EXPRESSION OF HUMAN M2 MUSCARINIC RECEPTORS IN SF9 CELLS - CHARACTERIZATION AND RECONSTITUTION WITH G-PROTEINS

The gene for the human m2 muscarinic receptor was expressed in Sf9 cel ls using the baculovirus expression system. As assessed by [H-3]NMS bi nding, Sf9 cells expressed receptor at levels of 3.3 pmoles/mg protein . The receptor was identified on western blots using an anti-muscarini c receptor antibody and was shown to have the pharmacological characte ristics of an m2 muscarinic receptor. Membranes from Sf9 cells were ex amined to identify endogenous G-proteins by immune-blotting and by ADP -ribosylation, indicating the presence of Gq, and a pertussis-toxin su bstrate which was not recognized by antibodies raised against the alph a-subunits of Gi1, Gi2, Gi3 or Go. Gsa was not detected, neither were there any cholera toxin substrates in Sf9 membranes. Sf9 membranes exp ressing m2 receptors did not show carbachol-stimulated GTP gamma S bin ding to endogenous G-proteins; however, when membranes were reconstitu ted with a mixture of purified Gi and Go, a maximum 8-fold stimulation of GTP gamma S binding was observed in response to carbachol that cou ld be reduced by atropine. These data show that the human muscarinic m 2 receptor expressed in Sf9 cells is functional.