Blocking a Nerve Cell Receptor Could Make Morphine Work Better, Study Finds

Morphine has long been the cornerstone of treatment for moderate-to-severe chronic pain, but its usefulness is undermined by a stubborn clinical problem: tolerance. Patients who take opioids repeatedly need progressively higher doses to achieve the same relief, and that escalation brings a heightened risk of side effects, dependence, and overdose. Now, a team of researchers publishing in Advanced Science has identified a molecular mechanism that drives this loss of efficacy, and their findings point to a surprising new target for keeping morphine working longer. The key player is EphB1, a receptor that belongs to the largest family of receptor tyrosine kinases in the body and that, until recently, was best known for its role in wiring the developing nervous system.

The study, led by researchers at the Southern University of Science and Technology, began with a deceptively simple observation. When mice received a spinal injection of morphine, the drug rapidly increased phosphorylation, the biochemical switch that activates EphB1 receptors, in both the dorsal root ganglia, clusters of sensory neurons outside the spinal cord, and the spinal cord itself. In the dorsal root ganglia, morphine also raised levels of ephrinB2, the ligand that binds and switches on EphB1. That increase, the researchers showed, depended on the mu-opioid receptor, the very molecule through which morphine produces pain relief. In other words, morphine appears to trigger its own weakening by activating a signaling pathway that sits alongside its primary target.

To test whether EphB1 signaling actually interferes with pain relief, the team administered ephrinB2-Fc, a laboratory reagent that activates EphB1 receptors, directly into the spinal canal of mice. Even at a low dose that did not alter pain sensitivity on its own, the activator significantly blunted the analgesic effect of morphine in both the tail-flick and hot-plate tests, standard measures of pain response in rodents. Crucially, the dampening effect was selective. EphrinB2-Fc interfered with the analgesia produced by DAMGO, a drug that specifically activates mu-opioid receptors, but left untouched the effects of DPDPE and U69593, which act on the delta and kappa opioid receptor subtypes. That selectivity told the researchers that EphB1 signaling is functionally intertwined with the mu-opioid receptor in particular, not with opioid signaling in general.

The next step was to remove EphB1 and see what happened. Using genetic tools, the team created mice lacking EphB1 receptors specifically in neurons that express Vglut2, a marker of excitatory glutamatergic neurons found both in the dorsal root ganglia and in the spinal dorsal horn, the region where pain signals from the body are processed. When these mice received morphine, the analgesic effect was markedly stronger than in normal animals, while baseline pain sensitivity remained unchanged. The enhancement again applied only to DAMGO, reinforcing the link to mu-opioid receptors. The same pattern held in mouse models of disease: in animals with partial sciatic nerve ligation, a model of neuropathic pain, and in animals implanted with lung carcinoma cells in the tibia, a model of bone cancer pain, deleting EphB1 significantly strengthened morphine’s ability to reverse mechanical allodynia and thermal hyperalgesia.

To separate the peripheral and central contributions, the researchers ran parallel experiments. Crossing mice with an inducible Cre driver called Advillin allowed them to delete EphB1 exclusively in dorsal root ganglion neurons, and this too enhanced morphine analgesia. Meanwhile, injecting a Cre-expressing virus directly into the lumbar spinal cord deleted the receptor only in dorsal horn Vglut2-positive neurons, with the same result. Imaging studies supported the anatomical logic: EphB1 and the mu-opioid receptor are co-expressed in small-diameter sensory neurons, on sensory axon terminals, and in excitatory interneurons of the dorsal horn, where roughly thirty percent of Vglut2-positive neurons carried both Ephb1 and Oprm1, the gene encoding the mu-opioid receptor. Inhibitory neurons showed far less overlap.

Electrophysiology then revealed how removing EphB1 changes the behavior of individual neurons. Opioids quiet pain pathways partly by shutting down voltage-gated calcium channels in sensory neurons, which reduces the release of pain-signaling neurotransmitters. In cultured dorsal root ganglion neurons lacking EphB1, DAMGO suppressed calcium influx, measured by calcium imaging, and calcium currents, measured by patch clamp, more powerfully than in control neurons. In spinal cord slices, DAMGO’s inhibition of spontaneous excitatory synaptic currents in dorsal horn Vglut2-positive neurons was deeper without EphB1, and the drug also produced larger outward potassium currents, a hallmark of opioid receptor activation, in those neurons. Every measure pointed the same way: without EphB1, mu-opioid receptors simply work better.

The mechanism behind that improvement turned out to be receptor trafficking. When mu-opioid receptors are phosphorylated at a specific amino acid, serine 375, by enzymes called G protein-coupled receptor kinases, the adaptor protein beta-arrestin is recruited and drags the receptor off the cell surface into the interior of the cell, where it can no longer respond to morphine. The researchers found that activating EphB1 with ephrinB2-Fc pushed mu-opioid receptors out of the membrane and into the cytoplasm of cultured cells, even without any opioid drug present, and amplified the internalization triggered by DAMGO. EphB1 activation also increased phosphorylation of the receptor at serine 375, both in cell culture and in the dorsal root ganglia and spinal cords of living mice. Conversely, deleting EphB1 blunted that phosphorylation in response to DAMGO, including in the neuropathic and cancer pain models.

The missing link was GRK2, one of the G protein-coupled receptor kinases. Co-immunoprecipitation experiments showed that EphB1 receptors physically associate with GRK2 in the dorsal root ganglia and spinal cord, and that all three proteins, EphB1, GRK2, and the mu-opioid receptor, form a complex. GRK2 is heavily expressed in the same nociceptor populations that carry EphB1 and the opioid receptor. When EphB1 was activated, GRK2 became phosphorylated on tyrosine residues, a modification known to boost the enzyme’s catalytic activity, and GRK2 bound more tightly to the mu-opioid receptor. Paroxetine, an antidepressant already approved by the US Food and Drug Administration that happens to be a potent and selective GRK2 inhibitor, completely reversed the EphB1-driven phosphorylation of the opioid receptor at serine 375 and blocked the receptor internalization that followed. That result anchors the entire pathway: EphB1 activates GRK2, GRK2 phosphorylates the mu-opioid receptor, and the receptor disappears from the cell surface.

The implications reach well beyond a single experiment. Because GRK2 helps terminate signaling for hundreds of G protein-coupled receptors, the ephrin-Eph system may turn out to be a broad regulator of GPCR trafficking in many contexts, a possibility the authors flag as an exciting direction for future work. More immediately, the study offers a concrete strategy against opioid tolerance: if blocking EphB1 signaling, or inhibiting GRK2 with drugs such as paroxetine, keeps mu-opioid receptors on the neuronal surface, patients might obtain durable pain relief at lower opioid doses, reducing both escalation and overdose risk. The findings also add to a growing body of evidence that receptor tyrosine kinases, including the insulin receptor and the epidermal growth factor receptor, crosstalk with opioid receptors and shape analgesia, dependence, and reward. Translating the mouse results into the clinic will require much more work, but the identification of a druggable node in the tolerance machinery gives researchers a clear place to start.

Subject of Research: EphB1 receptor regulation of mu-opioid receptor trafficking and morphine antinociception

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

Article References: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization. (n.d.). https://doi.org/10.1002/advs.78099

Image Credits: AI Generated

DOI: 10.1002/advs.78099

Keywords: EphB1, morphine, opioid tolerance, mu-opioid receptor, GRK2, receptor internalization, dorsal root ganglion, spinal cord, pain, analgesia, ephrinB2, paroxetine