Expression of functional human muscarinic M2 receptors in different insect cell lines.
Human M2 receptors were expressed using the baculovirus expression system in three different insect cell lines: Sf9, Sf21 and High5. The level of expression was slightly increased in Sf21 cells versus Sf9 cells. In contrast, High5 cells were not able to produce more recombinant protein than Sf9. We also show that in both Spodoptera frugiperda cell lines a peak of expression was reached after 6 days of infection, whereas in High5 cells, the maximum of expression occurred after 3 days. Immunodetection of m2 muscarinic receptor clearly shows that the expressed protein undergoes significant proteolysis in both the Sf9 and High5 cells, whereas in the Sf21 cells this phenomenon was less detectable. Additionally, we show that in all three cell lines, the expressed recombinant receptor was functional in that it was able to stimulate GTP gamma S binding in the presence of exogenous G-proteins. Analysis of the population of G-proteins (G alpha i, G alpha o and G beta common) in Sf21 and High5 cells is provided.
Heitz
F
F
Marion Merrell Research Institute, Strasbourg, France.
Nay
C
C
Guenet
C
C
D000818:Animals; D016367:Baculoviridae; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000032:analysis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007915:Lepidoptera; D010957:Plasmids / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism*; D018411:Spodoptera
D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D019204:GTP-Binding Proteins