In FY2009, we investigated the regulatory effects of GRK2 on D2 dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Over-expression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas siRNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D2 receptor as a phosphorylation-null receptor mutant was regulated similarly and over-expression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2, and several of its mutants, were able to co-immunoprecipitate with the D2 receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that over-expression of GRK2 attenuated the functional coupling of the D2 receptor and that this activity required the kinase activity of GRK2, but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγbinding activity of GRK2, but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates GPCR signaling in a manner independent of receptor phosphorylation. In FY2009, we continued working on our drug discovery project for allosteric ligands of the D2 receptor. G protein-coupled receptors (GPCRs) represent the largest family of therapeutic drug targets and account for the mechanisms of action of >60% of all FDA-approved drugs. Receptors for the neurotransmitter dopamine are members of this GPCR super-family and are involved in the etiology and/or therapy of a number of neuropsychiatric and endocrine disorders. In fact, amongst the dopamine receptors (DARs), the D2 subtype is arguably one of the most validated drug targets in neurology and psychiatry. For instance, all receptor-based antiparkinsonian drugs work via stimulating the D2 DAR whereas all FDA-approved antipsychotic agents are antagonists of this receptor. The D2 DAR is also therapeutically targeted in other disorders such as restless legs syndrome, tardive dyskinesia, Tourettes syndrome, and hyperprolactinemia. Most drugs targeting the D2 DAR are problematic, however, either being less efficacious as desired or possessing limiting side effects, most of which are due to cross-GPCR reactivity. One pharmacological approach towards improved target specificity is to identify allosteric ligands which bind to less conserved regions of receptors and therefore have the potential to be much more selective. Ligands that bind to such allosteric sites may promote conformation changes in the receptor that can produce positive or negative effects with respect to activation by the endogenous agonist, or in some cases can exhibit functional efficacy (agonist or inverse agonist) of their own. The goal of this project is to use high throughput screening (HTS) approaches to identify and develop novel small molecule allosteric modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. To this end, we propose to develop two assays capable of large-scale, high throughput screening of small molecule libraries. One assay involves the cellular co-expression of the D2 DAR with a chimeric Gq protein thus enabling the receptor to stimulate Ca2+ mobilization, which is detected through the activation of an intracellular fluorescent dye. The ssecond assay measures the ability of D2 DARs to promote the flux of thallium ion through G protein-regulated inward rectifying potassium (GIRK) channels, again measured through the activation of an intracellular dye. These assays will be configured into HTS formats and evaluated through the generation/calculation of Z parameters. The superior assay will be submitted for consideration by the Molecular Libraries Screening Centers Network (MLSCN) program for interrogation of the NIH Molecular Libraries small molecule repository. Secondary and counter-screening assays for other DAR subtypes will also be developed and implemented as necessary to confirm and validate MLSCN-generated hits. If needed, limited medicinal chemistry efforts will be performed to enhance the potency or efficacy, or the bioavailability of the most promising hit compounds. Future studies will be directed at evaluating the therapeutic potential of D2 DAR allosteric modulators using proof-of-concept efficacy tests in animal models of Parkinsons disease and schizophrenia. In FY2009, we also initiated a second drug discovery project related to improving therapeutics targeting the D2 receptor. A novel approach for attaining greater selectivity of therapeutic action is to identify and develop ligands that exhibit functionally selective properties. The phenomenon of functional selectivity, also termed biased agonism, protean agonism, agonist-directed trafficking, or collateral efficacy, is a relatively new concept in pharmacology and can occur when a receptor is able to transduce signals through more than one intracellular pathway. In this case, most agonists, in particular the endogenous transmitter will activate all signaling pathways in parallel with equal efficacy. However, it is now recognized, that some synthetic agonists may preferentially activate one pathway over another. While the mechanisms underlying functionally selective phenomena are not known with certainty, one hypothesis is that receptors can adopt multiple functionally active conformational states that are either stabilized or induced by selective ligands. In this scenario, some ligand-specific active conformations will selectively engage different G proteins or other signaling transducers such as β-arrestin. This may allow for the development of novel ligands that differentially activate (or block) a subset of signaling pathways for a single receptor, thus optimizing therapeutic action. The goal of this project is to use high throughput screening (HTS) approaches to identify and develop functionally-selective modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. We are particularly interested in developing probes that are functionally selective for the D2-DAR-stimulated β-arrestin signaling pathway, as previously all ligands for this receptor have been functionally characterized using G protein-mediated signaling. To this end, we will implement a high-fidelity bioluminescence resonance energy transfer (BRET)-based interaction assay for measuring agonist-induced recruitment of β-arrestin-2 to the D2 DAR. The assay will be configured into HTS format and submitted to the MLPCN program for interrogation of the NIH Molecular Libraries small molecule repository. Counter-screening assays will be developed and implemented to confirm and validate MLPCN-generated hits and to establish functionally selective characteristics of lead compounds. Such lead compounds should selectively stimulate, or block, D2 DAR/β-arrestin-mediated signaling in vivo, thus allowing for the of the role of this pathway in dopamine-regulated behaviors. Future studies will also evaluate the therapeutic potential of these functionally selective ligands via efficacy tests in animal models Parkinsons disease and schizophrenia.
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