PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF NEUROPEPTIDE Y-2 RECEPTOR

Neuropeptide Y (NPY) receptors consist of three subtypes, designated N PY1, NPY2, and NPY3. The Y-1 receptor tor has been cloned. The present study reports the purification of the NPY-Y-2 receptor from porcine b rain and its biochemical characterization. NPY receptors were solubili zed and purified by sequential hydrophobic interaction, ion exchange, and NPY-affinity chromatography. By use of SDS-polyacrylamide gel elec trophoresis, high performance liquid chromatography gel permeation chr omatography, and chemical cross-linking studies, the affinity-purified brain NPY-Y-2 receptor was identified as a monomeric glycoprotein wit h a molecular mass of 60 kDa. Following deglycosylation, the molecular mass of the Y-2 receptor was decreased to 45 kDa. Although the I-125- NPY binding to the purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect. Therefore, the p urified NPY-Y-2 receptor is probably not associated with G-proteins, b ut may have intramolecular-free sulfhydryl groups. The specific activi ty of the isolated NPY-Y-2 receptor is 15.8 nmol/mg of protein. The is olated receptor retained its capacity to bind to I-125-NPY, specific t o NPY and peptide YY, and showed no cross-reactivity with any other pe ptides. Highly purified (10(9)-fold purification) NPY receptor from th e brain was identified as the Y-2 subtype as demonstrated by its affin ity to C-terminal fragments of NPY, including NPY (13-36).