Opiate and opioid compounds act on opioid receptors to produce pharmacological effects, most notably analgesia and euphoria. Multiple opioid receptors have been demonstrated and mu, delta and kappa opioid receptors have been cloned. The applicant's long-term objectives are to understand the structure-function relationships of these receptors at the molecular level. The goals of the proposed research are to determine the molecular basis of ligand binding selectivity and to understand conformational changes related to the activation of the mu opioid receptor. This goal will be accomplished by comparing residues exposed in the binding-site crevices by the substituted cysteine accessibility method among the mu, delta, and kappa opioid receptors and between wildtype and constitutively active mutants of the mu receptor.
The specific aims are as follows: (1) Assess whether ligand binding to mu, delta, and kappa opioid receptors is sensitive to treatment with sulfhydryl-specific methanesulonate (MTS) reagents and, if so, determine the cysteine residues within the TMHs that confer the sensitivity and construct an MTS-insensitive mutant for each receptor; (2) Determine and compare the residues in the TMHs 6 and 7 (which play important roles in ligand binding selectivity) that are accessible in the binding-site crevices of mu, delta, and kappa opioid receptors; (3) Generate and characterize constitutively active mutants of the mu opioid receptor, determine their sensitivity to the MTS reagents and, if necessary, construct MTS-insensitive mutants; (4) Determine and compare the residues in the TMHs 3,5, and 6 (which are important in receptor activation) accessible in the binding-site crevices of the wildtype and constitutively active mutants of the mu opioid receptor in order to identify conformational changes related to receptor activation. All data obtained will be interpreted in the context of current molecular models of GPCRs. Dr. Harel Weinstein of Mt. Sinai School of Medicine has agreed to collaborate on this aspect. With the integration of experimental data and model simulation, the proposed studies should yield information on the structure of the opioid receptors, structural basis of ligand binding selectivity and conformational changes related to receptor activation. In the absence of high-resolution three-dimensional crystal structure, the proposed approach represents an excellent method to obtain such precise information. The research will also determine whether distantly related receptors such as the opioid receptors and the D2 dopamine receptor, share similar transmembrane structures. These studies will advance our understanding of structures of not only opioid receptors, but also GPCRs in general. The information will provide insights into the structures of the binding-site crevices of opioid receptors, which will aid in the design of selective agonists and antagonists for prevention and treatment of drug abuse.
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