Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Bin ding of [H-3]diprenorphine to yeast spheroplasts was specific and saturable (K-d = 1 nM, B-max = 0.2-1 pmol.mg(-1) of membrane proteins). Inhibition o f [H-3]diprenorphine binding by antagonists and agonists with varying opioi d selectivities (mu, delta and kappa) occurred with the same order of poten cy as in mammalian tissues. Affinities of antagonists were the same with ye ast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of he terotrimeric G(i,o)-proteins purified from bovine brain shifted the mu-opio id receptor into a high-affinity state for agonists. Using individually pur ified G(alpha)-subunits re-associated with beta gamma-dimers, we showed tha t alpha o1, alpha o2, alpha i1, alpha i2 and alpha i3 reconstituted high-af finity agonist binding with equal efficiency. This suggests that the struct ural determinants of the mu-opioid receptor responsible for G-protein coupl ing are not able to confer a high degree of specificity towards any member of the G(i,o) family. The selective effects of opioid observed in specializ ed tissues upon opioid stimulation may be a result of regulation of G-prote in activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.