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Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

ABSTRACT: The ?-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-Hp? 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 ? < 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]GTP?S 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.

SUBMITTER: Bauer M

PROVIDER: S-EPMC3481297 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

Bauer Mark M Chicca Andrea A Tamborrini Marco M Eisen David D Lerner Raissa R Lutz Beat B Poetz Oliver O Pluschke Gerd G Gertsch Jürg J

The Journal of biological chemistry 20120905 44

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 (SALSDL ...[more]

PMID: 22952224

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Project description:The N-methyl-D-aspartate (NMDA) receptor family regulates various central nervous system functions, such as synaptic plasticity. However, hypo- or hyperactivation of NMDA receptors is critically involved in many neurological and psychiatric conditions, such as pain, stroke, epilepsy, neurodegeneration, schizophrenia, and depression. Consequently, subtype-selective positive and negative modulators of NMDA receptor function have many potential therapeutic applications not addressed by currently available compounds. We have identified allosteric modulators with several novel patterns of NMDA receptor subtype selectivity that have a novel mechanism of action. In a series of carboxylated naphthalene and phenanthrene derivatives, compounds were identified that selectively potentiate responses at GluN1/GluN2A [e.g., 9-iodophenanthrene-3-carboxylic acid (UBP512)]; GluN1/GluN2A and GluN1/GluN2B [9-cyclopropylphenanthrene-3-carboxylic acid (UBP710)]; GluN1/GluN2D [3,5-dihydroxynaphthalene-2-carboxylic acid (UBP551)]; or GluN1/GluN2C and GluN1/GluN2D receptors [6-, 7-, 8-, and 9-nitro isomers of naphth[1,2-c][1,2,5]oxadiazole-5-sulfonic acid (NSC339614)] and have no effect or inhibit responses at the other NMDA receptors. Selective inhibition was also observed; UBP512 inhibits only GluN1/GluN2C and GluN1/GluN2D receptors, whereas 6-bromo-2-oxo-2H-chromene-3-carboxylic acid (UBP608) inhibits GluN1/GluN2A receptors with a 23-fold selectivity compared with GluN1/GluN2D receptors. The actions of these compounds were not competitive with the agonists L-glutamate or glycine and were not voltage-dependent. Whereas the N-terminal regulatory domain was not necessary for activity of either potentiators or inhibitors, segment 2 of the agonist ligand-binding domain was important for potentiating activity, whereas subtype-specific inhibitory activity was dependent upon segment 1. In terms of chemical structure, activity profile, and mechanism of action, these modulators represent a new class of pharmacological agents for the study of NMDA receptor subtype function and provide novel lead compounds for a variety of neurological disorders.

Dataset Information

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

Publications

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

Similar Datasets

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