, plasma membrane localization is not 22,23 . Additionally, this work supports a growing amount of evidence supporting that targeting specifically intracellular GPCR populations or the downstream signaling cascades activated by these pools might lead to improved therapeutic strategies for treating cancer, cardiovascular disease, and pain management 6,7,40 . HEK293T cells were obtained from ATCC and grown in DMEM (Corning) media supplemented with 10% FBS.…”

PI3K class II α regulates δ-opioid receptor export from thetrans-Golgi network

“…To visualize intracellular δR retention, we used the expression of an N-terminally FLAG-tagged δR in cultured neuroendocrine cells. This expression system has been highly useful to study many GPCRs, including δR, and we and others have extensively confirmed that tagged receptors are functional ( Guan et al , 1992 ; Kim and von Zastrow, 2003 ; Arttamangkul et al , 2008 ; Puthenveedu et al , 2010 ; Soohoo and Puthenveedu, 2013 ; Vistein and Puthenveedu, 2013 ; Bowman et al , 2015 ; Shiwarski et al , 2017 ). To confirm proper localization of this construct, we expressed FLAG-tagged δR in cultured adult mouse trigeminal ganglia (TG) neurons.…”

“…Image analysis and quantification demonstrated a significant increase in the percentage of δR fluorescence localized to the Golgi and in the percentage of cells that had Golgi-localized δR in PI3K C2A shRNA–expressing cells compared with cells that only expressed the FAP-tagged δR ( Figure 5, D and E ). To evaluate the functional consequence of PI3K C2A knockdown on δR function, we measured cAMP inhibition, as a readout for δR activation and Gi coupling, using the exchange protein directly activated by cAMP (EPAC)–based Förster resonance energy transfer (FRET) sensor ( DiPilato et al , 2004 ; Shiwarski et al , 2017 ). PC12 cells stably expressing FAP-δR were transiently transfected with the EPAC FRET sensor and siRNA against PI3K C2A.…”

“…PC12 cells stably expressing FAP-δR were transiently transfected with the EPAC FRET sensor and siRNA against PI3K C2A. Activation of δR was quantified based on inhibition of forskolin-stimulated cAMP using live-cell fluorescence imaging as previously described ( Shiwarski et al , 2017 ). Cells transfected with control siRNA showed an expected increase in cAMP after 5 µM forskolin, followed by inhibition after addition of the δR agonist DADLE (10 µM) and subsequent receptor internalization ( Figure 5F ).…”

“…Factors such as nerve growth factor (NGF), bradykinin, and δR activation itself can alter the sensitivity of neurons to δR agonists ( Patwardhan et al , 2005 ; Cahill et al , 2007 ; Bie et al , 2010 ; Mittal et al , 2013 ; Pettinger et al , 2013 ). Further, studies evaluating expression and localization of δR in cellular systems found that δR is localized to intracellular pools in neuronal cells, raising the exciting idea that the basal surface expression of δR is tightly controlled by physiological signals that regulate the surface delivery of δR ( Roth et al , 1981 ; Zhang et al , 1998 ; Petaja-Repo et al , 2000 ; Wang et al , 2001 ; Cahill et al , 2001a , b ; Bao et al , 2003 ; Kim and von Zastrow, 2003 ; Gendron et al , 2015 ; Shiwarski et al , 2017 ; St-Louis et al , 2017 ). The cellular mechanisms that regulate δR surface delivery downstream of extracellular pathways, however, are relatively unknown.…”