Research ArticleGPCR SIGNALING

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN

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Science Signaling  26 Apr 2016:
Vol. 9, Issue 425, pp. ra43
DOI: 10.1126/scisignal.aad0694
  • Fig. 1

    PEN binds and activates a GPCR in the brain. (A) Schematic representation of peptides derived from proSAAS processing. PEN sequences of mouse (mPEN), rat (rPEN), and human (hPEN) are in single-letter amino acid code. (B) Saturation binding with [125I]Tyr-rPEN in mouse hypothalamic membranes (30 μg). (C) The ability of mPEN and rlittleLEN to displace [125I]Tyr-rPEN (3 nM) binding in mouse hypothalamic membranes (30 μg). (D) The effect of mPEN on [35S]GTPγS binding in mouse hypothalamic membranes (20 μg). (E) The effect of mPEN on phospholipase C (PLC) activity in mouse hypothalamic membranes (10 μg). (F) Specific binding of [125I]Tyr-rPEN in different mouse brain regions and peripheral tissues. (G) Displacement by mPEN of [125I]Tyr-rPEN (3 nM) binding to mouse hippocampal membranes (30 μg). (H) The effect of mPEN, Tyr-rPEN, or rlittlePENLEN on [35S]GTPγS binding in mouse hippocampal membranes (20 μg). (I) The effect of mPEN on adenylyl cyclase (AC) activity in mouse hippocampal membranes (2 μg). Data represent means ± SE (n = 3 to 8 individual experiments).

  • Fig. 2

    Effect of mPEN on synaptic activity of PVN neurons in rat hypothalamic slices. (A) Spontaneous excitatory postsynaptic current sEPSC traces (top) in a PVN neuron after bath application of 100 nM mPEN (scale bar, 5 pA and 200 ms). Cumulative plots for this neuron demonstrate the effect of mPEN on sEPSC amplitudes (left) and inter-sEPSC intervals (right). (B) Graphical representation of frequency (left) and mean amplitudes (right) of sEPSCs in PVN neurons after mPEN application. Each circle represents an individual neuron. (C) Dose response for change in evoked EPSC amplitude. Frequency (baseline, 5.8 ± 1.7 Hz; mPEN, 3.9 ± 1.3 Hz; P = 0.03; n = 8); amplitude (baseline, 11.8 ± 1.5 pA; mPEN, 12.5 ± 1.6 pA; P = 0.24; n = 8 individual neurons); and inset, example traces of evoked responses from one cell. Black, baseline; red, 10 nM mPEN; blue, 100 nM mPEN. Scale bars, 40 pA and 20 ms. n = 3 to 5 cells per dose. (D) The paired-pulse ratio in PVN neurons in response to 100 nM PEN. The data in blue are from the one neuron where the highest dose tested was 10 nM PEN. The data represent means ± SE (n = 3 to 8 individual experiments). *P < 0.05 [paired two-tailed t test for (B) and (D); for statistical analysis, see table S1].

  • Fig. 3

    PEN binds and activates a GPCR in Neuro2A cells. (A) The effect of mPEN, mbigLEN, or rSAAS (1 μM) on neurite outgrowth in Neuro2A cells. (B) Saturation binding with [125I]Tyr-rPEN in Neuro2A cell membranes (30 μg). (C) The ability of mPEN to displace [125I]Tyr-rPEN (3 nM) binding from Neuro2A cell membranes (50 μg). (D) The effect of mPEN on [35S]GTPγS binding in Neuro2A membranes (20 μg). (E) The effect of mPEN on intracellular cAMP levels in Neuro2A cells (10,000 per well). (F) The effect of mPEN on PLC activity in Neuro2A membranes (10 μg). (G) The effect of mPEN on intracellular Ca2+ release in Neuro2A cells. ATP (adenosine 5´-triphosphate) (1 μM) was used as a positive control. RFU, relative fluorescence units. (H) The effect of mPEN (1 μM) on MAPK (mitogen-activated protein kinase) phosphorylation at 5 and 30 min in Neuro2A cells. Data represent means ± SE (n = 3 to 6 independent experiments). ***P < 0.0001 [one-way analysis of variance (ANOVA) for (A) and two-way ANOVA for (H)]; for details of statistical analysis, see table S1.

  • Fig. 4

    Expression of GPR83 in heterologous cells confers PEN binding and signaling. (A) The effect of PEN (1 μM) on intracellular Ca2+ release in cells expressing hGPR83 along with a promiscuous chimeric hGα16/i3 protein. Scrambled peptide (1 μM), hPEN (1 μM), rlittleLEN (1 μM), mbigLEN (1 μM), or ATP (1 μM). (B) Saturation binding with [125I]Tyr-rPEN in CHO hGPR83 cell membranes (30 μg). (C) mPEN displacement of [125I]Tyr-rPEN (3 nM) binding to HEK-293 mGPR83 cells (50 μg). (D) The ability of mPEN, hPEN, and NPY to displace [125I]Tyr-rPEN (3 nM) binding to CHO hGPR83 cell membranes (50 μg). (E) The effect of mPEN on GTPγS binding to membranes (20 μg) from CHO hGPR83 cells (CHO83) or CHO alone. (F) The effect of pertussis toxin (PTX; 50 ng/ml) pretreatment on 1 μM PEN-mediated [35S]GTPγS binding in membranes (20 μg) from CHO hGPR83 cells versus absence of PTX. ***P < 0.0005 (t test). (G) The effect of mPEN on intracellular cAMP levels in CHO hGPR83 cells (10,000 cells per well). (H) The effect of PEN on PLC activity in membranes (10 μg) from CHO hGPR83 cells. (I) The effect of mPEN on IP3 levels in CHO hGPR83 cells (10,000 cells per well). (J) The effect of mPEN or hPEN on intracellular Ca2+ release in CHO hGPR83 cells expressing a promiscuous chimeric hGα16/i3 protein. ATP (1 μM) was used as a positive control. Data (A to J) represent means ± SE (n = 3 to 8 independent experiments). ***P < 0.001[one-way ANOVA for (A); t test for (F)]; details of the statistical analyses are in table S1.

  • Fig. 5

    Knockdown or knockout of GPR83 leads to reduced binding and signaling by PEN. (A) The effect of expressing GPR83 siRNA (50 to 200 pmol) on the levels of GPR83 in Neuro2A cells. (B) The effect of expressing GPR83 siRNA (200 pmol) in Neuro2A cells on specific binding of [125I]Tyr-rPEN (3 nM) to membranes (50 μg). Specific binding is defined as the difference in [125I]Tyr-rPEN bound in the absence and presence of 10 μM mPEN. cpm, counts per minute. (C) The effect of mPEN (1 μM) on GTPγS binding in membranes (20 μg) from Neuro2A cells transfected with 200 pmol of GPR83 siRNA. (D) The effect of mPEN (1 μM; 5 min) on phosphorylation of MAPK in untransfected Neuro2A cells (Cont) and in cells expressing 200 pmol of GPR83 siRNA. (E) The effect of mPEN (1 μM) on neuritogenesis in untransfected Neuro2A cells (Cont) and cells expressing GPR83 siRNA (200 pmol). Data (A to E) represent means ± SE (n = 3 to 6 individual experiments). *P < 0.05; **P < 0.001; and ***P < 0.0001 [two-way ANOVA for (A), t test for (B), and two-way ANOVA for (C), (D), and (E)]. (F) [125I]Tyr-rPEN binding to membranes (30 μg) from individual GPR83 knockout and wild-type (WT) mice. The number on the x axis denotes the individual mouse number. (G) The effect of mPEN (1 μM) on GTPγS binding in hypothalamic membranes (20 μg) from individual GPR83 knockout mice. (H) The effect of mPEN (100 nM) on PLC activity in hypothalamic membranes (20 μg) from WT and GPR83 knockout (KO) mice. Data (H) represent means ± SE (n = 3 individual animal tissues); *P < 0.05 compared to WT without PEN (two-way ANOVA). In (H), the difference in the presence and absence of mPEN is not significant in the GPR83 knockout mouse tissue. For details of statistical analysis, see table S1.

  • Fig. 6

    GPR83 functionally interacts with GPR171. (A) The effect of expressing mGPR171 on [125I]Tyr-rPEN binding to membranes (30 μg) from CHO hGPR83 cells. (B) The effect of expressing mGPR171 on surface abundance of hGPR83 in CHO hGPR83 cells. (C) The effect of expressing mGPR171 on PEN-mediated GTPγS binding in CHO hGPR83 cells. (D) The effect of shRNA-mediated knockdown of GPR171 on [125I]Tyr-rPEN binding to Neuro2A cells. The GPR171 shRNA has been described previously (13). (E) The effect of shRNA-mediated knockdown of GPR171 in Neuro2A cells on PEN-mediated GTPγS binding. (F) The effect expressing mGPR171 in CHO hGPR83 cells (2 × 105 cells) on 100 nM mPEN-mediated hGPR83 internalization. (G) The effect of expressing hGPR83 in CHO mGPR171cells (2 × 105 cells) on bigLEN (100 nM)–mediated mGPR171 internalization. Data (A to G) represent means ± SE (n = 3 to 6 independent experiments). **P < 0.01; ***P < 0.001 [t test for (B); for details of statistical analysis, see table S1].

  • Fig. 7

    GPR83 and GPR171 are colocalized in the PVN and close enough to interact. (A) PLA to determine the interaction of GPR83 and GPR171 in CHO cells coexpressing mGPR83 and mGPR171. (B) Quantification of the PLA signal, n = 30 cells. (C) Coronal section stained for GPR83 (green) in the PVN. (D) Immunohistochemical localization using the antibody recognizing GPR83 (green) and the antibody recognizing GPR171 (red) to determine colocalization of GPR83 and GPR171 in the PVN. Magnification, ×10. (E) Quantification of 63× images from the PVN shown as a pie chart. n = 50 cells per field (two fields per mice) from three independent animals. (F) PLA to determine the interaction of GPR83 and GPR171 in the PVN; 40× image. (G) A ×63 magnification of (F). (H) A zoomed-in image of (F) showing PLA signal in a single cell. (I) Quantification of data in (G). n = 50 cells per field (two fields per mice) from three independent animals. (J) The effect of knockdown of GPR83 (GPR83 knockout) on bigLEN-mediated adenylate cyclase activity. Data represent means ± SE (n = 3 independent animals). **P < 0.01; ***P < 0.001 [one-way ANOVA for (B); for details of statistical analysis, see table S1].

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/425/ra43/DC1

    Fig. S1. Variable results with studies examining the effect of mPEN on adenylyl cyclase activity in hypothalamic membranes.

    Fig. S2. The binding of mPEN to hypothalamic membranes from individual male mice.

    Fig. S3. Variable results with studies examining the effect of mPEN on PLC activity in hippocampal membranes.

    Fig. S4. Evoked EPSC amplitude after sequential application of increasing concentrations of mPEN and washout.

    Fig. S5. mPEN-stimulated neurite outgrowth in Neuro2A cells.

    Fig. S6. Expression of GPR83 in heterologous cells confers PEN signaling and receptor endocytosis.

    Fig. S7. Quantitative RT-PCR to confirm the presence of GPR83 mRNA in Neuro2A cells.

    Fig. S8. Specificity of the GPR83 and GPR171 antibodies.

    Fig. S9. Confirmation of antibody specificity and accuracy of colocalization by analysis of the lateral septum.

    Table S1. Description of statistical analysis for different figures.

  • Supplementary Materials for:

    Identification of GPR83 as the receptor for the neuroendocrine peptide PEN

    Ivone Gomes, Erin N. Bobeck, Elyssa B. Margolis, Achla Gupta, Salvador Sierra, Amanda K. Fakira, Wakako Fujita, Timo D. Müller, Anne Müller, Matthias H. Tschöp, Gunnar Kleinau, Lloyd D. Fricker, Lakshmi A. Devi*

    *Corresponding author. Email: lakshmi.devi{at}mssm.edu

    This PDF file includes:

    • Fig. S1. Variable results with studies examining the effect of mPEN on adenylyl cyclase activity in hypothalamic membranes.
    • Fig. S2. The binding of mPEN to hypothalamic membranes from individual male mice.
    • Fig. S3. Variable results with studies examining the effect of mPEN on PLC activity in hippocampal membranes.
    • Fig. S4. Evoked EPSC amplitude after sequential application of increasing concentrations of mPEN and washout.
    • Fig. S5. mPEN-stimulated neurite outgrowth in Neuro2A cells.
    • Fig. S6. Expression of GPR83 in heterologous cells confers PEN signaling and receptor endocytosis.
    • Fig. S7. Quantitative RT-PCR to confirm the presence of GPR83 mRNA in Neuro2A cells.
    • Fig. S8. Specificity of the GPR83 and GPR171 antibodies.
    • Fig. S9. Confirmation of antibody specificity and accuracy of colocalization by analysis of the lateral septum.
    • Table S1. Description of statistical analysis for different figures.

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    Citation: I. Gomes, E. N. Bobeck, E. B. Margolis, A. Gupta, S. Sierra, A. K. Fakira, W. Fujita, T. D. Müller, A. Müller, M. H. Tschöp, G. Kleinau, L. D. Fricker, L. A. Devi, Identification of GPR83 as the receptor for the neuroendocrine peptide PEN. Sci. Signal. 9, ra43 (2016).

    © 2016 American Association for the Advancement of Science

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