High-level expression of G protein-coupled receptors with the aid of the Semliki Forest virus expression system.
The expression of two G protein-coupled receptors was studied in several cell lines using the Semliki Forest virus expression system. Human neurokinin-2 and dopamine D3 receptor cDNAs were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper RNA into BHK cells resulting in in vivo packaging of high titer SFV-NK-2 and SFV-D3 virus stocks, respectively. Infection of BHK, HOS and CHO cells with the recombinant NK-2 virus resulted in high levels of receptor expression as detected by metabolic labelling with [35S]-methionine. The expression of the NK-2 receptors in the cell membrane was demonstrated by Flow cytometry experiments on infected BHK and CHO cells with fluoresceinyl-NKA as the ligand. Saturation binding assays on membranes prepared from SFV-NK-2 infected CHO cells with [3H] GR100679 showed maximum receptor densities of 6.5 pmol receptor/mg protein. Additionally, the expressed NK-2 receptors were able to stimulate Ca2+ mobilization in CHO cells indicating functional coupling to G proteins. CHO cells infected with SFV-D3 also produced high levels of receptor as evidenced by both [35S]methionine labelling and [3H]spiperone binding.
Lundstrom
K
K
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Mills
A
A
Allet
E
E
Ceszkowski
K
K
Agudo
G
G
Chollet
A
A
Liljestrom
P
P
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018492:Dopamine Antagonists / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D007763:Lac Operon; D009842:Oligopeptides / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism; D050637:Receptors, Dopamine D3; D018041:Receptors, Neurokinin-2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics; D013134:Spiperone / Q000378:metabolism
C493593:DRD3 protein, human; D018492:Dopamine Antagonists; D009842:Oligopeptides; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D018041:Receptors, Neurokinin-2; C083380:GR 100679; D013134:Spiperone; D019204:GTP-Binding Proteins; D002118:Calcium