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, 23 (9), 3020-9

Novel Endogenous Peptide Agonists of Cannabinoid Receptors

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Novel Endogenous Peptide Agonists of Cannabinoid Receptors

Ivone Gomes et al. FASEB J.

Abstract

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1) . In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hpalpha) or two (VD-Hpalpha) additional amino acids, as well as a beta-hemoglobin-derived peptide with sequence similarity to that of hemopressin (VD-Hpbeta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hpalpha and VD-Hpalpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid- and peptide-derived signaling molecules.

Figures

Figure 1.
Figure 1.
Mass spectrometric identification of endogenous RVD-Hpα. Peptides were extracted from microwave-irradiated mouse brain, labeled with TMAB isotopic labels, purified by microfiltration using a 10-kDa exclusion unit, and analyzed by LC/MS. In this example, the tandem mass spectrum is shown for the quadruply charged ion with an m/z of 420.5 and a monoisotopic mass of 1423.79 Da (after subtraction of the mass of the hydrogenated form of the TMAB isotopic tag). Inset: an expanded y axis for the indicated m/z range. The observed ions represent b-series, y-series, and internal ions formed secondary to the cleavage of the D-P bond. Fragmentation ions usually lose the trimethylamine moiety. However, the doubly charged b3 ion shows a substantial peak for the fragment that retained the trimethylamine; this is indicated as b32+ + 59. Conversely, the parent ion that lost the trimethylamine moiety is indicated as MH4+ − 59. amu, atomic mass units.
Figure 2.
Figure 2.
Longer Hp peptides are able to increase CB receptor-mediated intracellular Ca2+ levels. HEK-293 cells (40,000 cells/well) coexpressing chimeric G16/Gi3 and individual receptors (except in the case of AT1 receptors) were treated with 1 μM concentrations of receptor agonists, RVD-Hpα, VD-Hpα, or VD-Hpβ, and intracellular Ca2+ levels were determined. RFU, relative fluorescence units.
Figure 3.
Figure 3.
Longer Hp peptide-mediated calcium release is blocked by the CB1 receptor antagonist. HEK-293 cells (40,000 cells/well) coexpressing chimeric G16/Gi3 and CB1 receptors were treated with 1 μM HU-210 (Hu), RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716 (SR), and intracellular Ca2+ levels were determined. Results are means ± se; n = 3. RFU, relative fluorescence units.
Figure 4.
Figure 4.
Longer Hpα peptides induce neurite outgrowth in Neuro 2A cells. Neuro2A cells were treated for 16 h with 1 μM Hu-210 (Hu), RVD-Hpα, or VD-Hpα in the absence or presence of 10 μM SR141716 (SR), and the percentage of cells extending neurites was determined. Results are means ± se; n = 3. **P < 0.01, ***P < 0.001; 1-way ANOVA.
Figure 5.
Figure 5.
Longer Hp peptides induce CB1 receptor internalization. CHO cells expressing myc-tagged CB1 receptors (1–2×105 cells/well) were treated with 1 μM Hu-210 (Hu), RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716 (SR) and subjected to ELISA using 1:1000 of anti-myc polyclonal antibody and 1:1000 of HRP-conjugated anti-rabbit secondary antibody, as described in Materials and Methods. Results are means ± se of 3 experiments in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA.
Figure 6.
Figure 6.
Longer Hpα peptides mediate ERK phosphorylation. A) HEK-293 cells expressing myc-tagged CB1 receptors, HA-tagged CB2 receptors, or GPR55 were treated with 1 μM Hu-210 (Hu), RVD-Hpα, or VD-Hpα in the absence or presence of 10 μM SR141716 (SR), and levels of pERK1/2 were determined. Results are means ± se; n = 3. *P < 0.01, **P < 0.001; 1-way ANOVA. B) HEK-293 cells expressing CB1 receptors were treated for 0–20 min with 1 μM Hu-210 or RVD-Hpα in the absence or presence of PTX, and levels of phosphorylated ERK1/2 were determined as described in Materials and Methods. Results are means ± se of 3 experiments in triplicate. **P < 0.01; ***P < 0.001. Representative blots from 3 independent experiments are shown.
Figure 7.
Figure 7.
Longer Hpα peptides activate a signal transduction pathway distinct from the pathway activated by classic CB1 receptor agonists: [35S]GTPγS binding. Striatal or cerebellar membranes (10 μg) were subjected to a [35S]GTPγS binding assay using the indicated concentrations of Hu-210 (Hu), RVD-Hpα, or VD-Hpα, as described in Materials and Methods. Results are means ± se; n = 3.
Figure 8.
Figure 8.
Longer Hpα peptides activate a signal transduction pathway distinct from the pathway activated by classic CB1 receptor agonists: calcium release. A) Neuro 2A cells (7–18 cells) were treated 1 min before data recording with 1 μM Hu-210, RVD-Hpα, VD-Hpα, or 2-AG. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 7–18 cells. B) Neuro 2A cells were treated 1 min before data recording with 1 μM Hu-210, RVD-Hpα, or Hpα in the absence or presence of 10 μM SR141716. Data represent means ± se of 7–18 cells. C) Neuro 2A cells were treated in the presence or absence of PTX before data recording with 1 μM RVD-Hpα. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 7–18 cells. D) Neuro 2A cells were treated in the presence or absence of Ca2+-free medium 1 min before data recording with 1 μM RVD-Hpα. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 18–22 cells.

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