High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.
Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.
Walker
P
P
Division of Hypertension, University Hospital, Lausanne, Switzerland.
Munoz
M
M
Combe
M C
MC
Grouzmann
E
E
Herzog
H
H
Selbie
L
L
Shine
J
J
Brunner
H R
HR
Waeber
B
B
Wittek
R
R
D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D017476:Receptors, Neuropeptide Y / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014616:Vaccinia virus / Q000502:physiology*
D004274:DNA, Recombinant; D004279:DNA, Viral; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; D004247:DNA; D013937:Thymidine Kinase