Non-peptide opioid drugs like morphine and fentanyl bind to opioid receptors (ORs) on the neuronal cell membrane, just as naturally occurring peptide OR ligands do, to bring about analgesia. As such, it’s often been assumed that each group of compounds acts via the same cellular mechanisms to provide pain relief. Yet without adequate tools to detect OR activation in living neurons, whether this is actually the case remained an open question.
Now, using a new “nanobody” biosensor that can bind to ORs activated by non-peptide drugs or endogenous opioid peptides, a team led by Mark von Zastrow, University of California, San Francisco (UCSF), US, reveal a unique activation pattern, in time and in space: While peptides first activate ORs in the plasma membrane—and subsequently in endosomes, too, contrary to prevailing assumptions—non-peptide drugs also activate the receptors in another subcellular compartment, the Golgi apparatus.
William Schmidt, NorthStar Consulting, Davis, US, an expert on drug development who was not involved in the study, says the paper is a “real eye-opener,” with great implications for future analgesic drug development.
“This offers a new way of thinking about how opioid peptides versus alkaloid-related drugs may interact with cells, finally allowing us to better understand the similarities and differences we see between them,” he said.
The study was published June 6, 2018, in Neuron, along with an accompanying Preview by Dong Wang, Daniel Berg, and Grégory Scherrer, Stanford University, Palo Alto, US.
Questioning the conventional wisdom
ORs are G protein-coupled receptors (GPCRs) spanning the cell membrane. Once an opioid, of the endogenous or exogenous variety, binds to the OR and activates it, the receptor undergoes endocytosis, a process where the cell “ingests” the receptor into specialized membrane-bound compartments called endosomes.
“One of the functions of this endocytosis process is to change the number of receptors available for binding at the cell surface—and, over the long term, change the total number of receptors present in the cell,” said von Zastrow. “With opioids, this process happens really quickly, more quickly than we see with other GPCRs.”
Conventional wisdom held that endogenous and exogenous opioids worked by the same cellular mechanisms in neurons. But given that opioid users report feeling such robust, pleasurable effects from the drugs, many scientists wondered if there was more to the story.
“The idea is that these very different chemicals mimic the action of endogenous ligands. And there’s some good evidence for that,” says von Zastrow. “But it doesn’t explain why there is so much anecdotal evidence suggesting that opioid drugs produce stronger, more rewarding effects, and why these drugs so often lead to addiction.”
Von Zastrow wondered if perhaps the rapid movement of receptors into the endosomes after ligand binding might offer some clues to help explain these differences between endogenous and exogenous opioids. But he needed the right tools to look.
A nanobody biosensor to detect receptor activation inside and outside the cell
Aashish Manglik, also at UCSF, had developed a “nanobody” biosensor—a single domain antibody fused to a fluorescent protein—that can sense when GPCRs are activated after different compounds bind to them in vitro (Manglik et al., 2017). So, in collaboration with von Zastrow’s team, including first author Miriam Stoeber, Manglik adapted this nanobody to work with mu and delta ORs.
The nanobody acts as a conformational biosensor that can detect changes to the structure of the receptor when a natural opioid or opioid drug binds to it, and without negatively affecting receptor function. The nanobody fusion protein allows researchers to observe receptor activation using total internal reflection fluorescence microscopy (TIRFM), a technique that can selectively detect fluorescence at the plasma membrane. The researchers also used time-lapse confocal microscopy to follow the activated receptors as they made their way into the interior of cells.
Prior research had suggested that OR activation started and ended at the plasma membrane for both endogenous peptide ligands and exogenous non-peptide compounds. But using the biosensor in human embryonic kidney (HEK) cells, the researchers made a surprising discovery: While endogenous opioid peptides activated receptors on the cell membrane, OR activation did not stop there. For instance, they found that DAMGO, an OR peptide agonist designed to mimic natural opioids, as well as natural compounds such β-endorphin and met-enkephalin, not only activated mu and delta ORs on the cell membrane but also, minutes later, those within endosomes, reaching a receptor recruitment plateau at about 20 minutes.
When the researchers used naloxone, a competitive antagonist ligand for ORs, they were able to reverse OR sensor localization to endosomes within 30 seconds, confirming that the activation of ORs in endosomes was dependent on the presence of agonist ligand. Furthermore, DADLE, another OR peptide agonist, strongly inhibited cyclic adenosine monophosphate (cAMP), a downstream signal of OR activation within the cell, even after agonist washout. This suggested that activated ORs in endosomes could influence cell signaling.
Taking a trip to the Golgi apparatus
In subsequent experiments using cultured medium striatal spiny neurons that are known to express MORs and DORs endogenously, the researchers again followed receptor activation after the addition of DAMGO. They discovered that, within seconds of application, DAMGO activated ORs both on the plasma membrane and on endosomes, which extended out toward the soma and the dendrites of the neurons.
But non-peptide opioid drugs, including etorphine and morphine, also activated ORs in the Golgi apparatus, an organelle in the soma responsible for intracellular vesicular transport. The researchers also saw activated ORs in so-called Golgi outposts, which are Golgi-related membrane clusters, in dendrites. What’s more, the drugs moved easily across the cell membrane and activated the receptors within seconds. In contrast, endogenous opioids were unable to produce any activation in these Golgi-localized pools of receptors, even after extended exposure.
“This clearly shows a striking difference in where these different compounds will activate these receptors, and that influences signaling,” said Manglik. “The opioid drugs are getting into the cell and activating these cryptic pools of ORs, places where endogenous opioids aren’t activating receptors at all—and it’s not something we ever would have anticipated.”
Von Zastrow added that these differences in OR activation patterns could explain some of the adverse outcomes of opioid drugs.
“This raises the possibility that these drugs, in addition to mimicking many of the actions of natural opioids, may be producing additional effects by their natural tendency to access internal membrane locations,” says von Zastrow. “One could speculate that the pathological effects of these drugs, including their addictive properties, could be a consequence of those activations.”
A paradigm shift for opioid drug development?
Schmidt says the findings offer new insights on why current opioid drugs can have such detrimental side effects, how to improve those drugs, and how to better develop new, safer analgesics.
“This is entirely new, and it’s an idea that could drive new drug development,” he said. “There’s been a suggestion, for instance, that peptide-related analgesics might interact differently with ORs. Maybe they produce less tolerance, less dependence, or less respiratory depression. But we could only talk about that on a theoretical basis. This new information may give us the tools we need to go back and take a more careful look—as well as design safer drugs in the future.”
Von Zastrow agrees. He plans to follow up on the new study by comparing and contrasting the OR activation patterns of different opioid drugs, ranging from oxycodone to fentanyl, as well as investigating how endosome and Golgi apparatus OR activation may affect cellular signaling and associated analgesic effects.
“We would like to understand if there are differences in the consequences downstream of activating receptors in the endosomes and Golgi apparatus beyond the more prolonged nature of the signaling we’ve already seen,” says von Zastrow. “But these data inspire a great deal of excitement about the potential of developing very new and different therapies for pain relief. They provide strong support for the idea that we could achieve more control and more potential for therapeutic benefit by exploring these very basic aspects of how receptors are activated by these drugs.”
Kayt Sukel is a freelance writer based outside Houston, Texas.
Image credit: guniita/123RF Stock Photo
