We propose to characterize structurally and functionally the mu type opioid receptor, which we have recently purified to homogeneity in our laboratory. Specifically, we will: (1) reconstitute the receptor into a membrane environment, and attempt to demonstrate restoration of both opioid binding and opioid-receptor mediated function; (2) raise monoclonal and polyclonal antibodies to the receptor, and use them to (a) map the receptor's distribution in the brain; (b) determine the role of different portions of the receptor on binding and function; (c) construct an immunoaffinity column for purifying large quantities of the receptor; and (d) clone the receptor (see also aim #4, below); (3) to run several critical tests of receptor models and to study the molecular mechanisms of opiate binding to its receptor, including (a) determining whether negative cooperativity occurs during opiate binding; (b) use thermodynamic analysis of opiate binding, to determine the molecular mechanism of agoinst and antagonist interactions; (c) determining the regulatory effects of ions, GTP, and lipids on opioid-receptor interactions; (d) determining whether the mu type opioid receptor can inter- convert to other types, such as delta or kappa; and (e) determining by physical methods whether receptor conformational changes occur during opiate binding; and (4) clone the opiate receptor, by synthesis of oligodeoxynucleotide probes corresponding to the N- terminal sequence of the receptor, use of these to isolate opioid receptor mRNA, and insertion of the corresponding DNA into a cloning vector. In addition, the availability of receptor antibody should provide an alternative means for receptor cloning (see specific aim #2, above). These studies will make it possible to clarify the role opioid receptors play in analgesia and other clinically important effects, and to determine the biochemical processes by which these effects are produced.
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