title: Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.
abstract: The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, National Center of Competence in Research TransCure, University of Bern, CH 3012 Bern, Switzerland.
authors.forename: Mark
authors.initials: M
authors.lastname: Bauer
authors.forename: Andrea
authors.initials: A
authors.lastname: Chicca
authors.forename: Marco
authors.initials: M
authors.lastname: Tamborrini
authors.forename: David
authors.initials: D
authors.lastname: Eisen
authors.forename: Raissa
authors.initials: R
authors.lastname: Lerner
authors.forename: Beat
authors.initials: B
authors.lastname: Lutz
authors.forename: Oliver
authors.initials: O
authors.lastname: Poetz
authors.forename: Gerd
authors.initials: G
authors.lastname: Pluschke
authors.forename: Jürg
authors.initials: J
authors.lastname: Gertsch
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D063385:Cannabinoid Receptor Modulators / Q000097:blood / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D006224:Cricetinae; D003511:Cyclohexanols / Q000378:metabolism; D018604:Epitope Mapping; D005260:Female; D018922:HL-60 Cells; D006454:Hemoglobins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008814:Mice, Inbred NZB; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000097:blood / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D034421:Sus scrofa; D053719:Tandem Mass Spectrometry
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D063385:Cannabinoid Receptor Modulators; D003511:Cyclohexanols; D006454:Hemoglobins; D010446:Peptide Fragments; C000590773:RVD-hemopressin, human; D043884:Receptor, Cannabinoid, CB1; C473218:hemopressin; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol