Coupling Efficacy and Selectivity of the Human μ‐Opioid Receptor Expressed as Receptor—Gα Fusion Proteins in Escherichia coli

• Published in: Journal of neurochemistry Vol. 75; no. 3; pp. 1190 - 1199
• Main Authors: Stanasila, Laura, Lim, William K., Neubig, Richard R., Pattus, Franc
• Format: Journal Article
• Language: English
• Published: Oxford UK Blackwell Science Ltd 01-09-2000; Blackwell
• Subjects: Biological and medical sciences; Cell receptors; Cell structures and functions; Escherichia coli; Fundamental and applied biological sciences. Psychology; G protein‐coupled receptors; Molecular and cellular biology; Neuropeptide receptors; Pharmacology; Receptor‐G protein fusion; μ‐Opioid receptor; Human; Signal transduction; Escherichia coli; μ Opioid receptor; Bacteria; Selectivity; Coupling; Enterobacteriaceae; Biological receptor; G protein
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2000.0751190.x

: Two constructs encoding the human μ‐opioid receptor
(hMOR) fused at its C terminus to either one of two Gα subunits,
Gαo1 (hMOR‐Gαo1) and Gαi2
(hMOR‐Gαi2), were expressed in Escherichia coli at
levels suitable for pharmacological studies (0.4‐0.5 pmol/mg). Receptors fused
to Gαo1 or to Gαi2 maintained high‐affinity
binding of the antagonist diprenorphine. Affinities of the μ‐selective
agonists morphine,
[D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin
(DAMGO), and endomorphins as well as their potencies and intrinsic activities
in stimulating guanosine
5′‐O‐(3‐[35S]thiotriphosphate)
([35S]GTPγS) binding were assessed in the presence of added
purified Gβγ subunits. Both fusion proteins displayed high‐affinity
agonist binding and agonist‐stimulated [35S]GTPγS binding. In
the presence of Gβγ dimers, the affinities of DAMGO and
endomorphin‐1 and ‐2 were higher at hMOR‐Gαi2 than at
hMOR‐Gαo1, whereas morphine displayed similar affinities at
the two chimeras. Potencies of the four agonists in stimulating
[35S]GTPγS binding at hMOR‐Gαo1 were
similar, whereas at hMOR‐Gαi2, 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 Gαo1 and Gαi2 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.