Cross-linking of neuropeptide Y to its receptor on rat brain membranes

The receptor for neuropeptide Y (NPY) was identified on rat brain membranes after covalent labeling with ¹²⁵I-NPY using the homobifunctional cross-linkers disuccinimido suberate and discuccinimido dithiobis(propionate) and the heterobifunctional photoactive cross-linker succinimido 4-azidobenzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of two bands at M(r) 62,000 and 39,000. Both species showed the same high affinity for ¹²⁵I-NPY. Exposure to reducing agents did not change the migration of these bands. When the NPY receptor complex was solubilized from the membranes with 1% Triton-X-100 and analyzed by gel filtration chromatography, it eluted from a Fractogel TSK 55F column as a peak at ~ 65 kDa. This peak was asymmetric with a shoulder of radioactivity that probably reflects the smaller receptor species. These data indicate that the NPY receptor on rat brain membranes is a monomeric 58-kDa unit (62 kDa minus the mass of the cross-linked NPY) without covalently or noncovalently linked subunits. The smaller 39-kDa species may be an immature form of the 62-kDa species, a second distinct receptor, or a degradation product of the 62-kDa band.