Activation of (kappa) opioid receptors produces many effects, including analgesia, dysphoria and water diuresis and kappa agonists may be useful as analgesics, water diuretics and antipruritic drugs. Kappa opioid receptors are coupled through pertussis toxin-sensitive G proteins to affect a variety of effectors. Repeated or continuous administration of Kappa agonists leads to tolerance and dependence. Part of tolerance can be accounted for at the receptor level. The applicant's long term objectives are to understand at the molecular level the biochemical events that occur following chronic exposure to opioid drugs. After exposure to the selective agonist U50, 488H, the human kappa opioid receptor (hkor) undergoes internalization and the internalized receptors are recycled to plasma membranes or sorted to lysosomes and proteasomes for degradation. Whether there are signals directing the sorting of the internalized receptors is not well understood. The central hypothesis of this application is that the hkor is associated with EBP50/NHERF-1 and GABARAPL1 and undergoes ubiquitination, all of which play important roles in the regulation, trafficking and signaling of the receptor.
The specific aims are as follows. (1) To investigate the interaction of the hkor with EBP50/NHERF-1, a PDZ domain-containing protein. The residues in the hkor involved in the interaction with EBP50/NHERF-1 will be determined by mutagenesis studies. Effects of receptor phosphorylation and ubiquitination on its association with EBP50/NHERF-1 will be assessed and the role of EBP50/NHERF in the U50,488H-induced stimulation of Na+,H+-exchange will be examined. (2) To examine the interaction of the hkor with GABARAPL 1. Whether there are interactions between GABARAPL1 and tubulin, N-ethylmaleimide-sensitive factor, EBP50/NHERF-1, rab5 or rab7 will be investigated. The regions of GABARAPL 1 and the hkor involved in the interaction will be determined. The functional significance of the hkor-GABARAPL1 interaction will be studied in terms of trafficking, surface expression, ligand binding and signal transduction of the hkor. (3) To delineate the role of ubiquitination in the signaling and trafficking of the hkor. Effects of different agonists and receptor phosphorylation on ubiquitination of the hkor will be examined. The sites of ubiquitination in the hkor will be determined by mutagenesis. The impact of ubiquitination on the functional properties, regulation and trafficking of the hkor will be investigated. Elucidation of the functional significance of EBP50/NHERF-1 and GABARAPL 1 and receptor ubiquitination in the trafficking and signaling of the hkor will provide better understanding of cell biology of the hkor and will have implications for development of kappa agonists and antagonists as therapeutic agents. In addition, such understanding will have important ramifications for other G protein coupled receptors since these molecules may play similar roles for other receptors.
Showing the most recent 10 out of 58 publications
