G-protein coupling of mu-opioid receptors (OP3): elevated basal signalling activity.

To determine mu-opioid receptor (OP(3)) signalling activity, guanosine 5'-[gamma-[(35)S]thio]triphosphate (GTP[(35)S]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with mu-opioid receptor (HEK-mu). GTP[(35)S] binding to HEK-mu membranes was significantly elevated compared with HEK-293 control membranes (without OP(3)), and this was abolished by pertussis-toxin pretreatment. The irreversible antagonist beta-chlornaltrexamine (beta-CNA) dose-dependently decreased elevated basal G-protein coupling of HEK-mu to control levels in cells devoid of OP(3). This characterizes beta-CNA as an inverse OP(3) agonist. Immunoprecipitation of solubilized G-proteins with G(i3)alpha antisera demonstrated that basal GTP[(35)S] binding to G(i3)alpha was also substantially elevated in HEK-mu membranes over the control, whereas G(i3)alpha protein levels were unchanged. Basal GTP[(35)S] binding to G(i1)alpha/G(i2)alpha and G(o)alpha was also increased twofold in HEK-mu membranes over the control. Morphine further increased coupling to each of these Galpha proteins with similar potency, but not to G(q)/(11)alpha or G(s)alpha. These results indicate that the wild-type OP(3) can couple constitutively to endogenously expressed G(i3)alpha, G(i1)alpha/G(i2)alpha and G(o)alpha subunits of G-proteins in HEK-293 cells.