Molecular characterization of the human neuropeptide Y Y2-receptor

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.