HIGH-LEVEL EXPRESSION OF THE HUMAN NEUROKININ-1 RECEPTOR IN MAMMALIAN-CELL LINES USING THE SEMLIKI FOREST VIRUS EXPRESSION SYSTEM

Human neurokinin-1 receptor cDNA was introduced into the pSFV1 Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electr oporated into BHK cells with pSFV-Helper RNA. This procedure resulted in the packaging of a high-titer SFV-NK-1 virus stock containing appro ximately 5X10(9) infective units/ml. Infection of baby hamster kidney, COS-7, Chinese hamster ovary and human osteosarcoma cells yielded hig h levels of human neurokinin-l receptor expression as assessed by [H-3 ]substance P binding. The maximal receptor expression level obtained w as 4X10(6) receptors/cell and studies of the post-infection time indic ated that a high level of receptor expression was observed 10-24 h pos t-infection. The human neurokinin-1 receptor expressed in infected bab y hamster kidney, COS-7 and Chinese hamster ovary cells was able to st imulate Ca2+ mobilization indicating functional coupling to guanine-nu cleotide-binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of l arge quantities of receptor protein for both pharmacological and struc tural studies.