Kappa opioid receptors (KORs) mediate many effects of opioid drugs, including analgesia, dysphoria and water diuresis. Kappa agonists may be useful as analgesics, water diuretics and antipruritic agents. Repeated or continuous administration of kappa agonists leads to tolerance, part of which 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. The following are the hypotheses for proposed studies. Following chronic exposure to an agonist, the human KOR undergoes desensitization, sequestration and down-regulation. Desensitization is due to agonist- induced phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs) followed by binding of beta-arrestin, leading to the uncoupling of the receptor from G proteins. Binding of beta-arrestin to phosphorylated receptors initiates receptor sequestration into cells via clathrin-coated vesicles. Some internalized receptors are degraded resulting in down-regulation. Certain amino acid residues or sequence motifs in the receptor play important roles in these. Kappa agonist treatment regulates levels of certain regulators of G protein signaling (RGS) proteins, which are negative regulators of the KOR signaling.
Specific aims are as follows. (1) To determine whether there are differences between the human and rat KORs in agonist-induced desensitization, sequestration, down-regulation and phosphorylation. (2) To determine the roles of GRKs, protein kinase A and protein kinase C in agonist-induced desensitization and phosphorylation of the human KOR by expression of GRKs, beta-arrestin GRK mutants and activation and inhibition of protein kinases A and C. (3) To determine the amino acid residue(s) of the human KOR that are phosphorylated upon desensitization by site-directed mutagenesis studies. (4) To investigate molecular mechanisms of agonist-induced sequestration of the kappa receptor by expression of beta-arrestin and dynamin mutants and to determine the motif important for sequestration by site-directed mutagenesis studies. (5) To determine the amino acid residue(s) of the kappa receptor that are important in down-regulation by site-directed mutagenesis studies. (6) To determine whether RGS proteins regulate signal transduction of the KOR. Elucidation of the mechanisms involved in agonist-induced regulation of the KOR will lead to a better understanding of tolerance to kappa agonist, which will help in the development of better kappa agonists as useful drugs.
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