High‐level Expression of the Human Neurokinin‐1 Receptor in Mammalian Cell Lines using the Semliki Forest Virus Expression System

Abstract

Human neurokinin-1 receptor cDNA was introduced into the pSFV1 Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electroporated into BHK cells with pSFV-Helper RNA. This procedure resulted in the packaging of a high-titer SFV-NK-1 virus stock containing approximately 5×10⁹ infective units/ml. Infection of baby hamster kidney, COS-7, Chinese hamster ovary and human osteosarcoma cells yielded high levels of human neurokinin-1 receptor expression as assessed by [³H]substance P binding. The maximal receptor expression level obtained was 4×10⁶ receptors/cell and studies of the post-infection time indicated that a high level of receptor expression was observed 10–24 h post-infection. The human neurokinin-1 receptor expressed in infected baby hamster kidney, COS-7 and Chinese hamster ovary cells was able to stimulate Ca²⁺ mobilization indicating functional coupling to guanine-nucleotide-binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of large quantities of receptor protein for both pharmacological and structural studies.