Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors

• Published in: The Journal of biological chemistry 2012-10, Vol.287 (44), p.36944-36967
• Main Authors: Bauer, Mark, Chicca, Andrea, Tamborrini, Marco, Eisen, David, Lerner, Raissa, Lutz, Beat, Poetz, Oliver, Pluschke, Gerd, Gertsch, Jürg
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Amino Acid Sequence; Animals; Antagonist; Antibodies, Monoclonal, Murine-Derived - biosynthesis; Antibody; Binding, Competitive; Brain - metabolism; Cannabinoid Receptor Modulators - blood; Cannabinoid Receptor Modulators - chemical synthesis; Cannabinoid Receptor Modulators - immunology; Cannabinoid Receptor Modulators - metabolism; Cannabinoids; CB Cannabinoid Receptors; CB1; CHO Cells; Cricetinae; Cyclohexanols - metabolism; Endocannabinoid System; Epitope Mapping; Female; Hemoglobin; Hemoglobins - biosynthesis; Hemoglobins - chemical synthesis; Hemoglobins - chemistry; Hemoglobins - immunology; Hemoglobins - metabolism; HL-60 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NZB; Molecular Sequence Data; Monoclonal Antibodies; Negative Allosteric Modulators; Neurobiology; Pepcans; Peptide Endocannabinoids; Peptide Fragments - biosynthesis; Peptide Fragments - blood; Peptide Fragments - chemical synthesis; Peptide Fragments - immunology; Peptide Fragments - metabolism; Peptides; Protein Binding; Protein Transport; Rats; Receptor, Cannabinoid, CB1 - agonists; Receptor, Cannabinoid, CB1 - antagonists & inhibitors; Receptor, Cannabinoid, CB1 - metabolism; Signal Transduction; Sus scrofa; Tandem Mass Spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.382481

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). 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 CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [3H]CP55,940 and [3H]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 [3H]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 CB1 receptors. Competition binding studies revealed Ki 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, [35S]GTPγS binding, and CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling. Background: The α-hemoglobin-derived peptide RVDPVNFKLLSH was found to interact with cannabinoid CB1 receptors. Results: We generated mAbs against RVDPVNFKLLSH and identified a new family of endogenous peptides (Pepcans) that act as negative allosteric modulators at CB1 receptors. Conclusion: Allosteric ligands for CB1 receptors are present in the brain. Significance: Pepcans are a novel class of endogenous modulators of endocannabinoid signaling.