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Coupling Efficacy and Selectivity of the Human μ‐Opioid Receptor Expressed as Receptor—Gα Fusion Proteins in Escherichia coli
Author(s) -
Stanasila Laura,
Lim William K.,
Neubig Richard R.,
Pattus Franc
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0751190.x
Subject(s) - damgo , g protein , chemistry , μ opioid receptor , agonist , stereochemistry , receptor , g protein coupled receptor , opioid receptor , guanosine , gtp' , fusion protein , affinities , intrinsic activity , biochemistry , gene , recombinant dna , enzyme
Two constructs encoding the human μ‐opioid receptor (hMOR) fused at its C terminus to either one of two Gα subunits, Gα o1 (hMOR‐Gα o1 ) and Gα i2 (hMOR‐Gα i2 ), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4‐0.5 pmol/mg). Receptors fused to Gα o1 or to Gα i2 maintained high‐affinity binding of the antagonist diprenorphine. Affinities of the μ‐selective agonists morphine, [D‐Ala 2 , N ‐Me‐Phe 4 ,Gly 5 ‐ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5′‐ O ‐(3‐[ 35 S]thiotriphosphate) ([ 35 S]GTPγS) binding were assessed in the presence of added purified Gβγ subunits. Both fusion proteins displayed high‐affinity agonist binding and agonist‐stimulated [ 35 S]GTPγS binding. In the presence of Gβγ dimers, the affinities of DAMGO and endomorphin‐1 and ‐2 were higher at hMOR‐Gα i2 than at hMOR‐Gα o1 , whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [ 35 S]GTPγS binding at hMOR‐Gα o1 were similar, whereas at hMOR‐Gα i2 , endomorphin‐1 and morphine were more potent than DAMGO and endomorphin‐2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Gα o1 and Gα i2 and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.