Molecular Characterization of the Human Neuropeptide Y Y2-Receptor

• Published in: Biochemistry (Easton) Vol. 38; no. 21; pp. 6897 - 6902
• Main Authors: Ingenhoven, Nikolaus, Eckard, Christophe P, Gehlert, Donald R, Beck-Sickinger, Annette G
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.05.1999
• Subjects: Amino Acid Sequence; Animals; Blotting, Western; CHO Cells; Cricetinae; Cross-Linking Reagents - metabolism; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y - chemical synthesis; Neuropeptide Y - metabolism; Photoaffinity Labels - metabolism; Photochemistry; Receptors, Neuropeptide Y - chemistry; Receptors, Neuropeptide Y - genetics; Receptors, Neuropeptide Y - metabolism; Transfection; Tumor Cells, Cultured
• DOI: 10.1021/bi982831b
• ISSN: 0006-2960

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), [N α-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [N α-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]NPY (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.