The overall goal of this project is to isolate and characterize the receptors specific for endogenous opioid peptides, particularly beta- endorphin and dynorphinA, and to compare them to the conventional opioid receptor classes, mu, delta, and kappa. The approaches we will use included (1) purification, sequencing and cloning of these receptors; (2) preparation of monoclonal and polyclonal antibodies to the receptors, and their use in determine structure and function of the receptors; (3) determination of the second messenger systems associated with these receptors; and (4) determining the role of these receptors in opioid tolerance and dependence. Receptors will be purified by cross-linking beta-endorphin and dynorphin to brain membranes in vitro, and isolating the cross-linked products by HPLC and other fractionation methods. In preliminary studies, we have identified three protein species cross-lined to 125I-beta-endorphin, and developed an HPLC procedure capable of purifying them. Cloning of the proteins identified by cross-linking will be achieved by classical procedures, including (a) N-terminal microsequencing; (b) synthesis of oligonucleotide probes corresponding to these sequences; (c) use of these probes to identify opioid peptide binding proteins present in a rat brain cDNA library; (d) transfection of the cDNA into bacteria, using a phage vector; and (e) screening of he transfected bacterial colonies using the oligonucleotide probes. Monoclonal and polyclonal antibodies to the purified proteins will be raised by standard procedures, and screened by their ability to immunoprecipitate the purified proteins, or to inhibit opioid peptide binding to the purified proteins and to brain membranes. The antibodies will be used to map the distribution of the opioid peptide binding proteins n brain and to determine their functional domains. They will also be used to construct immunoaffinity columns for rapid, large-scale purification of these binding proteins. Opioid receptors in the periaqueductal gray (PAG), which our previous work has indicated are of the mu type, will be purified by immunoaffinity columns, and will be characterized by comparison with opioid peptide binding proteins. We have also shown that these receptors are associated with a GTP-binding protein, and may be coupled with adenylate cyclase. We will further characterize this association, as well as determine whether other second messenger systems, such as phosphatidylinositol PI turnover, are involved in the action of these receptors. Finally, we will determine the role of opioid peptide binding proteins in opioid tolerance and dependence. We will determine the ability of antibodies to these proteins to block the development of opioid tolerance at several levels of the nervous system, including the PAG and spinal cord; and isolate beta-endorphin and dynorphin receptors from tolerant and withdrawn animals, and study their binding characteristics and associated second messenger systems.
