CHARACTERIZATION OF THE HUMAN Y1 NEUROPEPTIDE-Y RECEPTOR EXPRESSED ININSECT CELLS

We have expressed the human Y1 NPY receptor in insect cells using a re combinant 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 fo r NPY (K-d = 0.38 +/- 0.8 nM) was identical to the natural receptor. W e used a foreign epitope to characterize, immunopurify, and localize t he recombinant protein. Cross-linking experiments identified a 65 kDa band as the major NPY binding species. Confocal microscopy indicated t hat although some recombinant proteins are detectable as early as 12 h post-infection, significant expression at the cell surface is only se en 24-48 h post-infection. We also describe a procedure to treat infec ted 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.