title: The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.
abstract: Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.
authors.affiliation: Division of Preclinical Research, Boehringer Ingelheim Pharma KG, Biberach, Germany.
authors.forename: Heike A
authors.initials: HA
authors.lastname: Wieland
authors.forename: Richard M
authors.initials: RM
authors.lastname: Söll
authors.forename: Henri N
authors.initials: HN
authors.lastname: Doods
authors.forename: Dirk
authors.initials: D
authors.lastname: Stenkamp
authors.forename: Rudolf
authors.initials: R
authors.lastname: Hurnaus
authors.forename: Bärbel
authors.initials: B
authors.lastname: Lämmle
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001069:Appetite Regulation / Q000502:physiology*; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007231:Infant, Newborn; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism; D064446:Orexin Receptors; D000068797:Orexins; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins