Site-Directed Mutagenesis Study

Structural elements of the rat 1.t-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 ~ 8, and K receptors (Asn 15°t Ala, His297 to Ala, and Tyr326 to Phe) and wo de igned to test for ~i/8 selectivity (lie198 to Val and Va1202 to lie). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-iabeied c-A1a2,NMePhe4,Giy-o15-enkephalin), [3H]bremazocine, or [3H]ethyiketocyciazocine. Mutation of Asn15° in TM3 produces a threeto 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, ~3-endorphin 1_31, JOM-13, deltorphin Ii, dynorphin1_13, and U50,488, with no change in the binding of antagonists such as naloxone, naitrexone, naitrindole, and nor-binaltorphamine. in contrast, the Tyr 326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of ~u, 8, and K agonists and antagonists. Altering Va1202 to lie in TM4 produced no changeon hgand affinity, but lie198 to Val resulted in a fourto fivefold decreased affinity for the ~i agonists morphine and DAMGO, with no change in the binding affinities of K and 8 igands.