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M G Caron

Publications and source records attributed to M G Caron.

At least 19 recordsLinked to original sources
PubMed1998

Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs.

Genetic loss or pharmacological inhibition of monoamine oxidase A (MAOA) in mice leads to a large increase in whole-brain levels of serotonin (5-HT). Excess 5-HT in mouse neonates prevents the normal barrel-like clustering of thalamic axons in the somatosensory cortex. Projection fields of other neuron populations may develop abnormally. In the present study, we have analyzed the localization of 5-HT immunolabeling in the developing brain of MAOA knock-out mice. We show numerous atypical locations of 5-HT during embryonic and postnatal development. Catecholaminergic cells of the substantia nigra, ventral tegmental area, hypothalamus, and locus ceruleus display transient 5-HT immunoreactivity. Pharmacological treatments inhibiting specific monoamine plasma membrane transporters and genetic crosses with mice lacking the dopamine plasma membrane transporter show that the accumulation of 5-HT in these catecholaminergic cells is attributable to 5-HT uptake via the dopamine or the norepinephrine plasma membrane transporter. In the telencephalon, transient 5-HT immunolabeling is observed in neurons in the CA1 and CA3 fields of the hippocampus, the central amygdala, the indusium griseum, and the deep layers of the anterior cingulate and retrosplenial cortices. In the diencephalon, primary sensory nuclei, as well as the mediodorsal, centrolateral, oval paracentral, submedial, posterior, and lateral posterior thalamic nuclei, are transiently 5-HT immunolabeled. The cortical projections of these thalamic nuclei are also labeled. In the brainstem, neurons in the lateral superior olivary nucleus and the anteroventral cochlear nucleus are transiently 5-HT immunolabeled. None of these structures appear to express the monoamine biosynthetic enzyme L-aromatic amino acid decarboxylase. The administration of monoamine plasma membrane transporter inhibitors indicates that the 5-HT immunolabeling in these structures is attributable to an uptake of 5-HT by the 5-HT plasma membrane transporter. This points to neuron populations that form highly precise projection maps that could be affected by 5-HT during specific developmental stages.

Animals↗
PubMed1998

Control of myocardial contractile function by the level of beta-adrenergic receptor kinase 1 in gene-targeted mice.

We studied the effect of alterations in the level of myocardial beta-adrenergic receptor kinase betaARK1) in two types of genetically altered mice. The first group is heterozygous for betaARK1 gene ablation, betaARK1(+/-), and the second is not only heterozygous for betaARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a betaARK1 COOH-terminal inhibitor peptide, betaARK1(+/-)betaARKct. In contrast to the embryonic lethal phenotype of the homozygous betaARK1 knockout (Jaber, M., Koch, W. J., Rockman, H. A., Smith, B., Bond, R. A., Sulik, K., Ross, J., Jr., Lefkowitz, R. J., Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), betaARK1(+/-) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the betaARK1(+/-) and betaARK1(+/-)betaARKct mice with the greatest level observed in the betaARK1(+/-)betaARKct animals. Contractile parameters were measured in adult myocytes isolated from both groups of gene-targeted animals. A significantly greater increase in percent cell shortening and rate of cell shortening following isoproterenol stimulation was observed in the betaARK1(+/-) and betaARK1(+/-)betaARKct myocytes compared with wild-type cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of betaARK1 activity. This has important implications in disease states such as heart failure (in which betaARK1 activity is increased) and suggests that betaARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of betaARK1 activity enhances beta-adrenergic receptor signaling leading to improved functional catecholamine responsiveness.

Adrenergic beta-Agonists↗
PubMed1998

Role of dopamine transporter in methamphetamine-induced neurotoxicity: evidence from mice lacking the transporter.

The role of the dopamine transporter (DAT) in mediating the neurotoxic effects of methamphetamine (METH) was tested in mice lacking DAT. Dopamine (DA) and serotonin (5-HT) content, glial fibrillary acidic protein (GFAP) expression, and free radical formation were assessed as markers of METH neurotoxicity in the striatum and/or hippocampus of wild-type, heterozygote, and homozygote (DAT -/-) mice. Four injections of METH (15 mg/kg, s.c.), each given 2 hr apart, produced 80 and 30% decreases in striatal DA and 5-HT levels, respectively, in wild-type animals 2 d after administration. In addition, GFAP mRNA and protein expression levels, extracellular DA levels, and free radical formation were increased markedly. Hippocampal 5-HT content was decreased significantly as well (43%). Conversely, no significant changes were observed in total DA content, GFAP expression, extracellular DA levels, or free radical formation in the striatum of DAT -/- mice after METH administration. However, modest decreases were observed in striatal and hippocampal 5-HT levels (10 and 17%, respectively). These observations demonstrate that DAT is required for, and DA is an essential mediator of, METH-induced striatal dopaminergic neurotoxicity, whereas serotonergic deficits are only partially dependent on DAT.

Animals↗
PubMed1998

Role for G protein-coupled receptor kinase in agonist-specific regulation of mu-opioid receptor responsiveness.

The G protein-coupled mu-opioid receptor (mu OR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of mu OR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate mu OR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the mu OR. Although both etorphine and morphine effectively activate the mu OR, only etorphine elicits robust mu OR phosphorylation followed by plasma membrane translocation of beta-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause mu OR internalization, morphine is unable to either elicit mu OR phosphorylation or stimulate beta-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce mu OR phosphorylation, accompanied by the rescue of beta-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote mu OR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

Cell Line↗
PubMed1998

The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin.

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Animals↗
PubMed1998

Visualization of dynamic trafficking of a protein kinase C betaII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization.

Protein kinase C (PKC) links various extracellular signals to intracellular responses and is activated by diverse intracellular factors including diacylglycerol, Ca2+, and arachidonic acid. In this study, using a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII), we demonstrate a novel approach to study the dynamic redistribution of PKC in live cells in response to G protein-coupled receptor activation. Agonist-induced PKC translocation was rapid, transient, and selectively mediated by the activation of Gqalpha- but not Gsalpha- or Gialpha-coupled receptors. Interestingly, although the stimuli were continuously present, only one brief peak of PKC membrane translocation was observed, consistent with rapid desensitization of the signaling pathway. Moreover, when GFP-PKCbetaII was used to examine cross-talk between two Gqalpha-coupled receptors, angiotensin II type 1A receptor (AT1AR) and endothelin A receptor (ETAR), activation of ETARs resulted in a subsequent loss of AT1AR responsiveness, whereas stimulation of AT1ARs did not cause desensitization of the ETAR signaling. The development of GFP-PKCbetaII has allowed not only the real time visualization of the dynamic PKC trafficking in live cells in response to physiological stimuli but has also provided a direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.

Biological Transport↗
PubMed1998

Profound neuronal plasticity in response to inactivation of the dopamine transporter.

The dopamine transporter (DAT) plays an important role in calibrating the duration and intensity of dopamine neurotransmission in the central nervous system. We have used a strain of mice in which the gene for the DAT has been genetically deleted to identify the DAT's homeostatic role. We find that removal of the DAT dramatically prolongs the lifetime (300 times) of extracellular dopamine. Within the time frame of neurotransmission, no other processes besides diffusion can compensate for the lack of the DAT, and the absence of the DAT produces extensive adaptive changes to control dopamine neurotransmission. Despite the absence of a clearance mechanism, dopamine extracellular levels were only 5 times greater than control animals due to a 95% reduction in content and a 75% reduction in release. Paradoxically, dopamine synthesis rates are doubled despite a decrease of 90% in the levels of tyrosine hydroxylase and degradation is markedly enhanced. Thus, the DAT not only controls the duration of extracellular dopamine signals but also plays a critical role in regulating presynaptic dopamine homeostasis. It is interesting to consider that the switch to a dopamine-deficient, but functionally hyperactive, mode of neurotransmission observed in mice lacking the DAT may represent an extreme example of neuronal plasticity resulting from long-term psychostimulant abuse.

Animals↗
PubMed1998

A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor.

The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination. We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.

Cell Line↗
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

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↗
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↗