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

To determine mu-opioid receptor (OP3) signalling activity, guanosine 5'-[gamma-[S-35]thio]triphosphate (GTP[S-35]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with mu-opioid receptor (HEK-mu). GTP[S-35] binding to HEK-mu membranes was significantly elevated compared with HEK-293 control membranes (without OP3), 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 OP3. This characterizes beta-CNA as an inverse OP3 agonist. Immunoprecipitation of solubilized G-proteins with G(13)alpha antisera demonstrated that basal GTP[S-35] binding to G(13)alpha was also substantially elevated in HEK-mu membranes over the control, whereas G(13)alpha protein levels were unchanged. Basal GTP[S-35] binding to G(11)alpha G(12)alpha and G(0)alpha was also increased twofold in HEK-mu membranes over the control. Morphine further increased coupling to each of these G alpha proteins with similar potency, but not to G(q)/(11)alpha or G(s)alpha. These results indicate that the wild-type OF, can couple constitutively to endogenously expressed G(13)alpha, G(11)alpha/G(12)alpha and G(0)alpha subunits of G-proteins in HEK-293 cells.