High-Level Expression of G Protein-Coupled Receptors with the Aid of the Semliki Forest Virus Expression System

Abstract

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.