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.
Moon
H E
HE
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Cavalli
A
A
Bahia
D S
DS
Hoffmann
M
M
Massotte
D
D
Milligan
G
G
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