Dopamine (DA) storage and release, as well as DA D2 receptor binding potential, are altered in the striatum of patients with schizophrenia, and D2R antagonism remains the unifying property of all current antipsychotic drugs. Remarkably, the mechanisms by which D2R blockers exert these therapeutic actions are still unknown, both at the level of the receptor signaling unit, and with regard to the signaling events downstream of the receptor. Thus, with regard to the signaling unit, D2R has been shown to homomerize and to heterodimerize with multiple G protein-coupled receptor (GPCR) partners. Although the stability and physiological importance of such interactions are not yet clear, the prospect of developing drugs that target defined GPCR heteromers for increased regional and functional specificity has engendered much excitement. We propose single- molecule imaging, energy transfer, and computational studies to achieve a mechanistic understanding of these properties, with the goal being to determine the molecular composition of the receptor signaling unit that interacts with G proteins or arrestins, thus clarifying the role of oligomerization in GPCR functio and the potential for exploiting receptor heteromers as drug targets. With regard to the signaling events downstream of the receptor, it has become clear that compounds can act as both agonists and antagonists of different effectors downstream of the same receptor-a process known as functional selectivity or biased agonism- thereby providing hope for better tuning of therapeutic and side effects of drugs. Although bias can emerge from differences in G protein and arrestin activation, it can also occur between effectors thought to be downstream of G protein activation. We have obtained preliminary data using a novel G protein-based biosensor technology, which show that differences in the Gbeta subtype present in the G protein heterotrimer can lead to drug-dependent differences in G protein activation~ this biosensor technology will be used to probe systematically the roles of Galphabetagamma subtypes in determining biased agonism, both in cells and in brain slice preparations using a variety of ex vivo measures of endogenous D2R function. The achieved understanding of the molecular underpinnings of receptor interactions, crosstalk and functional selectivity will advance development of new and improved GPCR-targeted drugs with enhanced specificity and efficacy. Thus, we propose the following specific aims: 1. To determine the dynamics and signaling properties of dopamine D2R homo- and hetero-dimers in living cells and their modulation by membrane-dependent processes using single- molecule imaging, energy transfer studies, and computational simulations in membrane systems. 2. To uncover molecular mechanisms of functional selectivity at D2R by using a combination of biochemical and biophysical methods applied in heterologous cells and in brain slice preparations to probe the effects of specific drug action at defined G protein heteromers and arrestin, as well as at selected downstream effectors.
Dopamine D2 receptors are the targets of all antipsychotic drugs currently available, but we lack fundamental knowledge as to which specific signaling pathways in the brain must be blocked to achieve antipsychotic effects and which contribute to side effects. The molecular studies in cells and in brain tissue will lead to improved understanding of how these receptors function and how their function on different pathways can be selectively inhibited and activated, paving the way to improved medications.
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|Lambert, Nevin A; Javitch, Jonathan A (2014) Rebuttal from Nevin A. Lambert and Jonathan A. Javitch. J Physiol 592:2449|
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|Clayton, Cecilea C; Donthamsetti, Prashant; Lambert, Nevin A et al. (2014) Mutation of three residues in the third intracellular loop of the dopamine D2 receptor creates an internalization-defective receptor. J Biol Chem 289:33663-75|
|Rives, Marie-Laure; Javitch, Jonathan A (2013) Sensing conformational changes in metabotropic glutamate receptors. Proc Natl Acad Sci U S A 110:5742-3|
|Gavalas, Anthony; Lan, Tien-Hung; Liu, Qiuju et al. (2013) Segregation of family A G protein-coupled receptor protomers in the plasma membrane. Mol Pharmacol 84:346-52|
|Michino, Mayako; Donthamsetti, Prashant; Beuming, Thijs et al. (2013) A single glycine in extracellular loop 1 is the critical determinant for pharmacological specificity of dopamine D2 and D3 receptors. Mol Pharmacol 84:854-64|
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