Dawn Thompson

Altered Ratio of D1 and D2 Dopamine Receptors in Mouse Striatum Is Associated with Behavioral Sensitization to Cocaine

Background Drugs of abuse elevate brain dopamine levels, and, in vivo, chronic drug use is accompanied by a selective decrease in dopamine D2 receptor (D2R) availability in the brain. Such a decrease consequently alters the ratio of D1R∶D2R signaling towards the D1R. Despite a plethora of behavioral studies dedicated to the understanding of the role of dopamine in addiction, a molecular mechanism responsible for the downregulation of the D2R, in vivo, in response to chronic drug use has yet to be identified. Methods and Findings Ethics statement: All animal work was approved by the Gallo Center IACUC committee and was performed in our AAALAC approved facility. In this study, we used wild type (WT) and G protein coupled receptor associated sorting protein-1 (GASP-1) knock out (KO) mice to assess molecular changes that accompany cocaine sensitization. Here, we show that downregulation of D2Rs or upregulation of D1Rs is associated with a sensitized locomotor response to an acute injection of cocaine. Furthermore, we demonstrate that disruption of GASP-1, that targets D2Rs for degradation after endocytosis, prevents cocaine-induced downregulation of D2Rs. As a consequence, mice with a GASP-1 disruption show a reduction in the sensitized locomotor response to cocaine. Conclusions Together, our data suggests that changes in the ratio of the D1R∶D2R could contribute to cocaine-induced behavioral plasticity and demonstrates a role of GASP-1 in regulating both the levels of the D2R and cocaine sensitization.

Highly Potent 5-Aminotetrahydropyrazolopyridines: Enantioselective Dopamine D3 Receptor Binding, Functional Selectivity, and Analysis of Receptor−Ligand Interactions

Heterocyclic dopamine surrogates of types 5 and 7 were synthesized and investigated for their dopaminergic properties. The enantiomerically pure biphenylcarboxamide (S)-5a displayed an outstanding K(i) of 27 pM at the agonist-labeled D(3) receptor and significant selectivity over the D(2) subtype. Measurement of [(35)S]GTPγS incorporation in the presence of a coexpressed PTX-insensitive G(α0-1) subunit indicated highly efficient G-protein coupling. Comparison of ligand efficacy data from cAMP accumulation and [(3)H]thymidine incorporation experiments revealed that ligand biased signaling is exerted by the test compound (S)-5a. Starting from the D(3) crystal structure, a combination of homology modeling and site directed mutagenesis gave valuable insights into the binding mode and the intermolecular origins of stereospecific receptor recognition. According to these data, the superior affinity of the eutomer 5a is caused by the favorable binding energy that results from interaction between the ligands central ammonium unit and the aspartate residue in position 3.32 of the receptor.

Changes in G Protein-coupled Receptor Sorting Protein Affinity Regulate Postendocytic Targeting of G Protein-coupled Receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant β2 adrenergic receptor and a mutant μ opioid receptor that have previously been described as lacking “recycling signals” due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.

Differential Regulation of Behavioral Tolerance to WIN55,212-2 by GASP1

Cannabinoid agonists have shown some promise clinically as analgesics, in particular for cancer pain, in which they have the additional benefit of decreasing nausea. However, as for most other drugs, the long-term use of cannabinoids is limited by the development of tolerance. Several molecular mechanisms have been proposed to explain drug tolerance, including receptor downregulation. The cannabinoid 1 (CB1) receptors can be downregulated in vitro through an interaction with the G-protein-coupled receptor-associated sorting protein1, GASP1, that targets CB1 receptors for degradation after their agonist-mediated endocytosis. To investigate whether GASP1-mediated postendocytic sorting of the CB1 receptor contributes to tolerance to cannabinoid drugs in vivo, we generated a mouse with a disruption of GASP1. In wild-type mice, repeated administration of the cannabinoid agonist WIN55,212-2 promoted downregulation of CB1 receptor levels and concomitant tolerance to the effects of drug on antinociception, motor incoordination, and locomotor hypoactivity. In contrast, GASP1 knockout mice did not develop tolerance to any of these effects and showed no significant receptor downregulation. Taken together, this study provides evidence that GASP1 regulates CB1 receptor downregulation in vivo, and that postendocytic receptor trafficking has a key role in the development of tolerance to WIN55,212-2.

Development of a Bivalent Dopamine D2 Receptor Agonist

Bivalent D₂ agonists may function as useful molecular probes for the discovery of novel neurological therapeutics. On the basis of our recently developed bivalent dopamine D₂ receptor antagonists of type 1, the bivalent agonist 2 was synthesized when a spacer built from 22 atoms was employed. Compared to the monovalent control compound 6 containing a capped spacer, the bis-aminoindane derivative 2 revealed substantial steepening of the competition curve, indicating a bivalent binding mode. Dimer-specific Hill slopes were not a result of varying functional properties because both the dopaminergic 2 and the monovalent control agent 6 proved to be D₂ agonists substantially inhibiting cAMP accumulation and inducing D₂ receptor internalization. Investigation of the heterobivalent ligands 8 and 9, containing an agonist and a phenylpiperazine-based antagonist pharmacophore, revealed moderate steepening of the displacement curves and antagonist to very weak partial agonist properties.

The G-protein Coupled Receptor Associated Sorting Protein GASP-1 Regulates the Signalling and Trafficking of the Viral Chemokine Receptor US28

Human cytomegalovirus (HCMV) encodes the seven transmembrane (7TM)/G‐protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand‐independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR‐associated sorting protein‐1 (GASP‐1). We find that GASP‐1 binds to US28 in vitro and that disruption of the GASP‐1/US28 interaction by either (i) overexpression of dominant negative cGASP‐1 or by (ii) shRNA knock‐down of endogenous GASP‐1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post‐endocytic degradation. Furthermore, we found that GASP‐1 affects US28‐mediated signalling. The knock‐down of endogenous GASP‐1 impairs the US28‐mediated Gαq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor–κB (NF‐κB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP‐1 enhances both IP accumulation and transcription factor activity. Thus, GASP‐1 is an important cellular determinant that not only regulates the post‐endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1

The D3 dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D3 dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D3 and D2 that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D2 and D3 dopamine receptors. In addition, mutation of the D2 and D3 receptor C termini to resemble the D4, which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D4 to resemble that of the D2 and D3 facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D4 receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.

Trafficking properties of the D5 dopamine receptor.

Dopamine receptors are important for diverse biological functions and are important pharmacological targets in human medicine. Signal transduction from the dopamine receptors is controlled at many levels, including by the process of receptor trafficking. Little is known regarding the endocytic and postendocytic trafficking properties of the D5 dopamine receptor. Here, we show that endocytosis of the D5 receptor can be achieved both homologously, through direct receptor activation by agonist, and also heterologously, due to independent activation of protein kinase C (PKC). In contrast, the D1 receptor is endocytosed only in response to agonist but not PKC activation. We have identified the residue in the third intracellular loop of the D5 receptor that is both necessary for PKC‐mediated endocytosis of the D5 receptor and sufficient to induce PKC‐mediated endocytosis when introduced to the D1 receptor. In addition, we show that endocytosis of D5 through both pathways is dependent on clathrin and dynamin but that only agonist‐induced endocytosis engages β‐arrestin 2. Together, these data show that the D5 receptor shows a trafficking profile distinct from that of any of the other dopamine receptors.

The targeting of US28 is dependent on GASP-1

Background The human cytomegalovirus (HCMV) encodes the seven transmembrane (7 TM)/G protein-coupled receptor (GPCR) US28. US28 is able to endocytose and signal in a constitutive i.e. ligand-independent manner and is located predominantly in the membranes of intracellular organelles, especially late endosomes/lysosomes and multivesicular bodies (MVBs). It was suggested that the virions of HCMV may be budding into the membranes of these MVBs, where the viral receptors are then incorporated into the viral membranes during the final stages of virus assembly.