ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
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 Gi1 or Go1, 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 [3H]naltrindole with high affinity. d-ala2,d-leu5 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-ala2,d-leu5 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-ala2,d-leu5 enkephalin was more than three times greater for Gi1α than for Go1α. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of Gi1α compared with Go1α. An equivalent fusion protein between the human µ opioid receptor-1 and Gi1α produced a similar d-ala2,d-leu5 enkephalin-induced GTP turnover number as the δ opioid receptor−Gi1α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1α.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1471-4159.2001.00196.x
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