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Biology of G Protein–Coupled Receptors and Neurotransmitter Transporters
Summary: Marc Caron is interested in the structure-function relationships and regulatory mechanisms of receptor and transporter proteins that mediate the effects of catecholamines, and their role in normal and abnormal physiology.
Our laboratory is interested in understanding the fundamental mechanisms underlying the actions of hormones and neurotransmitters. We focus on two biochemical processes: how cells, through specific receptor proteins called G protein–coupled receptors, receive and transduce signals in a dynamic and regulated way and how another class of proteins, called transporters, function in brain cells essentially to terminate the cellular communication process, or neurotransmission. In particular, we study how these proteins mediate the actions of the monoamines dopamine, norepinephrine, and serotonin. Aberrant actions of monoamines are postulated to underlie disorders such as schizophrenia, depression, Parkinson's disease, hypertension, and heart failure. Our goals are to determine in molecular terms the structure-function relationships and regulatory mechanisms of receptor and transporter proteins and to examine their role in normal and abnormal physiology.
Molecular Characterization and Regulation of Catecholamine Receptors
G protein–coupled receptors (GPCRs) mediate the physiological effects of a whole series of signaling molecules in the body, from the perception of light, odor, and taste to the effects of hormones and neurotransmitters such as the catecholamines. Binding of an agonist ligand (e.g., dopamine) to a site delineated by the seven transmembrane domains of the receptor activates a subset of G proteins, which in turn leads to the activation of a diverse set of intracellular effectors (e.g., enzymes) that modulate the biochemistry of the receptor-bearing cell. After these initial reactions, intracellular biochemical events rapidly dampen the cellular response, in a phenomenon referred to as desensitization. The responsiveness of an individual cell to an extracellular signal is governed by the delicate balance between the generation and desensitization of the response, as well as the ability of the cell to reestablish normal responsiveness.
Desensitization is a normal physiological process that can severely limit the actions of agonist drugs. For GPCRs, desensitization is mediated by the agonist-dependent phosphorylation of the receptor by a family (seven different members) of receptor-specific kinases (G protein–coupled receptor kinases, GRKs). This receptor phosphorylation then allows members of another family of proteins, the arrestins, to interact with the receptor and turn off its conventional G protein–mediated signaling function. Receptor phosphorylation and arrestin binding not only limit the signaling function of the receptor but also initiate the process by which normal responsiveness is reestablished. Thus, by interacting with components of the clathrin-coated vesicle endocytic machinery such as AP2 and clathrin, arrestins initiate internalization of the receptors to endosomes, where they can be dephosphorylated and recycled back to the plasma membrane as signaling-competent receptors.
The stability of the receptor-arrestin complex, which depends on the nature and extent of the phosphorylation sites on the intracellular domains of the receptor, determines the kinetic parameters of the resensitization process. Receptors that dissociate from arrestin at or near the plasma membrane are rapidly dephosphorylated and recycled, whereas receptors that remain associated with arrestin within endosomes are only slowly dephosphorylated and recycled. The physiological relevance of this is corroborated by the observation that some naturally occurring loss-of-function mutant GPCRs present aberrant recycling properties. A mutant vasopressin receptor (V2R) associated with nephrogenic diabetes insipidus is complexed with arrestin in endocytic vesicles, even in the absence of the hormone, and is thus unresponsive to the hormone. A similar "constitutively desensitized" cellular phenotype underlies the loss-of-function sphingosine-1-phosphate receptor mutant, which is associated with cardia bifida in the miles apart zebrafish. Therefore, inappropriate formation of GPCR-arrestin complexes, leading to a constitutive desensitization phenotype, may underlie the pathology of several GPCR-based conditions. (A grant from the National Institutes of Health provided partial support for these projects.)
Cellular studies have suggested that GRK-dependent phosphorylation of GPCRs and subsequent interaction with arrestin play an important role in regulating responses to neurotransmitters and hormones, but little is known about their overall roles in physiological processes. To address this issue we have developed, in collaboration with Robert Lefkowitz (HHMI, Duke University), a series of mouse lines in which the genes for the various GRKs and arrestins have been genetically inactivated. Using physiological and behavioral paradigms in isolated tissues and whole animals, we have shown that these GPCR regulatory mechanisms play an essential role not only in modulating the responsiveness of tissues to signaling molecules but also in determining their basal function. For example, functional deletion of the barrestin-2 gene in mice, which prevents μ-opiate receptor desensitization, results in a remarkable potentiation and prolongation of the analgesic effect of morphine. Absence of μ-opiate receptor desensitization in these mice leads to the demonstration that two hallmark properties of opiate action, tolerance and physical dependence, are dissociable phenomena. Mice lacking the barrestin-2 gene lose the ability to develop antinociception tolerance to chronic morphine but still develop physical dependence on the drug.
In another series of studies, we have demonstrated that functional inactivation of the gene encoding one of the GRKs, GRK6, leads to enhanced behavioral responses to various psychostimulants. This enhanced responsiveness correlates with an enhanced function of D2 subtypes of dopamine receptors. Inactivation of other members of the GRK family did not enhance the effect of psychostimulants. In an animal model of parkinsonism, in which neuronal dopamine is depleted pharmacologically, inactivation of GRK6 potentiates the effect of dopamine replacement therapy. Thus, interfering with the mechanisms of GPCR desensitization may ultimately result in beneficial therapeutic applications.
Neurotransmitter Transporters
Neurotransmitter transporters regulate the intensity and duration of synaptic transmission by removing transmitters from the synapse or repackaging neurotransmitters into synaptic vesicles. In this role they also function as the presumed site of action of drugs such as antidepressants, as well as drugs of abuse such as cocaine and amphetamine. The monoamine transporters are prototypic of the large Na+/Cl–-dependent transporter family that includes transporters for dopamine, norepinephrine, serotonin, proline, glycine, creatine, and betaine. The nonselective vesicular monoamine transporter (VMAT2) is responsible for the vesicular storage of all monoamines, including histamine, in the brain.
Biochemically, monoamine transporters function as oligomeric complexes. For example, for the dopamine transporter (DAT), oligomerization is important for the proper synthesis and trafficking of the protein to the plasma membrane. In addition, a series of transporter-interacting proteins have been identified that appear to regulate various functions of transporters, such as cellular trafficking and targeting to neuronal terminals.
To understand the physiological importance of neurotransmitter transporters and shed light on potentially novel roles of the monoamines, we generated lines of mice in which the dopamine and norepinephrine transporter genes as well as the gene for the VMAT2 are ablated. Mice carrying these gene deletions present several overt phenotypes that implicate these transporters as critical components controlling monoamine homeostasis in the brain. These mice have adapted to the absence of the monoamine transporters by undergoing profound down-regulation of the GPCRs that normally mediate the action of the monoamines, as well as marked changes in the synthesis, degradation, storage, and release of the neurotransmitters, indicating that the transport process functions as a sort of master switch controlling the action of the neurotransmitter. This profound neuronal plasticity of the monoamine systems translates into all three genetically modified mouse lines being sensitized (i.e., hyperresponsive) to the action of psychostimulants or direct dopamine receptor agonists. We have capitalized on this phenotype to use a genome-wide approach to identify common mechanisms that may underlie sensitization to psychostimulants. Our results indicate that changes in fast glutamatergic neurotransmission are most important in elaboration of this phenotype.
In mice lacking the dopamine transporter (DATKO), the typical hyperactivity phenotype, due to high levels of extracellular dopamine is sporadically replaced by severe motor dysfunction. This is accompanied by a loss of striatal GABAergic neurons and the appearance of markers of both overstimulation of dopamine receptors and neurodegeneration. These findings suggest that persistently elevated extracellular dopamine may contribute to the selective degeneration of striatal neurons.
These genetically altered mice are also being used as animal models to explore which neuronal pathways are involved in the elaboration of symptoms that recapitulate those of certain human disorders.
Last updated June 09, 2003
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