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

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding 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 of [H-3]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric G(i,o)-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified G(alpha)-subunits re-associated with beta gamma-dimers, we showed that alpha o1, alpha o2, alpha i1, alpha i2 and alpha i3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling 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 specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.