Opiate and opioid compounds act on opioid receptors to produce pharmacological effects. Multiple opioid receptors have been demonstrated and mu, delta and kappa opioid receptors have been cloned. The applicant's long-term objectives are to understand the structure-function relationships of opioid receptors at the molecular level. Mu opioid receptors mediate analgesic effects of many opiates and opioids. Opiates (such as morphine and heroin) are among the most widely abused drugs, mainly because of their actions on the mu opioid receptor to produce euphoria. Development of tolerance and dependence to opiates is mediated largely by the mu receptor. In this application, the applicant proposes to examine palmitoylation of the cloned mu opioid receptor and the functions that his covalent modification may serve. Palmitoylation is an important post-translational modification of many G protein-coupled receptors. In palmitoylation studies conducted on other receptors, this covalent modification appears to play important roles in certain receptor functions, such as coupling G proteins, or agonist-induced receptor desensitization, internalization or downregulation. Our preliminary results showed that the mu receptor was palmitoylated.
Specific aims of the proposed study are as follows: (1) To determine whether the mu opioid receptor is palmitoylated by assessing the incorporation of [3H]palmitic acid into the mu receptor and to examine the effect of agonist exposure on palmitoylation and de-palmitoylation; (2) To determine the amino acid residue(s) that is (are) palmitoylated in the mu opioid receptor by site-directed mutagenesis studies; (3) To determine functional role(s) of palmitoylation in the mu opioid receptor by comparing wildtype and nonpalmitoylated mutant mu receptors in coupling to G proteins and agonist-induced receptor desensitization, phosphorylation, internalization and downregulation. It is important to understand the role(s) of this covalent modification in mu opioid functions since G protein-coupling is the first step in activating effectors and agonist-induced desensitization, internalization and downregulation are important regulatory mechanisms, which are parts of development of opioid tolerance. The proposed studies will enable us to understand the functional significance of palmitoylation of the mu receptor, which will help us better understand opioid actions. This information will be useful in the development of better drugs as well as in the prevention and treatment of opiate abuse.
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