Expression of Human M2 Muscarinic Receptors in SF9 Cells: Characterisation and Reconstitution with G-Proteins

Abstract

The gene for the human m2 muscarinic receptor was expressed in Sf9 cells using the baculovirus expression system. As assessed by [³H]NMS binding, Sf9 cells expressed receptor at levels of 3.3 pmoles/mg protein. The receptor was identified on western blots using an anti-muscarinic receptor antibody and was shown to have the pharmacological characteristics of an m2 muscarinic receptor. Membranes from Sf9 cells were examined to identify endogenous G-proteins by immuno-blotting and by ADP-ribosylation, indicating the presence of Gq, and a pertussis-toxin substrate which was not recognised by antibodies raised against the α-subunits of Gi1, Gi2, Gi3 or Go. Gsα was not detected, neither were there any cholera toxin substrates in Sf9 membranes. Sf9 membranes expressing m2 receptors did not show carbachol-stimulated GTPγS binding to endogenous G-proteins; however, when membranes were reconstituted with a mixture of purified Gi and Go, a maximum 8-fold stimulation of GTPγS binding was observed in response to carbachol that could be reduced by atropine. These data show that the human muscarinic m2 receptor expressed in Sf9 cells is functional.