Ligand-selective activation of mu-oid receptor: demonstrated with deletion and single amino acid mutations of third intracellular loop domain.

The mechanism for the differential regulation of the mu-opioid receptor by agonists is investigated by identifying the receptor domains used to define the relative efficacies of three mu-opioid receptor-selective agonists: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and [N-MePhe3,D-Pro4]-morphiceptin (PL017) to inhibit forskolin-stimulated intracellular cAMP production in human embryonic kidney 293 cells. This was accomplished by systematically deleting four to five amino acids clusters within the third intracellular loop of rat mu-opioid receptor, Arg258 to Arg280, followed by Ala substitution and scanning studies of the 276RRITR280 sequence, the putative G protein-coupling motif. Deletion of the four to five amino acid clusters resulted in differential effects on the affinities of the agonists and antagonists, and also on the potencies and coupling efficiencies of the three opioid agonists. Ala scanning studies of the 276RRITR280 sequence revealed also the differences between [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and PL017. Substitution of Arg276 or Ile278 with Ala reduced the potency of DAMGO but not that of morphine PL017. Meanwhile, mutation of Thr279 to Ala increased the potencies of morphine and PL017 but not that of DAMGO. The I278A mutation decreased the DAMGO coupling efficiency but increased the PL017 coupling efficiency. The R280A mutation resulted in the increase in PL017 potency and coupling efficiency without altering those of DAMGO and morphine. Thus, these mutation studies suggested that the activation of mu-opioid receptor and interaction between the critical domains such as RRITR within third intracellular loop and the G proteins are agonist-selective.