Coupling Efficacy and Selectivity of the Human m-Opioid Receptor Expressed as Receptor–Ga Fusion Proteins in Escherichia coli

Abstract: Two constructs encoding the human m-opioid receptor (hMOR) fused at its C terminus to either one of two Ga subunits, Gao1 (hMOR–Gao1) and Gai2 (hMOR– Gai2), were expressed in Escherichia coli at levels suit-able for pharmacological studies (0.4–0.5 pmol/mg). Re-ceptors fused to Gao1 or to Gai2 maintained high-affinity binding of the antagonist diprenorphine. Affinities of the m-selective agonists morphine, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 59-O-(3-[35S]thiotriphosphate) ([35S]GTPgS) binding were assessed in the presence of added purified Gbg subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [35S]GTPgS bind-ing. In the presence of Gbg dimers, the affinities o