The complex group of opioid receptors is biologically and clinically important in mediating the action of endogenous and exogenous opioids. Despite the clinical advantages of developing drugs which serve as specific agonists and antagonists for opioid receptor subtypes, studies to date have been limited by the inability to satisfactorily isolate and characterize these multiple opioid receptors. This project is aimed at better defining the biochemical and molecular basis of opioid receptor system and actions. The opioid receptor system consists of at least three parts: an opioid ligand binding receptor protein, a guanine nucleotide regulatory protein (G-protein) that regulates the receptor affinity and serves as a signal transducer, and an effector protein that produces cellular biochemical responses such as inhibition of adenylate cyclase, ion channel regulation or neurotransmitter release. The biochemical structure of opioid receptor proteins remain unsolved. The main obstacle in deducing the amino acid sequences for opioid receptors seems to lie in the difficulty in obtaining purified receptor in sufficient quantity for characterization and verification. Previous efforts often ended up with receptor preparations that were denatured or in such minute quantity that a primary sequence study was impossible. During the past granting period the importance of sodium ion on opioid receptor binding was re-examined and this effect was utilized to design chromatography procedures to purify of opioid receptor from rat brain to homogeneity. The purified material appears as a single diffused band of 62 kD on SDS gel, most likely a glycoprotein. It shows stereospecific and saturable binding for opioids, has an average specific binding activity of 18.8+/-2.3 pmol/mu g protein, and is predominantly of the mu-type. Most importantly, the receptor preparation is sensitive to guanine nucleotide regulation of its agonist binding affinity when reconstituted with purified G proteins in phospholipid vesicles. This proposal is to purify enough quantity of purified receptor for partial amino acid sequence determination and cloning of opioid receptor genes. Information generated from this proposal will aid in the elucidation of the molecular structure of the opioid receptor system and the mechanism of opioid actions.
