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Key Residues Defining the μ‐Opioid Receptor Binding Pocket: A Site‐Directed Mutagenesis Study
Author(s) -
Mansour Alfred,
Taylor Larry P.,
Fine Jeffrey L.,
Thompson Robert C.,
Hoversten Mary T.,
Mosberg Henry I.,
Watson Stanley J.,
Akil Huda
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68010344.x
Subject(s) - damgo , naltrindole , chemistry , opioid receptor , stereochemistry , enkephalin , binding site , receptor , mutagenesis , ligand (biochemistry) , site directed mutagenesis , (+) naloxone , biochemistry , opioid , mutation , mutant , gene
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 (Asn 150 to Ala, His 297 to Ala, and Tyr 326 to Phe) and two designed to test for μ/δ selectivity (Ile 198 to Val and Val 202 to Ile). Mutation of His 297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [ 3 H]DAMGO ( 3 H‐labeled d ‐Ala 2 , N ‐Me‐Phe 4 ,Gly‐ol 5 ‐enkephalin), [ 3 H]bremazocine, or [ 3 H]ethylketocyclazocine. Mutation of Asn 150 in TM3 produces a three‐ to 20‐fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β‐endorphin 1–31 , JOM‐13, deltorphin II, dynorphin 1–13 , and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor‐binaltorphamine. In contrast, the Tyr 326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val 202 to Ile in TM4 produced no change on ligand affinity, but Ile 198 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.