Browsing by Subject "GPCR"

Now showing 1 - 3 of 3
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    Dynamic regulation of R7BP (R7 Binding Protein) containing R7 RGS (R7 Regulators of G protein Signaling) protein complexes: role in controlling neuronal dopamine and opioid signaling in the striatum.
    (2010-02) Anderson, Garret R.
    G protein-coupled receptor (GPCR) signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior and nociception in mammals. One member of the R7 RGS family, RGS9-2 has been previously implicated as an essential modulator of signaling through neuronal dopamine and opioid G protein coupled receptors. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with R7BP (R7 RGS Binding Protein), which we have found to ultimately affect several critical properties of RGS9-2. First, it is this interaction with R7BP which is necessary for determining the subcellular targeting of RGS9-2 to the plasma membrane and to the specialized neuronal compartment of excitatory synapses, the postsynaptic density. Secondly, R7BP plays a selective role amongst the R7 RGS family in determining the proteolytic stability of RGS9-2. Further characterization of R7 RGS complexes in the striatum revealed that two equally abundant R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit which is present in complex predominantly with RGS9-2 rather than with RGS7. However, upon changes in neuronal activity the subunit composition of these complexes in the striatum undergoes rapid and extensive remodeling. Changes in the neuronal excitability or oxygenation status result in extracellular calcium entry, uncoupling RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to cytoplasmic RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multi-subunit complexes. The physiological consequence of this remodeling process appears to play a role in determining the signaling sensitivity to dopamine stimulation. Considering that upon the genetic elimination of RGS9, all available R7BP is funneled towards complex formation with RGS7, not only are RGS9 controlled GPCR signaling pathways affected, but those controlled by RGS7 as well. RGS9 knockout mice have an increased sensitivity to dopamine and opioid receptor stimulation and consequently display altered motor and reward behavior. The question arises as to the role of modulation of RGS7 function in controlling these behaviors. Since the function of RGS9-2 is controlled by its association with R7BP, we would predict that the elimination of R7BP would lead to similar alterations in striatal physiology for RGS9 controlled pathways. While at the same time, RGS7 would be largely unaffected by the elimination of R7BP, thus RGS7 controlled pathways would predictably remain unaltered. Using this rationale, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration consistent with the essential role of RGS9 in controlling these behaviors and the critical role played by R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not exhibit higher sensitivity to locomotor-stimulating effects of cocaine, suggesting a role for RGS7 in controlling dopamine sensitivity. Using a striatum-specific knockdown approach, we demonstrate that the sensitivity of motor stimulation to cocaine is indeed dependent on RGS7 function. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.
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    Patterns of drug-related behavior: role of VTA DA neurons and inhibitory GPCR-dependent signaling
    (2022-04) DeBaker, Margot
    Ventral tegmental area (VTA) dopamine (DA) neurons play an important role in modulating activity in the mesocorticolimbic system in response to reward. Output from VTA DA neurons is critical for mediating drug-related behaviors, as inhibition of these neurons blocks drug induced stimulation. Drugs of abuse act on a diverse set of molecular targets, but they share the ability to enhance DA levels throughout the mesocorticolimbic system. Enhanced DA levels engage negative feedback mechanisms on VTA DA neurons by activating inhibitory G protein-dependent signaling pathways, mediated by GABAB receptors (GABABRs) and D2 DA receptors (D2Rs). GABABR- and D2R-dependent feedback in VTA DA neurons is further modulated by Regulator of G Protein Signaling (RGS) proteins, which act to enhance the deactivation of G protein signaling. The R7 family of RGS proteins, including RGS6, is known to modulate neuronal G protein signaling preferentially via Gao, and has been implicated in drug-related behaviors. One goal of the work in this thesis was to assess how changes in VTA DA neuron inhibition may alter drug-related behaviors.In order to assess the relative influence of GABABRs and D2Rs on drug sensitivity, we characterized a neuron-specific CRISPR/Cas9 approach to ablate GABABRs or D2Rs in VTA DA neurons. Our ablation technique prevented VTA DA neuron somatodendritic responses to the GABABR agonist baclofen or the D2R agonist quinpirole in a receptor specific manner. Loss of VTA DA neuron D2R-dependent signaling resulted in enhanced cocaine-induced motor stimulation in both male and female mice, whereas loss of VTA DA neuron GABABR-dependent signaling resulted in enhanced cocaine-induced motor stimulation only in males. Neither GABABR nor D2R ablation had an effect on morphine-induced motor stimulation. These data suggest that VTA DA neuron inhibitory G protein-dependent feedback modulates behaviors in a drug- and sex-specific way. We also wanted to evaluate the role of RGS6 in modulating VTA DA neuron inhibitory feedback and drug-related behaviors. We showed that RGS6 and GB5, the binding partner of R7 RGS proteins, are expressed in the majority of VTA DA neurons. Additionally, both the GABABR and D2R can signal through Gao, the preferred substrate of RGS6, suggesting that RGS6 may modulate both GABABR- and D2R-dependent signaling. Indeed, constitutive RGS6–/– mice exhibited enhanced amplitude of somatodendritic VTA DA neuron D2R-dependent signaling and prolonged deactivation of GABABR-dependent signaling. Next, we utilized the CRISPR/Cas9 approach characterized previously to assess VTA DA neuron specific effects of RGS6–/–. As with constitutive RGS6–/–, VTA DA neuron specific RGS6 ablation enhanced D2R-dependent current amplitude. Further, VTA DA neuron specific RGS6 overexpression reduced D2R-dependent current amplitude. These data suggest a negative regulatory role for RGS6 in VTA DA neuron G protein-dependent signaling. We also report that both male and female constitutive RGS6–/– mice display decreased binge alcohol consumption, but that only female VTA DA neuron specific RGS6–/– display decreased binge alcohol consumption. Together these results suggest that RGS6 modulates VTA DA neuron G protein-dependent inhibition in a receptor-dependent manner, resulting in a sex-dependent influence on alcohol consumption. Most humans using drugs of abuse have co-occurring substance use patterns, so in addition to assessing independent drug mechanisms it is also important to assess how drug-related behavior patterns change during drug co-consumption. As alcohol and nicotine are the most frequently co-used drugs of abuse, the second goal of this work was to assess how abstinence from either nicotine or alcohol, after weeks of concurrent consumption, affects intake of the remaining drug. We did this by utilizing a 3-bottle choice model of concurrent alcohol, nicotine, and water consumption. When we removed the nicotine bottle after 3 weeks of concurrent consumption, we did not observe a change in levels of alcohol consumed, suggesting that mice do not compensate for the absence of nicotine by increasing alcohol consumption. When we instead added the aversive tastant quinine to the alcohol bottle, we saw an acute decrease in alcohol consumption in addition to an increase in the levels of nicotine consumed, suggesting that mice compensate for the absence of alcohol by increasing nicotine consumption. Further, chronically increasing quinine concentration in the alcohol bottle resulted in enhanced nicotine compensation in females but not in males. These results have important implications for treating patients with substance co-use disorders, as they suggest the order of drug abstinence may affect overall treatment outcomes. Collectively, work presented in this thesis provides novel insights about how drug-related behavioral outcomes are affected by drug mechanism, underlying inhibitory architecture, sex of the subject, and drug availability. These insights highlight the importance of gaining a greater understanding of both individual and co-substance use disorders in order to inform patient-specific treatment strategies.
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    Quantification of cell surface Mu and Delta opioid receptor and elucidation of factors governing surface receptor pool.
    (2009-10) Hareid, Jonathan Alexander
    We developed a method of quantifying cell surface and total cell receptor in individual cells by separately labeling these populations of receptor with different fluorescent antibodies, acquiring images with fluorescence microscopy, and quantifying fluorescence for each cell using Image J software. Using this method, we studied the surface-to-total ratios for transfected mu opioid receptor (MOR) and delta opioid receptor (DOR) in PC12 cells and HEK cells. We found that the average surface-to-total ratio was higher for DOR than for MOR in both cell types, and regardless of NGF differentiation of the PC12 cells, indicating the existence of receptor-specific installation mechanisms for these receptors. For both receptors, we found little relationship between the quantities of cell surface and total cell receptor, but surface-to-total ratio decreases at high levels of total receptor, suggesting that the mechanisms of receptor installation are saturable. In NGF-differentiated PC12 cells, we found that either depolarization using potassium chloride or co-expression of the Substance P precursor pre-pro-tachykinin increased the surface-to-total ratio of DOR but not MOR, consistent with a highly specific role for the regulated secretory pathway in cell surface delivery of DOR. We found that the DOR agonist deltorphin could depress DOR surface-to-total ratio for significantly longer than would be expected based on normal trafficking kinetics, showing that agonist treatment can cause receptor down-regulation not only by direct internalization but also by producing a longer-lasting change in receptor membrane trafficking. Finally, we found that in PC12 cells co-expressing DOR and MOR, treatment with the DOR agonist deltorphin for 30 minutes caused a rapid, significant increase in cell surface MOR, but treatment with the MOR agonist DAMGO for the same time period did not cause an increase in cell surface DOR. These experiments point to a multitude of receptor-specific factors that determine the quantity of DOR and MOR on the cell surface.