PROBING OF THE NEUROPEPTIDE Y-Y-1-RECEPTORS INTERACTION WITH ANTIRECEPTOR ANTIBODIES

The Y-1 receptor, which belongs to the family of rhodopsin-like GTP-bi nding protein-coupled, seven-transmembrane helix-spanning receptors, b inds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar aff inity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y-1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cel ls as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as inves tigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Wester n blotting and staining with the antibodies revealed two proteins of 7 3 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is st rongly inhibited in the presence of the native ligand. Using photoacti vatible analogues, it could be demonstrated that the competition effic iency strongly depends on the position of the crosslinker within the l igand, Based on these studies, a model for the ligand-receptor interac tion is suggested. These antibodies represent novel tools for the stru ctural characterization of the Y-1 receptor and its interaction with N PY and antagonists as well as for localization studies.