Differential activation of G-proteins by μ-opioid receptor agonists

1 We investigated the ability of the activated mu-opioid receptor (MOR) to differentiate between myristoylated G(alpha i1) and G(alpha oA) type G(alpha) proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G(alpha) protein. 2 Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The G(alpha) subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G(alpha) protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[S-35] GTP(gamma)S exchange was then compared for G(alpha i1) and G(alpha oA). 3 Activation of MOR by DAMGO produced a high-affinity saturable interaction for G(alpha oA) (K-m = 20 +/- 1 nM) but a low-affinity interaction with G(alpha i1) (K-m = 116 +/- 12 nM). DAMGO, metenkephalin and leucine-enkephalin displayed maximal G(alpha) activation among the agonists evaluated. Endomorphins 1 and 2, methadone and beta-endorphin activated both G(alpha) to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. 4 Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between G(alpha i1) and G(alpha oA). Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G(alpha). Differences in maximal activity and potency, for G(alpha i1) versus G(alpha oA), are both indicative of agonist selective activation of G-proteins in response to MOR activation. 5 These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects.