To determine μ-opioid receptor (OP3) signalling activity, guanosine 5ʹ-[γ-[35S]thio]triphosphate (GTP[35S]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with μ-opioid receptor (HEK-μ). GTP[35S] binding to HEK-μ membranes was significantly elevated compared with HEK-293 control membranes (without OP3), and this was abolished by pertussis-toxin pretreatment. The irreversible antagonist β-chlornaltrexamine (β-CNA) dose-dependently decreased elevated basal G-protein coupling of HEK-μ to control levels in cells devoid of OP3. This characterizes β-CNA as an inverse OP3 agonist. Immunoprecipitation of solubilized G-proteins with Gi3α antisera demonstrated that basal GTP[35S] binding to Gi3α was also substantially elevated in HEK-μ membranes over the control, whereas Gi3α protein levels were unchanged. Basal GTP[35S] binding to Gi1α/Gi2α and Goα was also increased twofold in HEK-μ membranes over the control. Morphine further increased coupling to each of these Gα proteins with similar potency, but not to Gq/11α or Gsα. These results indicate that the wild-type OP3 can couple constitutively to endogenously expressed Gi3α, Gi1α/Gi2α and Goα subunits of G-proteins in HEK-293 cells.

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