Nearly 100 years ago, physician David Macht wrote, “If the entire materia medica were limited to the choice and use of only one drug, I am sure … the majority of us would choose opium.” Gavril Pasternak, of Memorial Sloan-Kettering Cancer Center in New York, US, speaking at the Experimental Biology meeting held 20-24 April 2013 in Boston, Massachusetts, US, said that the statement holds true today despite intense efforts to develop new analgesic drugs. And although opiate drugs have been “widely used and very useful,” their many side effects still limit their use, Pasternak said. Opiates’ analgesic actions, and their side effects, stem from activation of μ (mu) opioid receptors (MORs), and in a session dedicated to sources of diversity in MOR signaling, researchers presented new work about genetic splice variations and heterodimerization of opioid receptors. The results suggest novel approaches to better MOR-targeted drugs.
Opiate drugs beget a barrage of side effects including respiratory depression, constipation, pain hypersensitivity, itch, and a rewarding euphoria that can lead to powerful addiction. Tolerance also presents a significant problem: With use, a drug’s analgesic effect diminishes, even when side effects remain or worsen. A given opiate drug affects different people very differently, both in terms of its painkilling ability and its side effects. By the same token, one individual might react very differently—and unpredictably—to one opiate drug compared to another. These clinical observations led scientists to reason that the drugs must be acting on more than one subtype of the MOR, despite the understanding that a single gene, OPRM1, encodes the receptor.
Over the last 15 years, Pasternak and his colleagues, including Ying-Xian Pan, also at Memorial Sloan-Kettering, have built a case for alternative splicing as a source of MOR diversity (Pasternak, 2010). Over 90 percent of human and rodent genes are alternatively spliced, and the brain boasts the highest levels of alternative splicing, Pan said in his talk. So it is no surprise that alternative splicing of the OPRM1 gene could contribute to receptor diversity. Pan described unpublished work in which he analyzed the expression of OPRM1 splice variants in 10 brain regions from four different mouse strains. The data revealed a pattern of messenger RNA diversity that was region- and strain-specific, suggesting a genetic influence on alternative splicing. The researchers have previously described region-specific expression of several variant OPRM1 mRNAs in rats and humans as well (Xu et al., 2011; Xu et al., 2009).
In order to appreciate the ramifications of MOR diversity, Pasternak suggested, imagine the tune of “Row, Row, Row Your Boat” being played by a saxophone, trumpet, French horn, and tuba. In one version, the trumpet and French horn play loudly; in another, the saxophone and tuba are featured. “They are playing the same song. Do they sound the same? They do, but they don’t.” In different cells and brain regions, a slightly different orchestral makeup of receptors might be “playing” their own unique version of the song.
Changes small and large
Like most G protein-coupled receptors (GPCRs), opioid receptors contain seven membrane-spanning domains. Researchers got a first glimpse of the receptors’ molecular shape last year when crystal structures were revealed for all three opioid receptor isoforms—including κ (kappa) and δ (delta), in addition to μ—and the related orphanin FQ receptor (see PRF related news stories here and here). Those structures revealed a large, open drug-binding pocket, and answered many questions about how drugs and other ligands interact with the receptors.
One might think that structural variation in the binding area could explain differential drug effects, but Pan and Pasternak found that region to be conserved among alternatively spliced variants. Instead, they found a tremendous diversity in the variants in one small region at the very tip of the protein’s intracellular carboxy terminus. While these subtle changes might seem functionally insignificant, the team has found the opposite, with C-terminal alterations producing receptors that vary widely in their properties including signaling, trafficking, and interaction with other proteins. These functional changes have real consequences in vivo; Pan described unpublished work with genetically altered mice showing that the C-terminal variants contribute differently to drug tolerance and physical dependence of MOR agonists.
In addition to these subtle structural variations at the C-terminus, Pan and Pasternak have also shown evidence for a functional receptor variant with only six transmembrane (TM) domains, rather than the usual seven. The group showed that the MOR agonist IBNtxA acted at the truncated receptor to produce potent analgesia in mice, without some of morphine’s side effects such as respiratory depression and constipation. In addition, tolerance to IBNtxA built more slowly than to morphine, and the drug did not produce reward or physical dependence (Majumdar et al., 2011).
Perhaps even more surprising were unpublished findings in which the group demonstrated that a truncated variant containing only a single TM domain also had functional relevance. In an in-vitro cell-based system, they found the one-pass receptor could dimerize with the seven-TM MOR receptor in the endoplasmic reticulum (ER). Pan described the single-TM receptor as having a chaperone function, helping full-length MORs escape ER-associated degradation. This, in turn, led to increased expression of MOR protein at the cell surface—which in itself might represent a therapeutic strategy. Indeed, antisense oligonucleotides directed at the single-TM variant delivered to mice decreased morphine analgesia, possibly because the loss of this chaperone function led to decreased surface expression of MOR. “This interaction between truncated and full-length variants,” Pan said, “provides yet another layer of [receptor] regulation.”
Mix and match
What does this receptor multiplicity mean for drug development? It may turn out that while some variants mediate analgesia, others might be responsible for opiates’ nefarious side effects. This opens the possibility of differentially targeting receptor subtypes to relieve pain without addiction or tolerance. For instance, recent data suggest that the C-terminal splice variant MOR1D is responsible for morphine-induced itch (see PRF related news story). The itch-producing variant MOR1D forms an odd couple with the unrelated GPCR gastrin-releasing peptide receptor (GRPR). Pan said he sees that as “a clear indication that different splice variants can dimerize not just within the opiate receptor family, but beyond to other GPCRs.”
The idea of heteromerization producing receptor diversity isn’t exactly new. Another presenter at the session, Lakshmi Devi, a neuroscientist at Mt. Sinai Hospital in New York, US, has long studied the way MOR dimerizes with the δ- and κ-opioid receptor (DOR and KOR) isoforms (Costantino et al., 2012). “She’s a pioneer in the dimerization story,” Pan said of Devi.
Previous observations have indicated that an interaction between MOR and DOR might contribute to drug tolerance, because the association targets MOR for degradation (He et al., 2011). Devi used biochemistry techniques to demonstrate that the proteins interact, and then developed an antibody that specifically recognized the MOR-DOR interaction (Rozenfeld and Devi, 2011). Devi next used the antibody to develop a high-throughput screening assay for compounds that activate the heteromeric complex. Six compounds emerged as MOR-DOR selective ligands; the investigators have recently focused on one in particular. In animals, the compound produced anti-nociception but induced less tolerance than did morphine. The preliminary unpublished results, she said, show that “compounds targeting heteromers could be used as therapeutics to treat pain with reduced side effects.” Devi hypothesized that such analgesic compounds might disrupt the MOR-DOR interaction, thereby freeing the receptors to signal analgesia.
Together, the researchers described a newly appreciated variety in receptors, which arises through alternative splicing of the OPRM1 gene, heteromerization between variants and other opioid receptors, and even with GPCRs outside the opioid family. They agreed that in addition to explaining the complexity of the opiate experience, the variants also represent a new arsenal of drug targets. And that could bring them closer to “the Holy Grail,” said Devi: “a compound that retains analgesia but doesn’t have these side effects.”
Stephani Sutherland, PhD, is a freelance neuroscience writer based in Southern California.
