Characterization of the human Y1 neuropeptide Y receptor expressed in insect cells.
We have expressed the human Y1 NPY receptor in insect cells using a recombinant baculovirus (BacY1). Non-linear curve fitting of competition binding data indicates the presence of 500,000-750,000 saturable NPY binding sites per cell. The affinity of the recombinant Y1 receptor for NPY (Kd = 0.38 +/- 0.8 nM) was identical to the natural receptor. We used a foreign epitope to characterize, immunopurify, and localize the recombinant protein. Cross-linking experiments identified a 65 kDa band as the major NPY binding species. Confocal microscopy indicated that although some recombinant proteins are detectable as early as 12 h post-infection, significant expression at the cell surface is only seen 24-48 h post-infection. We also describe a procedure to treat infected Sf21 cells in such a way that they can be frozen and stored at -80 degrees C for many months before being used for binding studies.
Munoz
M
M
Division of Hypertension, CHUV, Lausanne, Switzerland.
Sautel
M
M
Martinez
R
R
Sheikh
S P
SP
Walker
P
P
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D005576:Colforsin / Q000494:pharmacology; D003432:Cross-Linking Reagents; D015925:Cryopreservation; D046508:Culture Techniques / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*
D003432:Cross-Linking Reagents; D000939:Epitopes; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins