The human δ opioid receptor activates G i1 α more efficiently than G o1 α

To assess the relative capacity of the human δ opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the α‐subunits of either G i1 or G o1 , containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in‐frame with the C‐terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [ 3 H]naltrindole with high affinity. d ‐ala 2 , d ‐leu 5 Enkephalin effectively inhibited forskolin‐stimulated adenylyl cyclase activity in intact cells in a concentration‐dependent, but pertussis toxin‐insensitive, manner. The high‐affinity GTPase activity of both constructs was also stimulated by d ‐ala 2 , d ‐leu 5 enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to d ‐ala 2 , d ‐leu 5 enkephalin was more than three times greater for G i1 α than for G o1 α. As the effect of agonist in both cases was to increase V max without increasing the observed K m for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G i1 α compared with G o1 α. An equivalent fusion protein between the human µ opioid receptor‐1 and G i1 α produced a similar d ‐ala 2 , d ‐leu 5 enkephalin‐induced GTP turnover number as the δ opioid receptor−G i1 α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G i1 α.