ABSTRACT
Human m-opioid receptor (H μ OR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 ± 0.7 and 6.52 ± 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by m-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by δ nor by κ selective agonist. Na+ (100 m M) and GTP (50 μ M) could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed Hμ OR. m-selective agonists DAGO and Ohm effectively stimulated [35S]GTP γS binding (EC50 = 2.7 n M and 6.9 n M) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 n M and 0.3 n M). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.
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Acknowledgements
This project was supported by the National Basic Research Program (G1999054002) of China, and the National Natural Science Foundation of China (No. 39630350). We express our sincere thanks to Dr. Jia Bei WANG (School of Medicine, Johns Hopkins University, Baltimore, MD) for providing the gene encoding the HμOR.
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WEI, Q., ZHOU, D., SHEN, Q. et al. Human μ-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins. Cell Res 10, 93–102 (2000). https://doi.org/10.1038/sj.cr.7290039
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DOI: https://doi.org/10.1038/sj.cr.7290039
