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

We investigated the ability of the activated μ ‐opioid receptor (MOR) to differentiate between myristoylated G α i1 and G α oA type G proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G protein. 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 subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G protein by CB 1 cannabinoid receptors. The ability of agonists to catalyse the MOR‐dependent GDP/[ 35 S]GTPS exchange was then compared for G α i1 and G α oA . Activation of MOR by DAMGO produced a high‐affinity saturable interaction for G α oA ( K m =20±1 n M ) but a low‐affinity interaction with G α i1 ( K m =116±12 n M ). DAMGO, met‐enkephalin and leucine‐enkephalin displayed maximal G activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β ‐endorphin activated both G to more than 75% of the maximal response, whereas fentanyl partially activated both G‐proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between G α i1 and G α oA . Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G. Differences in maximal activity and potency, for G α i1 versus G α oA , are both indicative of agonist selective activation of G‐proteins in response to MOR activation. 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.British Journal of Pharmacology (2006) 147 , 671–680. doi: 10.1038/sj.bjp.0706661