The human delta opioid receptor activates G(i1)alpha more efficiently than G(o1)alpha.
To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-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)alpha than for G(o1)alpha. 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)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha.
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
H E
HE
Moon
A
A
Cavalli
D S
DS
Bahia
M
M
Hoffmann
D
D
Massotte
G
G
Milligan
D000262:Adenylyl Cyclases / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D004174:Diprenorphine / Q000493:pharmacokinetics; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D016133:Polymerase Chain Reaction; D017465:Receptors, Opioid, delta / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004174:Diprenorphine; D005576:Colforsin; D009271:Naltrexone; D016308:Enkephalin, Leucine-2-Alanine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases; C055382:naltrindole