This is an application for a senior scientist award (K05). The applicant plans to continue his long standing research with the long term goal of elucidating the molecular basis of the acute and chronic effects of opiates. To this end we are studying the endogenous opioid system and the focus of our research is on the structure, function, and regulation of the different types of opioid receptors. An understanding of this endogenous neuropeptide/receptor system should ultimately have important implications for the treatment and prevention of drug abuse and improved treatment of pain.
The first aim i nvolves structural studies of the active mu opioid receptor protein (ORP) we have purified. Posttranslational modifications of the purified mu receptor, such as glycosylation, phosphorylation and disulfide bridges, will be studied by the powerful technique of mass spectrometry, in collaboration with Dr. Ron Beavis, a leading expert in this technology. This approach will also be useful for the exploration of other modifications such as palmitoylation of C- terminal cysteines and alkylation of cysteines by sulfhydryl reagents. The protein, present in earlier preparations, that gave rise to peptides not found in the sequence of the bovine mu opioid receptor we have recently cloned, nor in any database, will be purified and characterized, since it may be an important, novel regulator of mu receptor function. The role of cysteine residues in receptor function will be studied by site-directed mutagenesis and affinity labeling. The focus will be on residues involved in ligand binding and those in the C-terminal that may be palmitoylated. The role of fatty acylation will be examined by comparing the functions of non-palmitoylated mutants with those of wild type receptors and by studying the effects of agonists and antagonists and other receptor regulators on the dynamic palmitoylation rate of receptors. The Substituted Cysteine Accessibility Method (SCAM) will be employed to probe the hydrophilic access crevice of the opioid receptor binding site. Briefly, amino acids in or near the binding site will be replaced by cysteines. Inactivation of ligand binding by hydrophilic SH reagents is taken as evidence that the cysteine faces the hydrophilic access cleft.
The second aim proposed probing the interaction of opioid receptors with cytoskeletal elements and the existence of proteins that link receptors to the cytoskeleton. Such an interaction may be essential for correct receptor location and orientation and even for the regulation of receptor functions. In the final aim, aspects of signal transduction will be studied, including identification of receptor regions involved in G protein activation.
| Koob, George F; Simon, Eric J (2009) The Neurobiology of Addiction: Where We Have Been and Where We Are Going. J Drug Issues 39:115-132 |
| Onoprishvili, Irma; Andria, Matthew L; Kramer, Hal K et al. (2003) Interaction between the mu opioid receptor and filamin A is involved in receptor regulation and trafficking. Mol Pharmacol 64:1092-100 |
| Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L et al. (2002) Delta opioid activation of the mitogen-activated protein kinase cascade does not require transphosphorylation of receptor tyrosine kinases. BMC Pharmacol 2:5 |
