Functional role of the spatial proximity of Asp114(2.50) in TMH2 and Asn332(7.49) in TMH7 of the mu opioid receptor

We examined whether a proposed spatial proximity between Asp114(2.50) and A sn332(7.49) affected the functional properties of the mu opioid receptor. T he D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO an d CTAP, but not for naloxone and [H-3]diprenorphine; this mutation also abo lished agonist-induced increase in [S-35]GTP gamma S binding. The N332(7.49 )D mutation eliminated detectable binding of either [H-3]diprenorphine or [ H-3]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affi nity binding for [H-3]diprenorphine, CTAP and naloxone, and restored partia lly the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the resid ues are adjacent in space and that their chemical functionalities are impor tant for ligand binding and receptor activation. (C) 1999 Federation of Eur opean Biochemical Societies.