Coupling efficacy and selectivity of the human μ-opioid receptor expressed as receptor-Gα fusion proteins in Escherichia coli

Two constructs encoding the human mu-opioid receptor (hMOR) fused at its C terminus to either one of two G alpha subunits, G alpha(o1) (hMOR-G alpha(o1)) and G alpha(i2) (hMOR-G alpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg), Receptors fused to G alpha(o1) or to G alpha(i2) maintained high-affinity binding of the antagonist diprenorphine, Affinities of the mu-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[S-35]thiotriphosphate) ([S-35]GTP gamma S) binding were assessed in the presence of added purified G beta gamma subunits, Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [S-35]GTP gamma S binding. In the presence of G beta gamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-G alpha(i2) than at hMOR-G alpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [S-35]GTP gamma S binding at hMOR-G alpha(o1) were similar, whereas at hMOR-G alpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to G alpha(o1) and G alpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.