Key Residues Defining the μ‐Opioid Receptor Binding Pocket: A Site‐Directed Mutagenesis Study

• Published in: Journal of neurochemistry 1997-01, Vol.68 (1), p.344-353
• Main Authors: Mansour, Alfred, Taylor, Larry P., Fine, Jeffrey L., Thompson, Robert C., Hoversten, Mary T., Mosberg, Henry I., Watson, Stanley J., Akil, Huda
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Biological and medical sciences; Cell receptors; Cell structures and functions; COS Cells; Drug Residues - metabolism; Endorphins; Enkephalin, Ala-MePhe-Gly; Enkephalins - metabolism; Ethylketocyclazocine - metabolism; Fundamental and applied biological sciences. Psychology; G protein‐coupled receptors; Immunohistochemistry; Ligands; Models, Molecular; Molecular and cellular biology; Morphine; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Neuropeptide receptors; Opiate; Opioid receptor; Rats; Receptors, Opioid, mu - genetics; Receptors, Opioid, mu - metabolism
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.1997.68010344.x

: Structural elements of the rat μ‐opioid receptor important in ligand receptor binding and selectivity were examined using a site‐directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for μ/δ selectivity (Ile198 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H‐labeled d‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three‐ to 20‐fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β‐endorphin1–31, JOM‐13, deltorphin II, dynorphin1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor‐binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile198 to Val resulted in a four‐ to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.