Opioid peptides are found in many different sites in the nervous and endocrine systems where they mediate diverse types of responses. All numbers of this large family of peptides (more than 15) have been shown to be derived from 3 different precursors known as pro-opiomelanocortin, proenkephalin and prodynorphin. The receptors that opioid peptides bind to are also diverse in nature. They can be classified into 3 predominant types based on the differences in opioids they bind. Each type of receptor and opioid peptide has a somewhat different distribution in the brain and may mediate different kinds of responses. The major goal of this research is to further our understanding of the mechanisms that regulate expression of opioid peptides and opioid receptors. This goal will be accomplished by a multilevel approach employing methods of molecular biology, immunology and protein chemistry. The sites of synthesis of opioid peptides will be mapped in the brain by in situ hybridization techniques using DNA probes specific for each opioid precursor. The regulatory sites in the opioid genes will be defined by gene transfer techniques. This will be done by studing the effects of specific alterations of the opioid genes (site-specific in vitro mutagenesis) on transcriptional activity and on processing of the precursors in two different types of recipient cells (mammalian cell lines and frog oocytes). The first step in the study of opioid receptors will be to determine the structure of one type of receptor by recombinant DNA techniques. A relatively new approach called the hybridization selection procedure will be used to purify the mRNA species that code for the receptor. The frog oocyte expression system will be used to assay the mRNA for its capacity to direct the synthesis of the receptor proteins. cDNA will be prepared from the purified mRNA and cloned. From the sequence of the cDNA it will be possible to determine the number of subunits in the receptor and the structure of each subunit. The next step will be to use various receptor cDNA probes to determine the number of opioid receptor genes and how similar these genes are to one another.
