An important goal towards understanding how opioid drugs interact with their receptors is to discover the relationship between the primary sequence of the opioid receptor proteins and the receptor type-selectivity exhibited by the opioid drugs. The proposed research addresses the programmatic interest of NIDA in the neuroscience of opioids. Opioid receptors come in three types; mu, delta and kappa. Most clinically-used opioid analgesics, as well as abused opioids, mediate their effects by acting at mu opioid receptors (MOR). The proposed project is a competitive continuing application for an AREA grant that seeks to 1) obtain a novel dataset of vertebrate MOR cDNA clones, 2) express and characterize these novel opioid receptors in transfected cell lines, 3) use bioinformatic methods to identify cDNA sequences and individual amino acids that may confer mu opioid selectivity, and 4) validate identified amino acids by creating novel mutant MOR cDNA to characterize in transfected cell cultures.
These aims will be carried be using a PCR-based strategy to obtain novel opioid receptor-like cDNA from mRNA isolated from non-mammalian vertebrate brain tissue, by using state-of- the-art molecular pharmacology methods to transfect CHO cells with novel opioid receptor-like cDNA and characterize their type-selectivity using radioligand binding techniques, by applying methods of comparative bioinformatics analyses of opioid receptor types and individual amino acids, and by using methods to carry-out site-directed mutagenesis on novel MOR proteins. The expected results will provide testable hypotheses of the molecular evolution of vertebrate MOR receptors with regard to mu opioid selectivity correlated to primary amino acid sequence. This information is crucial for a complete understanding of how opioid drugs bind to their target receptors. This will address public health initiatives as the expected results will provide novel insights for the design of new opioid drugs for the clinic and for the treatment of opioid dependence. This project seeks to understand how opioid analgesic drugs, like morphine, are selective for their molecular targets in the brain, called opioid receptor proteins. As all proteins are made up of many amino acids strung together, understanding which amino acids are needed for morphine and other opioid analgesic drugs to bind to them is crucial for the design of better analgesics to treat pain. ? ? ?
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