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 [S-35]- 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 [H-3] 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 [S-35]methionine labelling and [H-3]spiperone binding.