Opioid Peptides and Receptors 235

Opioid Peptides and Receptors

B L Kieffer, IGBMC,CNRS/INSERM/ULP, Illkirch,France

ã 2009 Elsevier Ltd. All rights reserved.

The Opioid System: A Short History

Discovery of the opioid system stems from the useand abuse of opium in ancient history. Opium,extracted from poppy seeds (Papaver somniferum;see Figure 1(a)), has powerful pain-relieving proper-ties and produces euphoria. This substance has beenused both medicinally and recreationally for severalmillennia. Morphine, named after the godMorpheus,is the most active ingredient of opium. The compoundwas isolated in 1805 and rapidly became the clinicaltreatment of choice to alleviate severe pain. Heroinwas synthesized chemically bymorphine diacetylationin the late 1800s and was commercialized as the firstnonaddictive opiate to treat cough and asthma. Thestrong addictive properties of heroin were soonacknowledged, and both heroin and opium were pro-hibited in 1910. Today morphine remains the bestpain-killer in contemporary medicine, despite a widearray of adverse side effects (respiratory depression,constipation, tolerance, and dependence). Heroin is amain illicit drug of abuse, and heroin addiction repre-sents a major public health issue. Because of theirextraordinarily potent analgesic and addictive pro-perties, opiates have prompted scientists to seek tounderstand their mode of action in the brain.In 1973, three independent teams showed that opi-

ates bind to membrane receptors in the brain, andthese receptors were named m, d, and k a few yearslater. In 1975, two pentapeptides, Met- and Leu-enkephalin were isolated as the first endogenousligands for these receptors. Many peptides followed,forming the opioid peptide family. Three distinctgenes encoding the peptides were isolated in the late1970s and early 1980s; three other genes encodingthe receptors were cloned a decade later. In the mid1990s the entire endogenous opioid system was char-acterized at the molecular level (Figure 1(b)).

Molecular Components of the OpioidSystem

The Peptides

Opioid peptide genes encode large precursor proteins,which are further cleaved into shorter peptides. Allopioid peptides share a common N-terminal Tyr-Gly-Gly-Phe signature sequence, which interacts with

opioid receptors. The preproenkephalin gene encodesseveral copies of the pentapeptide Met-enkephalinand one copy of Leu-enkephalin. This gene alsoencodes longer versions of Met-enkephalin. Notably,one of them, the opioid peptide bam22, activates opi-oid receptors via its N-terminal sequence while acti-vating another receptor family known as sensoryneuron-specific (SNS) receptors via its C-terminalpart. The preprodynorphin gene also encodesmultipleopioid peptides, including dynorphin A, dynorphin B,and neo-endorphin. The preprodynorphin-derivedpeptides all contain the Leu-enkephalin N-terminalsequence and basic amino acid residues in theirC-terminal end. Finally the preproopioimelanocortingene encodes a single opioid peptide, b-endorphin,among other peptides with nonopioid biologicalactivity. b-endorphin is the longest opioid peptide(31 amino acids) and shows highly potent andlong-lasting analgesic properties.

The Receptors

Opioid receptors aremembrane receptorswith a seven-transmembrane topology that belong to the large G-protein-coupled receptor (GPCR) superfamily. Thisfamily comprises several hundred members within themammalian genome. m-, d-, and k-opioid receptorgenes constitute a GPCR subfamily, together with afourth member encoding the orphanin FQ/nociceptinreceptor. The last receptor and its endogenous ligandshare high structural homology with the opioid recep-tors and peptides. However, the receptor does not bindopioids and the peptide orphanin FQ/nociceptin showsno affinity toward m, d, or k receptors. All four receptorgenes display similar genomic organization, with con-served intron–exon junctions, suggesting a commonancestor gene. These genes have been characterizedthroughout vertebrates, but cannot be identified earlierin evolution. The four genes are referred as to Oprm1(m), Oprd1 (d), Oprk1 (k), and Oprl1 (orphanin FQ/nociceptin) (genomic information is available on theNCBI website). The encoded receptors are classicallynamed MOR, DOR, KOR, and ORL-1, respectively,throughout the literature. A novel nomenclature MOP(m), DOP (d), KOP (k), and NOP (orphaninFQ/nociceptin) has recently been proposed by the Interna-tional Union of Basic and Clinical Pharmacology(IUPHAR) to unify receptor abbreviations. Manysingle-nucleotide polymorphisms have been identifiedin both coding and regulatory sequences of humanopioid receptor genes, and large genetic investiga-tions are underway to associate haplotypes withneurological or psychiatric diseases. At present, coding

Opioidpeptides

mdk

PainHedonic status

StressOthers

a

b

PreproenkephalinPreprodynorphinPreproopiomelanocortin

Opioidreceptors

Opiates

Analgesiaaddiction

MorphineHO O

N

OH

CH3

Figure 1 The opioid system: (a) exogenous opiates activateopioid receptors; (b) the endogenous opioid system. In (a),

morphine is the main active ingredient of opium and has

strong analgesic and addictive properties, which result from a

direct interaction of the drug with opioid receptors. In (b), the

endogenous opioid system is a neuromodulatory system. The

three opioid receptors naturally interact with a family of opioid

peptides, produced by proteolytic cleavage of three large precur-

sor proteins. Receptors and peptides are broadly expressed

throughout the nervous system and control nociceptive pathways

(pain), mood, well-being (hedonic status), and responses to

stress. The system also controls respiration, gastroinstestinal

motility, and endocrine and immune functions.

236 Opioid Peptides and Receptors

polymorphisms as well as silent or noncoding poly-morphisms that may influence receptor expressionlevels in the brain are under study.

Physiology of the Opioid System In Vivo

Opioid peptides and receptors are strongly expressedin the limbic system, as well as in nociceptive path-ways and along the hypothalamic–pituitary–adrenalaxis. In these neural circuits, the opioid system reg-ulates addictive and emotional behaviors, and con-trols responses to pain or stress.

Pharmacology

Three decades of pharmacology have demonstratedan analgesic activity for all three m, d, and/or k ago-nists. Generally, opioid analgesics used in the clinicare m agonists. These compounds show strongest effi-cacy in the treatment of severe acute pain, but theiractivity remains variable in situations of chronic pain.k agonists are potential candidates in the treatment ofvisceral pain. Significantly, studies of m and k com-pounds have highlighted opposing activities of mand k agonists on mood. m agonists show potent

rewarding properties and abuse liability, whereas kagonists are strongly dysphoric. The latter activityhas hindered the development of centrally acting kagonists for pain control in the clinic. The pharma-cology of d agonists has shown slower progress. Thenotion that d agonists may represent useful analgesicswith low abuse liability has been proposed for manyyears and is currently being explored.

Genetics Approach

For a decade, mice lacking m, d, or k receptors, aswell as preproenkephalin, preprodynorphin, orb-endorphin, have been created by gene-targeting(knockout mice). Single mutant mice, or even thetriple receptor knockout mice, are viable and fertileand show no obvious developmental deficit, indicat-ing that the opioid system is not essential for survival.These mutant mice have been extensively analyzedeither for spontaneous behaviors or in response toopioid and nonopioid drugs. Parallel to the pharma-cology, this genetic approach has clarified the specificcontribution of each molecular component of theopioid system in opioid-controlled physiology andbehaviors in vivo. In particular, the comparative analy-sis of m, d, and k knockout mice has clearly shownvery distinct activity patterns for each receptor in vivo(Figure 2). Similar comparisons are currently under-way for peptide knockout mice. Main conclusionsfrom gene knockout mice are the following.

m receptors represent the primary molecular targetfor morphine in vivo and mediate both beneficial andadverse effects of the most broadly used opiate.m receptors also mediate the rewarding properties ofnonopioid drugs of abuse including cannabinoids,alcohol, and nicotine or even natural reinforcerssuch as social interactions. m receptors therefore rep-resent a key molecular trigger for reward, and mostlikely contribute to the initiation of addictive behav-iors. k receptors, as predicted by the pharmacology,mediate dysphoric activities of both k opioids andcannabinoids and oppose m receptors in regulatingthe hedonic tone. d receptors are less directly involvedin hedonic control. Very distinct from m and k recep-tors, d receptors regulate emotional responses andshow anxiolytic and antidepressant activity. This spe-cific function of d receptors is now confirmed both bygene knockout and pharmacological studies usingSNC80, the only commercially available highly selec-tive d compound. Further analysis of d knockout miceand the development of more selective compoundswill probably reveal other activities of delta receptors.

m, d, and k receptor-deficient mice all exhibitenhanced pain sensitivity. This indicates that the threereceptors, activated by endogenous opioid peptides,tonically inhibit nociceptive responses. Noticeably,

Analgesia

DysphoriaEmotional reactivityReward

d k

DynorphinsEnkephalinsb-Endorphin

U50488HSNC80Morphine

m

Figure 2 Physiological role of opioid receptors and peptides in vivo. Opioid peptides show some, although weak, selectivity toward m, d,and k receptors. A highly selective exogenous (morphine) or synthetic (SNC80 and U50488H) agonist is indicated for each receptor. Thecombination of pharmacological studies with gene-knockout approaches (targeted gene inactivation in mice) showed that each receptor

controls specific neural responses. m and k receptors produce euphoria or dysphoria, respectively, whereas d receptors control levels ofanxiety and depressive-like behaviors (emotional reactivity). The activation of all three receptors inhibits pain (analgesia), but each

receptor controls a distinct panel of pain modalities.

Opioid Peptides and Receptors 237

the phenotypes of mutant mice differ across painassays. In models of physiological or acute pain, mreceptors modulate mechanical, chemical, and supra-spinally controlled thermal nociception, whereas kreceptorsmodulate spinally mediated thermal nocicep-tion and visceral pain. Again, d receptors differ fromm and k receptors in that there is no obvious regulationof acute pain. In contrast, there is evidence for a role ofd receptors in reducing hyperalgesia in situations ofinflammatory and neuropathic pain. These data, com-binedwithmany pharmacological studies, clearly dem-onstrate a specific role for each receptor in regulatingthe broad diversity of pain modalities.Finally, due to the short half-life and structural

similarity of the peptides, identifying a specific rolefor each endogenous opioid in vivo has been difficult.Data from opioid peptide knockout mice do not nec-essarily follow data from receptor knockouts, consis-tent with the notion that multiple peptides probablyact at each receptor. Although affinity studies andpharmacological approaches suggest a relationshipbetween b-endorphin and m receptors, enkephalinand d receptors, and dynorphin and k receptors(Figure 2), knockout data provide a more complexpicture. It is likely that the specific anatomical loca-tion of receptors and peptide release largely driveopioidergic synapse functioning.

Receptor Structure

Receptor Binding

Overall m, d, and k receptors show a 60% amino acidsequence identity. Closest homology occurs withinthe seven-transmembrane helical core, which con-tains the opioid-binding pocket (Figure 3(b)). Extra-cellular domains, including three extracellular loopsand the N-terminal domain, determine m, d, and kselectivity. These domains differ strongly amongreceptors and probably act as a gate filtering m, d,and k agonists or antagonists entering the bindingpocket. Note that synthetic compounds have beendeveloped with high m, d, and k selectivity, whereasendogenous peptides show limited receptor preference(Figure 2).

Receptor Signaling

As in all GPCRs, opioid receptors convey extracellu-lar signals within the cell by activating heterotrimericG-proteins, which interact with cytoplasmic domainsof the receptor. Agonist binding modifies helicalpacking of the receptor, and a rearrangement in thepositioning of transmembrane domains 3, 6, and7 has been proposed to drive the transition betweeninactive and active conformations of the receptor.

m, d, k selectivity

Binding pocket

Signal transduction

Gi/o-protein binding

Regulation

Inside

Outside

a

b

cc

Figure 3 Opioid receptors: (a) receptors belong to the G-protein-coupled receptor superfamily with a seven-transmembrane topology;(b) three-dimensional computer model of the human d-opioid receptor; (c) direct visualization of d receptors in native neurons. As shown in(a), m, d, and k receptors are highly homologous. Each receptor domain contributes to receptor function, as indicated. Regulation occursimmediately after the receptor is activated by an opioid agonist and includes receptor phosphorylation, uncoupling from the G-protein, and

internalization. In (b), the seven-transmembrane helices are shown in red, and portions of extrahelical loops are in white. The binding

pocket penetrates halfway within the helical bundle, and side chains of amino acid residues forming the main binding site are shown in

blue and green. As shown in (c), hippocampal neurons extracted from a genetically engineeredmouse endogenously produce fluorescent

d-opioid receptors. The receptor is observed at the surface of both cell bodies and processes of the neuron (see continuous greenfluorescence on the plasma membrane, top). Exposure to the agonist produces receptor internalization, visible under the form of highly

fluorescent dots within the neuron. Note the concomitant fading of surface fluorescence (bottom).

238 Opioid Peptides and Receptors

This helical movement modifies the receptor’s intra-cellular structure, hence the receptor–G-protein inter-action. Intracellular loops of the receptor form a largepart of the receptor–G-protein interface. These intra-cellular receptor domains are almost identical acrossm, d, and k receptors, consistent with the fact that allthree receptors interact with inhibitory G-proteins ofthe Go/Gi type. G-protein subunits dissociate from theactivated receptor and, in turn, modulate intracellu-lar effectors and pathways.Several opioid-evoked signaling events have been

identified both in transfected cells and native tis-sues. Opioids inhibit voltage-dependent Ca2þ channelsor activate inwardly rectifying potassium channels,thereby diminishing neuronal excitability. Opioidsalso inhibit the cyclic adenosine monophosphate(cAMP) pathway and activate mitogen-activated pro-tein kinase (MAPK) cascades, both of which affectcytoplasmic events and transcriptional activity of thecell. Overall, opioids inhibit neurons by decreasing

either neuronal firing or neurotransmitter release,depending on the post- or presynaptic localization ofthe receptors. Finally, opioid receptors are expressedon both excitatory and inhibitory neurons and cantherefore exert inhibition or disinhibition withinneural circuits.

Regulation of Receptor Signaling

The C-terminal domain differs largely across opioidreceptors. Together with intracellular loops, thisreceptor domain contributes to receptor–G-proteincoupling. Significantly, both the C-terminus andintracellular loops also interact with other cellulareffectors following receptor activation. Effectorsinclude receptor-specific (G-protein-coupled receptorkinase (GRK)) or-nonspecific (protein kinase A (PKA)and protein kinase C (PKC)) kinases, arrestins thatact as adaptors between the receptor and the endocy-tic machinery, and other recently identified proteins.

Opioid Peptides and Receptors 239

These interacting proteins drive the desensitization ofreceptor signaling, which typically occurs as an adap-tive response after receptor stimulation. Desensitiza-tion mechanisms include receptor phosphorylationand uncoupling from the G-protein, as well as recep-tor endocytosis (Figure 3(c)) and redistributionbetween the cell surface and intracellular compart-ments. Largely determined by C-terminal epitopes,these dynamic phenomena differ across m, d, andk receptors. Many studies have addressed the mechan-isms of opioid receptor trafficking and desensitiza-tion in cellular models and, more recently, in vivo.At present, studies on native neurons suggest thatagonist-stimulated d receptors are mainly targetedtoward degradation pathways after endocytosis,whereas internalized m receptors are resensitized inearly endosomes and rapidly recycle back to the cellsurface. Understanding the dynamic aspects of recep-tor trafficking to and from the cell surface has becomea major field in GPCR research.

Chronic Opiates

Regulatory events that occur at the receptor level,as already described, represent adaptive mechanismsthat classically contribute to cellular homeostasisunder acute agonist stimulation. Chronic exposureto opiates, however, has long-term consequences,which lead to strong and perhaps irreversible altera-tions of brain functioning at the cellular, synaptic,and network levels. Behaviorally, these adaptationsare manifested by tolerance, defined as a reduced sen-sitivity to the drug effects, and dependence revealed bydrug craving and withdrawal symptoms. Many effortshave been made for several decades to understand themolecular basis of these elaborate responses to chronicopiates. Modifications of opioid receptor-associatedion channels and second-messenger pathways havebeen demonstrated in several brain areas. Also severalprotein kinases, including PKA, PKC, and calcium/calmodulin-dependent protein kinase (CaMK)II; gluta-mate receptors; cytoskeleton proteins or neurotrophicfactors are involved. Finally opposing neurotransmittersystems – called anti-opioid systems – are recruited.Integrating these observations into a coherent expla-nation for tolerance and dependence in vivo remainsa challenge.

Opioid Receptor Heterogeneity

The Existence of Opioid ReceptorPharmacological Subtypes

Opioid receptor pharmacology is complex and theexistence of multiple m, d, and k receptor types has

been proposed since the early 1970s. Gene cloning ledto the characterization of only three receptor genes,and the molecular basis for pharmacological diversityhas long remained a matter of debate. Alternativesplicing has been reported, but it has been extremelydifficult to establish the biological relevance of thesealternative transcripts in vivo and to correlate theirexistence with the multiple opioid receptor subtypesthat were described earlier by the pharmacology.Today, it is admitted that the three main Oprm1-,Oprd1-, and Oprk1-encoded receptors are highlydynamic proteins, which may indeed account for thewide diversity of pharmacological subtypes.

Three Receptor Proteins, Many More CellularResponses

There are several ways to explain pharmacologicalheterogeneity of opioid receptors. First, increasing evi-dence supports the notion that m, d, and k receptorsmay adoptmultiple active conformations.Mutagenesisdata suggest the existence of multiple binding modesforopioidswithin thebindingpocket. Further, signalingstudies in cellularmodels show that receptor activationand subsequent regulations are strongly drug-depen-dent. Hence the ligand–receptor complex, rather thanthe receptor itself, determines the ultimate physiologi-cal cellular response. A second source of heterogeneityis the direct cellular environment of the receptor.Heterotrimeric G-proteins differ across cell types, andthe number of possible G-protein-associated signalingpathways has expanded dramatically. The variablecombinations of G-protein subunits and the nature ofassociated signaling networks necessarily generateneuron-specific, or even neuron compartment-specific,responses. Also, many regulatory proteins directlyinteract with the receptor C-terminal tail (as alreadydiscussed) and potentially influence opioid-receptorpharmacology, as was demonstrated for several otherGPCRs. A third potential receptor modulator isanother receptor molecule. The possibility thatGPCRs exist as dimeric or oligomeric complexes hasgained evidence in the recent years. Co-expression datasuggest that the physical association ofopioidreceptorseither as homodimers or heterodimers, or even withother GPCRs, creates novel receptor entities withunique pharmacological properties, which increasesopioid receptor heterogeneity.Whether receptor dimer-ization truly modulates opioid pharmacology in vivoremains an important question.

In conclusion, molecular approaches have provideda novel view of opioid receptors. The receptor is nowconsidered a dynamic multicomponent unit rather thana single protein entity. It is likely that the complexityof opioid responses will extend beyond the previously

240 Opioid Peptides and Receptors

reported pharmacological subtypes as in vivo molecu-lar pharmacology develops. Rational design of opioidcompounds that activate a specific subset of m, d, or kreceptor-associated signaling pathways is a possiblestrategy for developing novel drugs of high therapeu-tic value and low adverse activities, but this stillremains a far distant goal.

Conclusion

The last decade in opioid research has led to abetter understanding of the genetic basis of opioid-controlled behaviors and physiology. It has alsoopened avenues toward the development of noveltherapeutic opioids in the field of chronic pain, emo-tional disorders, and addictive diseases. Recombinanttechnologies have allowed high-throughput screeningfor novel opiate compounds, the detailed molecularanalysis of receptor structure and function, and theidentification of associated proteins regulating receptorsignaling.Ongoing studies have returned to analyzing opioid

receptor and peptides operating in their physiologicalenvironment, using molecular and genetic tools as wellas high-resolution imaging techniques. There is a needto identify neural sites where opioid peptide releaseand subsequent opioid receptor activation controlsnociceptive, emotional, andmotivational circuits.Also,it is important to characterize signaling pathwaysthat are relevant to specific behavioral or physiologicalresponses to opioids in vivo. In addition, novel insightsinto brain function will arise from understanding theinteractions of the opioid system with other neuro-transmitter systems. For example, whether the opioidsystem interacts with the cannabinoid system or anti-opioid systems at themolecular, cellular, or circuit levelremains an open question. Last, the elucidation ofmolecular and network adaptations to chronic opiateswill undoubtedly shed light on the general mechanismsof brain plasticity.

See also: Addiction: Neurobiological Mechanism; Drug

Addiction: Behavioral Neurophysiology; Invertebrate

Neurohormone GPCRs; Neuropeptides: Pain; Single

Photon Emission Computed Tomography (SPECT):

Technique.

Further Reading

Akil H, Watson SJ, Young E, et al. (1984) Endogenous opioids:

Biology and function. Annual Review of Neuroscience 7:223–255.

Bailey CP and Connor M (2005) Opioids: cellular mechanisms oftolerance and physical dependence. Current Opinion in Phar-macology 5: 60–68.

Brownstein MJ (1993) A brief history of opiates, opioid

peptides and opioid receptors. Proceedings of the NationalAcademy of Sciences of the United States of America 90:5391–5393.

Chang KJ, Porreca F, and Woods JH (eds.) (2004) The DeltaReceptor. New York: Dekker.

Contet C, Kieffer BL, and Befort K (2004) Mu opioid receptor:

A gateway to drug addiction.Current Opinion in Neurobiology14: 370–378.

Devi LA (2001) Heterodimerization of G-protein-coupled recep-

tors: Pharmacology, signaling and trafficking. Trends in Phar-macological Sciences 22: 532–537.

Dickenson TH and Kieffer BL (2005) Opiates – basic. In:Koltzenburg M, and McMahon SB (eds.) Wall & Melzack’sTextbook of Pain, 5th edn., ch. 27. London: Elsevier.

Evans CJ (2004) Secrets of the opium poppy revealed. Neurophar-macology 47(supplement 1): 293–299.

Ikeda K, Ide S, Han W, et al. (2005) How individual sensitivity to

opiates can be predicted by gene analyses. Trends in Pharmaco-logical Sciences 26: 311–317.

Kieffer BL (1997) Molecular aspects of opioid receptors. In:

Dickenson A and Besson J-M (eds.) Handbook of Experi-mental Pharmacology, Vol. 130: The Pharmacology of Pain,pp. 281–303. Berlin: Springer.

Kieffer BL andGaveriaux-Ruff C (2002) Exploring the opioid system

by gene knockout. Progress in Neurobiology 66: 285–306.LaForge KS, Yuferov V, and Kreek MJ (2000) Opioid receptor

and peptide gene polymorphisms: Potential implicationsfor addictions. European Journal of Pharmacology 410:249–268.

Mansour A, Fox CA, Akil H, et al. (1995) Opioid-receptor mRNAexpression in the rat CNS: Anatomical and functional implica-

tions. Trends in Neuroscience 18: 22–29.Nestler EJ (1996) Under siege: The brain on opiates. Neuron 16:

897–900.Pierce KL, Premont RT, and Lefkowitz RJ (2002) Seven-transmem-

brane receptors. Nature Reviews Molecular Cell Biology 3:639–650.

Williams JT, Christie MJ, and Manzoni O (2001) Cellular andsynaptic adaptations mediating opioid dependence. Physiologi-cal Reviews 81: 299–343.

Relevant Website

http://www.ncbi.nlm.nih.gov – NCBI.

http://www.ncbi.nlm.nih.gov

Opioid Peptides and ReceptorsThe Opioid System: A Short HistoryMolecular Components of the Opioid SystemThe PeptidesThe Receptors

Physiology of the Opioid System In VivoPharmacologyGenetics Approach

Receptor StructureReceptor BindingReceptor SignalingRegulation of Receptor SignalingChronic Opiates

Opioid Receptor HeterogeneityThe Existence of Opioid Receptor PharmacologicalSubtypesThree Receptor Proteins, Many More Cellular Responses

ConclusionFurther ReadingRelevant Website

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