Opioid receptors belong to the large superfamily of seven transmembrane spanning (7TM) G protein-coupled receptors (GPCRs).

As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones.

They are activated both by endogenously produced opioid peptides and by exogenously administered opiate compounds, some of which are not only among the most effective analgesics known but also highly addictive drugs of abuse.

Morphine and heroin, have a high liability for inducing tolerance, dependence, and addiction.

Opium is an extract of the poppy plant, Papaver somniferum. In 1806, the German chemist Serturner isolated the opium alkaloids, one of them being morphine after Morpheus, the god of dreams. However, it was not until 167 years later that the pharmacology of morphine was defined at the receptor level.

To date, four opioid receptors have been cloned: The MOP ( µ=mu for morphine) The KOP (κ= kappa for ketocyclazocine) The DOP (δ= delta for deferens because it was first

identified in mouse) And the NOP-R [initially called LC132, ORL-1, or

nociceptin/orphanin FQ receptor].

Dendrogram (phylogenetic tree) of opioid receptors based on their amino acid identity.

The endogenous opioid peptides are mainly derived from four precursors.

Except for nociceptin/orphanin FQ, all peptides derived from the other precursors consist of a pentapeptide sequence TyrGlyGlyPheMet/Leu (YGGFM/L).

Selective opioid receptor ligands

The opioid receptors are about 60% identical to each other with greatest homology in the transmembrane helices and the greatest diversity in their N and C termini as well as their extracellular loops.

Extensive site-directed mutagenesis studies and studies on receptors have helped to determine specific domains involved in:

(a) ligand binding (b) G protein-effector activation (c) constitutive activity (d) receptor desensitization/endocytosis/down regulation.

It has been suggested that all opioid receptors share a common binding cavity that is situated in an inner interhelical conserved region comprising transmembrane helices 3, 4, 5, 6, and 7. This cavity is partially covered by the extracellular loops.

The highly divergent extracellular loops, together with residues from the extracellular ends of the TM segments, play a role in ligand selectivity, especially for peptides, allowing them to discriminate between the different opioid receptor types.

Smaller alkaloid agonists (such as morphine) interact predominantly with conserved residues in the bottom of the binding cavity.

Moreover, alkaloid antagonists (such as naloxone) are thought to shift slightly deeper in the binding pocket than agonists, thereby sterically hindering a shift of TM3 and TM7, and consequently preventing an active receptor conformation, thus leading to functional antagonism.

Opioid receptors are predominantly coupled to pertussis toxin-sensitive (PTX), heterotrimeric Gi/Go proteins, although coupling to PTX-insensitive Gs or Gz proteins has also been reported.

Upon receptor activation, both G-protein and subunits interact with multiple cellular effector systems, inhibiting adenylyl cyclases and voltage-gated Ca 2+ channels and stimulating G protein-activated inwardly rectifying K+ channels (GIRKs) and phospholipase C (PLC).

Point mutations that result in constitutive activity and/or altered agonist efficacy in the MOP-R have pinpointed the importance of certain side chains, which include residues in transmembrane helices 2 (Asp114 and Tyr106), 4 (Ser196), and 6 (His297), as well as in the conserved Asp164 of the DRY motif at the beginning of intracellular loop 2 and in intracellular loop 3. Mutation of Ser196 in TM4 is of particular interest because it has recently been shown to confer agonist properties to antagonists in vitro and in vivo.

Tissue-specific expression of such a receptor could possibly provide a new therapeutic strategy for the treatment of pain.

In the NOP-R, alanine replacement of Gln286, located on top of TM 6, yielded a mutant receptor that retained a binding profile like the wild-type receptor. However, this mutant appeared to be functionally inactive, indicating that residue Gln286 may play a pivotal role in transmitting a nociceptin signal through NOP-R

Mutation of Asn133 in TM3 activates G proteins two- to threefold over basal levels. Eleven other point mutations resulted in either wild-type or decreased G protein-coupling efficiency.

Receptor desensitization is defined as any process that alters the functional coupling of a receptor to its G-protein/second messenger-signaling pathway.

Endocytosis is defined as the translocation of receptors from the cell surface to an intracellular compartment.

Down regulation is defined as any process that decreases the number of ligand binding sites.

Following activation by alkaloid or peptide agonists, opioid receptors are regulated by multiple mechanisms, many of which have been implicated in receptor desensitization.

One of these is receptor phosphorylation. As for many GPCRs, signaling from opioid receptors is rapidly regulated by a well-characterized and highly conserved cascade of events involving receptor phosphorylation by G protein-coupled receptor kinases (GRKs) and subsequent β-arrestin recruitment.

These processes contribute directly to rapid receptor desensitization by facilitating the uncoupling of the receptor from its G protein. GRK- and β– arrestin mediated desensitization is a rapid process occurring often within minutes of receptor activation.

Many kinases phosphorylate opioid receptors, a number of which have been shown to desensitize receptors as well. These include not only GRK, but also protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin dependent protein kinase II.

Opioid receptors can also be tyrosine phosphorylated. This modification affects desensitization as well, as has been shown in a series of studies examining C-terminal residues in MOP-R, DOP-R and KOP-R.

Endocytosis is one feature that distinguishes GRK/β –arrestin mediated desensitization from other phosphorylation-dependent desensitization mechanisms.

Following desensitization by GRKs and β-arrestin, opioid receptors are rapidly endocytosed into an intracellular compartment.

This process occurs following even brief agonist exposure and independently of signal transduction.

The speed and conservation of this process is ideal for modulating signaling from endogenous ligands, such as neurotransmitters, that are released in a pulsatile manner.

Following their endocytosis, receptors can then be recycled back to the membrane, thereby restoring the functional complement of receptors, a process termed “resensitization.”

In contrast, chronic exposure of opioid receptors to agonist, for example, during exogenous drug administration, can also lead to receptor desensitization/uncoupling.

This involves alternate mechanisms, including PKA- and PKC-mediated phosphorylation. These desensitization processes most often require prolonged agonist treatment and are dependent on signal transduction.

Importantly and in contrast to GRK-phosphorylated receptors, PKA/PKC-phosphorylated receptors are not necessarily rapidly endocytosed. Therefore, receptors that have been desensitized by GRK-independent phosphorylation require a mechanism other than endocytosis to resensitize.

Following endocytosis, receptors can be recycled, thereby restoring the functional complement of receptors.

Alternatively, receptors that have been endocytosed can be targeted for degradation, thereby decreasing the functional complement of receptors ultimately resulting in receptor downregulation.

Although endocytosis and subsequent degradation of receptors are not the only means of producing receptor downregulation, they can produce receptor downregulation rapidly, even following brief exposure to agonist.

Apparent receptor downregulation can also be affected by alterations in rate of receptor synthesis and/or folding/secretion.

Whereas MOP-Rs are recycled following their endocytosis, DOP-Rs are transported deeper into the endocytic pathway, are rapidly degraded by the lysosome, and hence “downregulated.”

Truncation of the C-terminal tails of the DOP and MOP receptors has been shown to result in agonist-independent increased internalization and recycling rates that suggest the presence of regulatory elements critical for facilitating the receptor’s interaction with the endocytic machinery.

Recently, several groups have reported dimerization of several GPCRs, including opioid receptors. In fact, heterodimerization of opioid receptors has been shown to alter opioid ligand properties and affect receptor trafficking in cell culture model systems and in vivo.

There are also reports of heterodimerization of the opioid receptors with other classes of GPCR.

Opioid receptor activation by endogenous and exogenous ligands results in a multitude of effects, which include analgesia, respiratory depression, euphoria, feeding, the release of hormones, inhibition of gastrointestinal transit, and effects on anxiety.

In general, agonists selective for MOP or DOP receptors are analgesic and rewarding, whereas KOP-R-selective agonists are dysphoric.

Morphine and other opioids remain the analgesics of choice for the treatment of chronic pain.

Morphine and other opioids remain the analgesics of choice for the treatment of chronic pain. However the major limitation to their long-term use is the development of physiological “tolerance,” a profound decrease in analgesic effects observed in most patients during prolonged drug administration.

In addition to tolerance, physiological “dependence,” which results in the necessity for continued administration of increasing doses of drug to prevent the development of symptoms of opioid withdrawal, can ensue in some patients.

The development of tolerance to opioids is typically measured as a change in antinociceptive or analgesic responses. The two most common behavioral assays to assess such changes are the hot plate and the tail flick tests, where a heat source is applied to either the tail or hind paw of an animal.

Dependence is measured in morphine-tolerant animals by either withdrawal of the opioid agonist or the administration of an opioid antagonist, such as naloxone. The typical behaviors reflecting withdrawal/dependence symptoms in animals are increases in locomotion, jumping, and weight loss.

Reward is measured using a technique referred to as conditioned place preference, a method that monitors an animal’s ability to develop a preference for a certain environment when paired with a drug.

In addition to being unresponsive to MOP-R ligands, such as morphine, MOP-R-deficient mice show reduced reward in a conditioned place preference paradigm to multiple other drugs of abuse. This indicates that MOP-Rs may play a role in the neural circuitry of reward.

The DOP-R has been implicated in the development of morphine tolerance, and in fact, DOP-R-deficient mice do not develop morphine tolerance to the same extent as their wild-type animals.

Morphine-activated MOP-Rs are relatively unique in that they are not GRK phosphorylated nor do they efficiently recruit β-arrestin, even though they are in an active receptor conformation.

Additionally, morphine fails to promote endocytosis of the wild-type MOP-R in cultured cells and native neurons, whereas endogenous peptide ligands, such as endorphins and the hydrolysis-resistant form of enkephalin, D-Ala2-MePhe4-Gly5-ol (DAMGO) as well as several opioid drugs, such as methadone, readily promote receptor endocytosis.

Hence morphine-activated MOP-Rs generally elude an important, highly conserved regulatory mechanism designed to rapidly modulate receptor-mediated signaling.

MOP-R endocytosis is an independent functional property of the receptor-ligand pair. Agonist activity and receptor endocytosis have opposing effects on receptor-mediated signaling.

The net amount of signal transmitted to the cell is a function of both processes. This “net signal” has been termed RAVE, for relative activity versus endocytosis.

Morphine would have a particularly high RAVE value as a consequence of its inability to promote receptor desensitization and endocytosis.

In contrast, endorphins and opioid drugs that acutely signal with similar efficacy, yet induce receptor desensitization and endocytosis, would have lower RAVE values than morphine.

Even though opioids, such as morphine, remain the analgesic of choice in many cases, a major limitation to their long-term use is the development of physiological tolerance, a profound decrease in analgesic effect observed in all patients during prolonged administration of opioid drug.

In theory, downregulation of opioid receptors would lead to tolerance by reducing the number of receptors available for drug-mediated actions.

Taken together, the available data suggest that it is unlikely that receptor downregulation is solely responsible for the development of morphine tolerance. This view has lead to the idea that rather than becoming downregulated, MOP-Rs may instead become desensitized or, more precisely, uncoupled from downstream signaling pathways.

In this context, receptors could become desensitized without the loss of a single receptor.

Following chronic morphine treatment, cellular levels of cAMP are elevated, a phenomenon termed cAMP superactivation.

The elevated cAMP levels reflect cellular adaptive changes, which include increased expression of certain types of adenylyl cyclase, protein kinase A (PKA), and cAMP response element binding protein (CREB).

For example, phosphorylation of type II adenylyl cyclase isoforms can significantly increase their stimulatory responsiveness to Gsα and Gβγ. Importantly, any of these compensatory changes produces cells/animals that appear tolerant to morphine simply because cAMP levels are no longer as effectively regulated by morphine as they were in the naive state.

The elevated levels of cAMP are responsible for changes in gene expression as well as for alterations in neurotransmitter release. These changes are long term and difficult to reverse.

Chronic morphine increases concentrations of types I and VIII adenylyl cyclase (AC I and VIII), PKA catalytic (C) and several phosphoproteins, including CREB.

Chronic opiate administration selectively up-regulates two forms of adenylyl cyclase (types I and VIII) in these locus coeruleus neurons.

Up-regulation of the type VIII enzyme appears to be mediated by cAMP response element– binding protein (CREB), one of the major cAMP regulated transcription factors in the brain.

A reduction in CREB concentration (achieved by infusion of antisense oligonucleotides to CREB directly into this region) blocks the morphine-induced increase in this enzyme.

Accordingly, antisense oligonucleotide treatment partially attenuates the activation of locus coeruleus neurons seen during withdrawal, as well as the severity of certain opiate withdrawal behaviors.

Consistent with these observations in the locus coeruleus is the more general observation that mutant mice deficient in CREB, a deficiency expressed ubiquitously, show attenuated opiate withdrawal.

Regulation of opioid receptors by endocytosis serves a protective role in reducing the development of tolerance and dependence to opioid drugs. This model, termed the RAVE hypothesis, is outlined as follows.

First, as a consequence of endocytosis, cells are rapidly desensitized to agonist.

Second, following endocytosis, receptors can be recycled to the cell surface in a fully active state, thereby resensitizing cells to agonist. This highly dynamic cycle of receptor regulation may be designed to mediate the actions of endogenous opioid peptides, which are typically released in a phasic or pulsatile manner.

Opioid drugs such as morphine in contrast, with high RAVE values,, would have an enhanced propensity to produce cAMP superactivation. This phenomenon will ultimately result in tolerance and dependence precisely because the morphine activated opioid receptors signal for aberrantly long periods of time.

mutations of the MOP-R that enhance morphine-induced endocytosis do indeed ameliorate cAMP superactivation.

Conversely, mutations that prevent endocytosis of the MOP-R in response to ligands, such as methadone and DAMGO, enhance methadone- and DAMGO-evoked cAMP superactivation.

In another study, an inverse relationship between endocytosis and tolerance was shown: Although morphine was able to produce acute analgesic tolerance, DAMGO failed to do so. However, when an inhibitor of PKC activation was applied to inhibit DAMGO-induced MOP-R internalization, DAMGO was able to induce acute tolerance.

The degree to which the maximal effect of a given drug can be achieved without full receptor occupancy defines its intrinsic efficacy. Each opioid ligand has its own intrinsic efficacy.

The fraction of the total receptor pool not required for maximal effect is the receptor reserve (or spare receptors).

The receptor reserve is reduced in morphine-tolerant animals. Therefore, more receptors are required to produce the same analgesic effect in these tolerant animals.

As mentioned in the case of CREB, Transcription factors are clearly one potential mechanism for persistent drug-induced plasticity.

The Fos and Jun families of transcription factors have also been studied extensively within the context of addiction.

Because of their extraordinary stability, the ΔFosB isoforms accumulate in the brain after repeated drug treatment and thereby are candidates to serve as molecular switches for long-lived adaptations to drug exposure.

A biological understanding of addiction requires knowledge of how acute effects of drugs of abuse on the brain are transformed into progressively longer-lasting adaptations in specific brain regions.

Thus, while molecular and cellular models of dependence, tolerance, sensitization, and withdrawal have been developed, very little is known about longer-lived forms of sensitization as well as the drug craving and high risk for relapse seen after months and even years of abstinence.

Transgenic and knockout methodologies represent powerful tools with which to establish causal relations between molecular and behavioral aspects of drug addiction.

The ability of drugs of abuse to alter the brain depends in part on genetic factors: Acute drug responses as well as adaptations to repeated drug exposure can vary markedly, depending on the genetic composition of the individual.

It may one day be possible to identify individuals who are particularly vulnerable to addiction and stress and thereby target them with specific psychosocial interventions.