Expression of human M2 muscarinic receptors in Sf9 cells: characterisation and reconstitution with G-proteins.
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 alpha-subunits of Gi1, Gi2, Gi3 or Go. Gsalpha was not detected, neither were there any cholera toxin substrates in Sf9 membranes. Sf9 membranes expressing m2 receptors did not show carbachol-stimulated GTPgammaS binding to endogenous G-proteins; however, when membranes were reconstituted with a mixture of purified Gi and Go, a maximum 8-fold stimulation of GTPgammaS 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.
Heitz
F
F
Marion Merrell Dow Research Institute, Strasbourg, France.
McClue
S J
SJ
Harris
B A
BA
Guenet
C
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011976:Receptors, Muscarinic / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins