Opioid receptors, like adrenergic catecholamine receptors to which they are closely related, are fundamentally regulated in the CNS by phosphorylation and endocytosis. These mechanisms underlie physiological homeostasis of the endogenous opioid system, and can distinguish the effects of clinically relevant non-peptide drugs such as morphine. We are working to understand how chemically distinct opioid ligands produce different regulatory effects. Our efforts are directed both at molecular mechanism and physiological consequence. Work carried out during the previous funding period elaborated a chemical analytical approach, based on rapid receptor purification and quantitative mass spectrometry, to resolve discrete phosphorylated receptor forms produced in intact cells. Using this approach, we identified agonist-selective effects on phosphorylation of both adrenergic and opioid receptors. We also defined a particular phosphorylated form of the mu opioid receptor that discriminates the endocytic activity of morphine from that of opioid peptide. To further address mechanism, we implemented an unbiased screening strategy for discovering novel endocytic/recycling regulators in the human kinome. To more precisely investigate physiological consequence, we collaboratively developed two mouse models for measuring and manipulating opioid receptor phosphorylation and endocytosis in an acutely prepared brain slice preparation. Preliminary studies using these models suggest a specific requirement for GRK2, a receptor kinase known to modulate opioid receptor endocytosis in cultured cell models, in mediating a sustained component of opioid desensitization that is elicited by chronic morphine administration in vivo. The proposed studies seek to: (1) Resolve and quantify agonist-selective phosphorylation of opioid receptors in intact cells using analytical mass spectrometry;(2) Identify novel kinase(s) that regulate endocytosis of opioid and adrenergic receptors by unbiased RNAi screening;(3) Determine effects of defined phosphorylations and kinases on receptor endocytosis, surface insertion and signaling in HEK293 cells and cultured neurons;and (4) Assess functional consequences of defined phosphorylations and kinases on acute and chronic morphine regulation in an intact brain slice preparation. The proposed studies address the cell biological basis of addictive drug action, contribute more generally to understanding the nature of partial agonism and functional selectivity among drugs, and may identify new targets useful for pharmacotherapy of opiate tolerance or dependence.
Addictive opiate drugs such as morphine and heroin activate the same cellular receptors as endogenous opioid neuropeptides, yet produce pathological tolerance and dependence after prolonged or repeated administration. We seek to understand the underlying cell biology and biochemistry of these drug effects, and to thereby identify new therapeutic targets for treating opiate tolerance and addictive disorders.
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|Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark (2015) G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes. J Biol Chem 290:6689-96|
|Koliwer, Judith; Park, Minjong; Bauch, Carola et al. (2015) The golgi-associated PDZ domain protein PIST/GOPC stabilizes the Î²1-adrenergic receptor in intracellular compartments after internalization. J Biol Chem 290:6120-9|
|Leaf, Alison; Von Zastrow, Mark (2015) Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia. Elife 4:|
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