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 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 x 10(9) 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 x 10(6) 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.
Lundström
K
K
Department of Molecular Biology, Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Mills
A
A
Buell
G
G
Allet
E
E
Adami
N
N
Liljeström
P
P
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D018040:Receptors, Neurokinin-1 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins; D012672:Semliki forest virus / Q000235:genetics*; D013373:Substance P / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P