> Departments > Teams > Kieffer Group

Research interests

Previous activity

Three receptors classes, mu, delta and kappa, have been described by pharmacological approaches. We have cloned the first opioid receptor, a mouse delta receptor, by expression cloning (Kieffer et al, 1992 Proc. Natl. Acad. Sci. USA 89, 12048). We then have isolated cDNAs encoding human mu and kappa receptors using homology cloning techniques, and examined their pharmacological profiles and expression patterns (Simonin et coll. 1994 Mol. Pharm. 46, 1015; Simonin et coll. 1995 Proc. Natl. Acad. Sci. USA 92, 7006). We also have cloned an opioid-like orphan -or ORL- receptor (Matthes et al 1996 Mol Pharm. 50, 447-450), a receptor homologous to opioid receptors (see below). We have analyzed the expression pattern of the four receptors in the human central nervous system and immune cells (Gavériaux et coll. 1995 FEBS Lett. 369, 272). We finally have shown the existence of splice variants for delta and kappa receptors (Gavériaux et coll. 1997 Mol. Br. Res. 48, 298-304).

We have developed structure-activity studies of the delta receptor, in collaboration with B. Maigret (Nancy, France) et AstraZeneca (Montréal, Canada). Using site-directed mutagenesis, we have delimited an opioid binding site spanning helical domains III to VII and consisting of aromatic residues (Befort et coll. 1996 Mol. Pharm. 49, 216; Befort et coll. 1996 J. Biol. Chem. 271, 10161). We have set up a functional assay (Befort et coll. 1996 Neurochem. Res. 21, 1301) to explore delta receptor activation and found constitutive activity for several mutants of the binding site (Befort et coll. 1999, J. Biol. Chem. 274, 18574-81). We then have developed a random mutagenesis approach to further examine the mechanism of receptor activation. Our screen for constitutive activity mutants has led to the isolation of thirty point mutants. The study of their localization on a 3D model of the receptor has revealed an activation path originating from the third extracellular loop and propagating through tightly packed helices III/VI/VII down to a VI/VII cytoplasmic switch (Decaillot et al 2003 Nature Structural Biology 10, 629).

We have screened combinatorial peptide libraries on the four recombinant human receptors and discovered a peptide with dual mu agonist/ORL-1 antagonist properties of potential therapeutical interest (Becker et al, 1999 J. Biol. Chem. 274, 27513; Bigoni et al. 2000 Life Sci. 68, 233 ; Halab et coll. J. Med. Chem 45, 5353). We also have characterized a signaling-deficient mu receptor polymorphic variant (Befort et al, 2001 J. Biol. Chem. 276, 3130).

We have produced mice lacking mu, delta or kappa opioid receptor genes by homologous recombination to evaluate the molecular mechanism of action of classical, as well as newly developed opiates of clinical interest in vivo and to clarify the specific implication of each opioid receptor in adult physiology. We have shown that lack of mu receptors causes abolition of morphine-induced analgesia, place preference and physical dependence, as well as morphine respiratory depression and immunosuppression, demonstrating that mu receptors are essential in mediating both therapeutical and adverse effects of the prototypal opiate (Matthes et coll. 1996 Nature 383, 819; Gavériaux-Ruff et coll. 1998 Proc. Natl. Acad. Sci. USA 95, 6326; Matthes et coll. 1998 J. Neurosci. 18, 7285, Kieffer et coll. 1997 Médecine et Sciences 13, 232; Valverde et coll. 1998 CNS Drugs 10, 1). We also have found altered behavior of mu receptor knockout mice in response to alcohol, cannabinoids and nicotine, demonstrating that mu receptors modulate the activity of other drugs of abuse (Roberts et al, 2001 Clin. Exp. Res. 25, 1249 ; Ghozland et al. 2002 J. Neurosci. 22, 1146 ; Berrendero et al. 2002 J. Neurosci. 22, 10935). We have compared behavioral responses of mutant mice in several models of anxiety and depression. Our data show opposing phenotypes in mu and delta receptor knockout mice, which contrasts with the classical notion of alike activities of mu- and delta receptors. They also show consistent anxiogenic- and depressive-like responses in the delta mutant mice, indicating that delta receptor activity contributes to improve mood states (Filliol et coll. 2000 Nature Genetics, 25, 195). We have observed increased visceral pain in mice lacking the kappa receptor (Simonin et coll, EMBO J., 1998, 17, 886-897). We have further compared nociceptive thresholds of mutant mice in response to several type of noxious stimuli and data suggest tonic activity of endogenous mu, delta and kappa systems with a distinct implication of each receptor to various pain modalities (Martin et al. 2003 Eur. J. Neurosci. 17, 701). We have generated triple knockout animals, where the entire opioid system is deleted and we use them as a tool to study non-classical opioid responses. We have found that kappa2 receptor sites are , in fact, a combination of mu, delta and kappa receptors (Simonin et al. (2001) Eur. J. Pharm. 414, 189). We also have discovered that naltrindole act as an immunosuppressant via a non-opioid mechanism (Gavériaux-Ruff et al. 2001 J. Pharm. Exp. Ther. 298, 1193). We have summarized our data and data from other laboratories obtained from opioid receptor knockout mice in several review articles (Kieffer 1999, Trends Pharmacol. Sci. 20, 537 ; Kieffer et al. 2002 Prog. Neurobiol. 66, 285 ; Gavériaux-Ruff et al. 2002 Neuropeptides 36, 62).

N/OFQ is a newly discovered neuropeptide that has been recently identified as naturally occurring agonist of the opioid-like orphan receptor (ORL₁ or NOP). Despite the fact that N/OFQ and its receptor show structural similarities to the peptides and receptors of the opioid family, they are pharmacologically distinct from the opiods. N/OFQ and its receptor are widely distributed through the central nervous system and are densely expressed in corticolimbic regions (amygdaloid complex, hippocampus, periaqueductal gray, dorsal raphé). We have shown that activation of NOP receptor following systemic administration of selective agonist, Ro64-6198, elicits anxiolytic-like effects across various tests of anxiety (Jenck et al, 2000 PNAS 97, 4938-4943), as previously found after i.c.v. infusion of N/OFQ itself (Jenck et al., 1997 PNAS 94, 14854). These findings show that an important role of the N/OFQ is to act as an endogenous regulator of acute stress/anxiety responses. We have also investigated the behavioral consequences of endogenous N/OFQ deficits using mice with targeted disruption of the coding region for the N/OFQ within the peptide precursor gene. We found that environmental factors have an important impact on the phenotypic expression of the mutation in animals (Jenck et al, 2000 Molecular Psychiatry 5, 572-574). When housed individually, N/OFQ-deficient mice displayed normal responses in behavioral tests of emotional reactivity (behavioral despair, circadian activity, light-dark preference, acoustic startle) suggesting that efficient homeostatic mechanisms compensated for the N/OFQ deficit to ensure that essential adaptative responses to moderate stress remain functional. Interestingly, when housed in dense group for several days, N/OFQ-knockout male mice showed significantly more home-cage aggression and high levels of anxiety (Ouagazzal et al., 2003 Behav. Brain Res. 144, 111-117). Finally, recent findings from our group showed that systemic administration of Ro64-6198, impairs the acquisition and retrieval of fear conditioning in mice (Ouagazzal & Whichmann, 2003 Behavioral Pharmacol 14, S34). Altogether, these observations provide further evidence for the importance of N/OFQ system in the modulation of stress-coping behavior and suggest that dysfunction of N/OFQ system may confer a predisposition to higher reactivity to environmental stresses and, possibly, a higher vulnerability to anxiety and depression in man.

Main on-going projects :

Mu receptors: the gate for drug abuse.
We study cellular and in vivo mechanisms underlying the development of opioid tolerance and dependence (see also Evans and Kieffer, 2002 Cell 108, 1-20), as well as general molecular adaptations to drug abuse in the brain. To this aim, we use pharmacological, behavioral, imaging and gene profiling methods. Because mu receptors represent a key switch for addictive behaviors (Contet et al. 2004 Curr. Op. Neurobiol. 14, 1-9)., we also use mouse genetics and target the mu receptor gene by knock-in or conditional knockout approaches.

Delta receptors: a potential therapeutic target for the treatment of inflammation and emotional disorders.
Our first data from delta receptor knockout mice show that classically used delta agonists are weakly selective in vivo, and also activate mu receptors (Sherrer et al., 2004 Eur. J. Neurosci. 19, 2239-2248.) The comparative analysis of mu and delta knockout mice reveals increased anxiety and depressive behaviors in the delta mutant, and opposing phenotypes in the two mutant lines (Filliol et al. 2000). Besides, responses to acute noxious stimuli are modified in mu, but not in delta knockout mice (Martin et al. 2003). Together, the data suggest that the delta receptor is very different from the mu receptor. The physiological activity of delta receptors remains to be clarified. We examine the role of delta receptors in emotional responses and inflammation. We also study delta receptor trafficking in neurons. As for mu receptors, we combine pharmacological and knockout approaches.

N/OFQ System (Ouagazzal A.M.)

Our findings show that increased N/OFQ transmission not only reduces anxiety levels but also interferes with the acquisition and retrieval of conditioned fear responses. Our goal is to identify the neuroanatomical sites and the molecular mechanisms of action of N/OFQ that are yet poorly understood. Consideration will be given to clarify how this neuropeptide modulate intracellular signaling pathways (e.g. protein kinases (PKA, PKC, CaMKII) and their downstream targets (Erk-1/2, CREB, Elk-1)) which are known to play essential role in neuronal plasticity and behavioral processes related to memory formation and stress adaptation.