Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest virus system.
The 1.3 S biotinylatable subunit of Proprionibacterium shermanii transcarboxylase complex was fused to the C-terminus of the human neurokinin 1 receptor gene and introduced into the Semliki Forest virus expression vector pSFV1. RNA transcribed from pSFV1-NK1-biot and pSFV-Helper2 was coelectroporated into BHK cells permitting in vivo packaging of recombinant virus. Infection of BHK and CHO cells with SFV-NK1-biot virus yielded high level of the fusion receptor as detected by metabolic labeling, immunoblotting with streptavidin alkaline phosphatase and binding to substance P. Like native receptor, the biotinylated receptor fusion was able to stimulate Ca2+ mobilization in infected CHO cells, indicating functional coupling to guanine-nucleotide-binding proteins.
Lundstrom
K
K
Department of Molecular Biology, Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Vargas
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A
Allet
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B
D000818:Animals; D001710:Biotin; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D064729:Neurokinin-1 Receptor Antagonists; D010957:Plasmids; D011424:Propionibacterium / Q000201:enzymology; D011869:Radioligand Assay; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012672:Semliki forest virus; D014158:Transcription, Genetic; D014162:Transfection; D014166:Transferases / Q000096:biosynthesis
D046911:Macromolecular Substances; D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D011993:Recombinant Fusion Proteins; D001710:Biotin; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D002118:Calcium