L. Stanasila et al., Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-G alpha fusion proteins in Escherichia coli, J NEUROCHEM, 75(3), 2000, pp. 1190-1199
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(o
1)) 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 morp
hine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins
as well as their potencies and intrinsic activities in stimulating guanosin
e 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 prote
ins displayed high-affinity agonist binding and agonist-stimulated [S-35]GT
P gamma S binding. In the presence of G beta gamma dimers, the affinities o
f DAMGO and endomorphin-1 and -2 were higher at hMOR-G alpha(i2) than at hM
OR-G alpha(o1), whereas morphine displayed similar affinities at the two ch
imeras. Potencies of the four agonists in stimulating [S-35]GTP gamma S bin
ding at hMOR-G alpha(o1) were similar, whereas at hMOR-G alpha(i2), endomor
phin-1 and morphine were more potent than DAMGO and endomorphin-2. The intr
insic activities of the four agonists at the two fusion constructs were sim
ilar. 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.