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M G Caron

Publications and source records attributed to M G Caron.

At least 91 records · Page 5Linked to original sources
PubMed1998

Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter.

Amphetamine (AMPH) inhibits uptake and causes release of dopamine (DA) from presynaptic terminals. AMPH can act on both vesicular storage of DA and directly on the dopamine transporter (DAT). To assess the relative importance of these two processes, we have examined the releasing actions of AMPH in mice with a genetic deletion of the DAT. The sequence of actions of AMPH has been determined by following the real time changes of DA in the extracellular fluid of intact tissue with fast scan cyclic voltammetry. In striatal slices from wild-type mice, AMPH causes a gradual (approximately 30 min) increase in extracellular DA, with a concomitant disappearance of the pool of DA available for depolarization-evoked release. Conversely, in slices from mice lacking the DAT, although a similar disappearance of electrically stimulated DA release occurs, extracellular DA does not increase. Similarly, microdialysis measurements of DA after AMPH in freely moving animals show no change in mice lacking the DAT, whereas it increases 10-fold in wild-type mice. In contrast, redistribution of DA from vesicles to the cytoplasm by the use of a reserpine-like compound, Ro4-1284, does not increase extracellular DA in slices from wild-type animals; however, subsequent addition of AMPH induces rapid (<5 min) release of DA. Thus, the DAT is required for the releasing action, but not the vesicle-depleting action, of AMPH on DA neurons, and the latter represents the rate-limiting step in the effects of AMPH. Furthermore, these findings suggest that in the absence of pharmacological manipulation, such as the use of amphetamine, endogenous cytoplasmic DA normally does not reach sufficient concentrations to reverse the DAT.

2H-Benzo(a)quinolizin-2-ol, 2-Ethyl-1,3,4,6,7,11b-↗
PubMed1998

Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase.

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and beta-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of beta-arrestin or dynamin, beta2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and beta-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.

Arrestins↗
PubMed1998

G protein-coupled receptor adaptation mechanisms.

G protein-coupled receptors (GPCRs) transduce extracellular signals that modulate the activity of a wide variety of biological processes, such as neurotransmission, chemoattraction, cardiac function, olfaction, and vision. However, GPCR signalling desensitizes rapidly as the consequence of receptor phosphorylation. G protein-coupled receptor kinase-mediated receptor phosphorylation promotes the binding of beta-arrestin proteins, which not only uncouple GPCRs from their cognate heterotrimeric G protein, but also target them for endocytosis. The sequestration (endocytosis) of desensitized GPCRs to endosomes is required for their dephosphorylation and subsequent resensitization to their pre-ligand exposed state. This review concentrates on the mechanisms underlying GPCR desensitization and resensitization.

Adaptation, Physiological↗
PubMed1998

Molecular mechanisms of G protein-coupled receptor desensitization and resensitization.

Beta-arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta2-adrenergic receptor (beta2AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta2AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta2AR sequestration to impair beta2AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta2AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying beta-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr2GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. Beta arr2GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. Beta arr2GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr2GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.

Animals↗
PubMed1998

Re-evaluation of the role of the dopamine transporter in dopamine system homeostasis.

Mice with a genetic deletion of the dopamine transporter (DAT) were used to assess its role in the function of dopamine (DA) neurons. Profound alterations in the homeostasis of the nigrostriatal DA system were induced by the absence of the DAT. Extracellular levels of DA were elevated and clearance of released DA was 300-times slower than in control mice. This was accompanied by a 20-fold decrease in tissue DA levels and a paradoxical doubling of the rate of DA synthesis. A crucial role is indicated for the DAT in maintenance of DA neuron presynaptic function, particularly in the control of storage mechanisms.

Animals↗
PubMed1998

Cocaine self-administration in dopamine-transporter knockout mice.

The plasma membrane dopamine transporter (DAT) is responsible for clearing dopamine from the synapse. Cocaine blockade of DAT leads to increased extracellular dopamine, an effect widely considered to be the primary cause of the reinforcing and addictive properties of cocaine. In this study we tested whether these properties are limited to the dopaminergic system in mice lacking DAT. In the absence of DAT, these mice exhibit high levels of extracellular dopamine, but paradoxically still self-administer cocaine. Mapping of the sites of cocaine binding and neuronal activation suggests an involvement of serotonergic brain regions in this response. These results demonstrate that the interaction of cocaine with targets other than DAT, possibly the serotonin transporter, can initiate and sustain cocaine self-administration in these mice.

Animals↗
PubMed1998

Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice.

The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/-) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/- mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/- and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/- mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/- and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/- mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/- and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/- mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/- and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/- mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1998

Dopamine receptors: from structure to function.

The diverse physiological actions of dopamine are mediated by at least five distinct G protein-coupled receptor subtypes. Two D1-like receptor subtypes (D1 and D5) couple to the G protein Gs and activate adenylyl cyclase. The other receptor subtypes belong to the D2-like subfamily (D2, D3, and D4) and are prototypic of G protein-coupled receptors that inhibit adenylyl cyclase and activate K+ channels. The genes for the D1 and D5 receptors are intronless, but pseudogenes of the D5 exist. The D2 and D3 receptors vary in certain tissues and species as a result of alternative splicing, and the human D4 receptor gene exhibits extensive polymorphic variation. In the central nervous system, dopamine receptors are widely expressed because they are involved in the control of locomotion, cognition, emotion, and affect as well as neuroendocrine secretion. In the periphery, dopamine receptors are present more prominently in kidney, vasculature, and pituitary, where they affect mainly sodium homeostasis, vascular tone, and hormone secretion. Numerous genetic linkage analysis studies have failed so far to reveal unequivocal evidence for the involvement of one of these receptors in the etiology of various central nervous system disorders. However, targeted deletion of several of these dopamine receptor genes in mice should provide valuable information about their physiological functions.

Animals↗
PubMed1998

[Behavioral, cellular and molecular consequences of the dopamine transporter gene inactivation].

Mice lacking the the plasma membrane dopamine transporter (DAT), following gene inactivation or knock out, show an increase in their spontaneous locomotor activity that is of the same magnitude than in normal mice treated with amphetamine or cocaine, known to increase levels of dopamine in the basal ganglia. Many adaptive responses have occurred in these animals than could not compensate for the hyper activity of the dopamine system. Surprisingly, while intracellular dopamine levels were of only 5%, extracellular dopamine levels were increased by 300%. We investigated the regulation of tyrosine hydroxylase (TH), the rate limiting enzyme of dopamine synthesis, and found that this enzyme is regulated at the levels of mRNA, protein, trafficking as well as in its regional, cellular and subcellular organization. Our results establish not only the central importance of the transporter as the key element controlling dopamine levels in the brain, but also its role in the behavioral and biochemical action of amphetamine, cocaine and morphine. In addition, these mice have provided key elements leading to possible clinical and social implications for illnesses such as Parkinson disease, attention deficit disorder and drug addiction.

Animals↗
PubMed1998

Cloning, gene structure and genomic localization of an orphan transporter from mouse kidney with six alternatively-spliced isoforms.

Two genes were identified and characterized that express cDNAs related to previously identified neurotransmitter and/or osmolyte transporters, but which are expressed specifically in the kidney. RNA transcribed from one of these two genes (XT2) was found to undergo an extensive degree of alternative splicing to generate six distinct isoforms. The intron-exon structure of the XT2 gene and the sites of alternative splicing were identified. Expression of the second gene (XT3) was found to be conserved in human kidney, and partial sequence was obtained from a human cDNA library. The expressions of both XT2 and XT3 RNAs were determined in mouse and human tissues, respectively, and the locations of the two genes within the mouse genome were identified. Screening experiments to identify the substrate(s) of these proteins failed to identify specific uptake with any of the tested compounds; however, immunofluorescent microscopy demonstrated that epitope-tagged variants of the protein products of the XT2 and XT3 cDNAs were present on the plasma membrane of transfected cells.

Alternative Splicing↗
PubMed1997

A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation.

G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a beta-arrestin2/green fluorescent protein conjugate (betaarr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-G protein-coupled receptor kinase or GPCR-arrestin interactions. Confocal microscopy demonstrates the translocation of betaarr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the beta-arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with beta-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active beta-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of beta-arrestin activity and GPCR signal transduction in living cells. The use of betaarr2-GFP as a biosensor to recognize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.

Animals↗
PubMed1997

A central role for beta-arrestins and clathrin-coated vesicle-mediated endocytosis in beta2-adrenergic receptor resensitization. Differential regulation of receptor resensitization in two distinct cell types.

G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether beta2-adrenergic receptor (beta2AR) resensitization was dependent upon beta-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the beta-arrestin 1-V53D dominant negative inhibitor of beta2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both beta2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired beta2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta2AR resensitization was restored in these cells following the overexpression of beta-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that beta2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that beta-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. beta-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization.

Adenylyl Cyclases↗
PubMed1997

G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization.

G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.

1-Methyl-3-isobutylxanthine↗
PubMed1997

HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor.

Although the human hCCR-5 chemokine receptor can serve as a co-receptor for both M-tropic (ADA and BaL) and dual-tropic (89.6) strains of human immunodeficiency virus type 1 (HIV-1), the closely related mouse mCCR-5 homolog is inactive. We used chimeric hCCR-5-mCCR-5 receptor molecules to examine the functional importance of the three extracellular domains of hCCR-5 that differ in sequence from their mCCR-5 equivalents. While this analysis revealed that all three of these extracellular domains could participate in the functional interaction with HIV-1 envelope, clear differences were observed when different HIV-1 strains were analyzed. Thus, while the ADA HIV-1 isolate could effectively utilize chimeric human-mouse CCR-5 chimeras containing any single human extracellular domain, the BaL isolate required any two human extracellular sequences while the 89.6 isolate would only interact effectively with chimeras containing all three human extracellular sequences. Further analysis using hybrid HIV-1 envelope proteins showed that the difference in co-receptor specificity displayed by the ADA and BaL isolates was due partly to a single amino acid change in the V3 loop, although this interaction was clearly also modulated by other envelope domains. Overall, these data indicate that the interaction between HIV-1 envelope and CCR-5 is not only complex but also subject to marked, HIV-1 isolate-dependent variation.

Amino Acid Sequence↗
PubMed1997

The dopamine transporter: a crucial component regulating dopamine transmission.

The dopamine system is implicated in the control of locomotion, cognition, and endocrine function. The relative contribution of the various dopamine-related components is not well established mainly because drugs that target the dopaminergic system often lack selectivity. The in vivo gene inactivation procedure, or knockout, enables the creation of new strains of mice lacking a specific gene. This technique has been applied recently to inactivate the expression of the plasma membrane dopamine transporter. Here we summarize the main findings obtained with these transgenic mice carrying this "genetic defect," leading to a better understanding of the relative contribution of the dopamine transporter regarding locomotor activity, regulation of the expression of peptides under the control of dopaminergic activity, and responses to various drugs targeting the dopamine system. Our results establish not only the central importance of the transporter as the key element controlling dopamine levels in the brain, but also its role as an obligatory target for the behavioral and biochemical action of amphetamine and cocaine. In addition, the genetically altered mice offer a unique model to test the specificity and selectivity of dopamine transporter-acting drugs and may provide important new concepts related to the clinical and social implications of conditions such as Parkinson's disease, schizophrenia, and drug addiction.

Amphetamines↗
PubMed1997

Cocaine and amphetamine elicit differential effects in rats with a unilateral injection of dopamine transporter antisense oligodeoxynucleotides.

We have developed an antisense oligodeoxynucleotide to the dopamine transporter and used it to discriminate the behavioral properties of amphetamine and cocaine. In SK-N-MC cells permanently transfected with the dopamine transporter complementary DNA, treatment with 5 mM antisense oligodeoxynucleotide reduced dopamine uptake by 25% when compared to sense control. Unilateral intranigral administration of dopamine transporter antisense (50 microM) twice daily in freely moving rats for 2.5 days was sufficient to reduce dopamine transporter messenger RNA by 70% as measured by in situ hybridization, but not protein levels as measured by [3H]mazindol binding. However, intranigral treatment via implanted osmotic minipump over a period of seven days produced reductions in both dopamine transporter messenger RNA and protein levels (32%) at a dose of 500 pmol/day. These results indicate a longer half-life for the dopamine transporter than expected. Potassium chloride depolarization of ipsilateral striatal slices showed a greater than 200% increase in dopamine overflow on the antisense-treated side compared to the control side. Since imbalance of dopamine tone is known to induce rotational activity, we tested this behavioral paradigm in rats treated with various oligodeoxynucleotides at different doses and time-points. We have found that antisense-treated animals did not rotate spontaneously under any experimental conditions. Using various psychostimulants that target the dopamine transporter and increase dopamine levels, we found that the antisense-treated animals consistently rotated contralaterally in response to amphetamine (2 mg/kg), but not to cocaine (10 mg/kg) or nomifensine (10 mg/kg). These results bring in vivo evidence for a different mode of action of amphetamine and cocaine on the dopamine transporter and lend direct support to the view that amphetamine acts as a dopamine releaser, whereas cocaine acts by blocking dopamine transport.

Amphetamine↗