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, 29 (11), 1619-33

Temporal cAMP Signaling Selectivity by Natural and Synthetic MC4R Agonists

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Temporal cAMP Signaling Selectivity by Natural and Synthetic MC4R Agonists

Brent M Molden et al. Mol Endocrinol.

Abstract

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain, where it controls energy balance through pathways including α-melanocyte-stimulating hormone (α-MSH)-dependent signaling. We have reported that the MC4R can exist in an active conformation that signals constitutively by increasing cAMP levels in the absence of receptor desensitization. We asked whether synthetic MC4R agonists differ in their ability to increase intracellular cAMP over time in Neuro2A cells expressing endogenous MC4R and exogenous, epitope-tagged hemagglutinin-MC4R-green fluorescent protein. By analyzing intracellular cAMP in a temporally resolved Förster resonance energy transfer assay, we show that withdrawal of α-MSH leads to a quick reversal of cAMP induction. By contrast, the synthetic agonist melanotan II (MTII) induces a cAMP signal that persists for at least 1 hour after removal of MTII from the medium and cannot be antagonized by agouti related protein. Similarly, in mHypoE-42 immortalized hypothalamic neurons, MTII, but not α-MSH, induced persistent AMP kinase signal, which occurs downstream of increased cAMP. By using a fluorescence recovery after photobleaching assay, it appears that the receptor exposed to MTII continues to signal after being internalized. Similar to MTII, the synthetic MC4R agonists, THIQ and BIM-22511, but not LY2112688, induced prolonged cAMP signaling after agonist withdrawal. However, agonist-exposed MC4R desensitized to the same extent, regardless of the ligand used and regardless of differences in receptor intracellular retention kinetics. In conclusion, α-MSH and LY2112688, when compared with MTII, THIQ, and BIM-22511, vary in the duration of the acute cAMP response, showing distinct temporal signaling selectivity, possibly linked to specific cell compartments from which cAMP signals may originate.

Figures

Figure 1.
Figure 1.
Chronic exposure to MTII induces profound loss of MC4R signaling in N2AHA-MC4R-GFP cells. A, Schematic diagram of the time course of the experiment. N2AHA-MC4R-GFP cells were prechallenged for the indicated time with 2nM MTII. Cells were washed and challenged acutely with 2nM MTII in the presence of IBMX. B, Amount of cAMP generated in response to chronic exposure to MTII (2nM) is expressed as percentage of that obtained in by acute MTII challenge in cells not pretreated with agonist.
Figure 2.
Figure 2.
MTII, unlike α-MSH, induces persistent cAMP signaling after agonist withdrawal. A, Dose-response curve to MTII. The amount of cAMP generated is measured by immunoassay as described in Materials and Methods. B, Schematic diagram of the experiments shown in C. C, N2AHA-MC4R-GFP cells were treated with 200nM α-MSH or 2nM MTII for 1 hour. Cells were then washed with the medium being replaced for a total of 3 times and incubated without any agonist. At the indicated time, cells previously treated with 500μM IBMX for 15 minutes were lysed, and intracellular cAMP was measured by ELISA. D, Live N2AHA-MC4R-GFP cells were incubated with 2nM MTII-Rh for 30 minutes at 37°C, transferred to a heated stage at 37°C to visualize the distribution of MTII-Rh and of HA-MC4R-GFP by confocal microscopy. E, Live N2AHA-MC4R-GFP cells were treated with 2nM MTII-Rh as in D (before wash) and then washed, with the medium being replaced for a total of 6 times. The distribution of MTII-Rh before the cell wash and at 5, 10, and 25 minutes after medium replacement is monitored by confocal microscopy. F and G, N2AHA-MC4R-GFP cells were transfected with TEpacVV and transferred to a heated stage at 37°C for confocal microscopy. Cells were treated with agonists as indicated followed by a wash step where the medium was replaced for a total of 6 times. The cAMP response was measured by a real-time FRET-based assay as described in Materials and Methods. The fluorescence of mTurquoise was excited at 458 nm, and the intensities of fluorescence emitted at 480–495 nm (mTurquoise) and at 535–565 nm (YFP) were recorded over time. In the graphs, the corrected ratio of the intensity of mTurquoise fluorescence to the intensity of YFP fluorescence is shown for a representative cell. The experiments in F and G were run in parallel using the same population of transfected cells.
Figure 3.
Figure 3.
Persistent cAMP signaling by MTII is not antagonized by AgRP. A and B, N2AHA-MC4R-GFP cells were transfected with TEpacVV. Cells were treated with α-MSH and MTII followed by a wash step where the medium was replaced for a total of 6 times before the addition of AgRP. Changes in corrected FRET ratio were monitored as in Figure 2C, normalized to that observed in response to the initial exposure to the agonist (Materials and Methods), and shown in the graphs as averages and SDs (n cells per condition = 5). Experiments in A and B were run in parallel using the same population of transfected cells.
Figure 4.
Figure 4.
HA-MC4R-GFP in a complex with MTII internalizes rapidly and at the same rate as the receptor in a complex with α-MSH or the ligand-free receptor. A, N2AHA-MC4R-GFP cells were transferred to a heated stage at 37°C for photobleaching/confocal microscopy. A, ROI is drawn around the HA-MC4R-GFP intracellular compartment of N2AHA-MC4R-GFP cells kept in basal conditions (t = 0, white circle). Photobleaching is carried out at the ROI (t = 2.5 min, white circle highlighted by the arrow). Fluorescence recovery is visualized at the ROI (t = 15 min, white circle). Scale bar, 10 μm. B–D, Integrated fluorescence intensity at the intracellular ROI is monitored over time in a cell either kept in basal conditions (A) or treated with α-MSH (C) and with MTII (D). The agonists, at a concentration of 200nM, were added to the cell medium 45 minutes before the start of the FRAP experiment. E, The graph shows the average half-life of FRAP in the selected ROIs (data are derived from ∼6 cells per condition).
Figure 5.
Figure 5.
Persistent cAMP signal by MTII is maintained after rapid redistribution of HA-MC4R-GFP to intracellular compartments by a dynamin inhibitor. A and B, N2AHA-MC4R-GFP cells were incubated at 37°C without and with the dynamin inhibitor, V 34–2 at 12.5μM, for the indicated time. Cells were fixed and stained with anti-HA (Cy3, red fluorescence). A, Merged image with both GFP and Cy3 fluorescence. Arrowhead indicates HA-MC4R-GFP at cytoplasmic vesicles clustered near the nucleus; arrows indicate HA-MC4R-GFP in scattered cytoplasmic vesicles. Scale bar, 10 μm. B, Cy3 fluorescence showing cell surface HA-MC4R-GFP in the same cells as in A. C, The graph shows the quantification of the experiments in A where the fraction of total HA-MC4R-GFP residing at the cell surface is monitored by measuring the ratio Cy3 pixel intensity to GFP pixel intensity for each individual cell (n cells = 60). D, N2AHA-MC4R-GFP cells were transfected with TEpacVV, transferred to a heated stage at 37°C for confocal microscopy to monitor changes in intracellular cAMP by real-time FRET assay. The time course of the experiment is shown above the graph. After addition of MTII (2nM), cells were incubated for approximately 5 minutes, washed, and incubated either with vehicle (black trace) or with V 34–2 (12.5μM, red trace), followed by forskolin (1μM) and IBMX (100μM). In the graph, the amount of cAMP being generated was derived from the corrected ratio of the intensity of mTurquoise fluorescence to the intensity of YFP fluorescence normalized to that induced by the addition of forskolin/IBMX as described in Materials and Methods. Data are shown in the graphs as averages and SEMs (n cells = 20 per condition from 2 independent experiments where the 2 conditions, vehicle and V 34–2, are run in parallel). E, N2AHA-MC4R-GFP cells were transfected as in D. MTII was added to the cells at t = 0 and kept in the incubation medium for approximately 3 minutes. The graph shows a representative experiment where the averages and SEMs of the amount of cAMP being generated, expressed as percentage of that induced by forskolin, are derived as in D (n cells = 3). The t1/2 of the cAMP response was derived by fitting the 1-phase association model to the data by using GraphPad Software.
Figure 6.
Figure 6.
MTII induces persistent AMPK signal in mHypoE-42 hypothalamic neurons. A, mHypoE-42 hypothalamic cells were treated as indicated by the schematic diagram. B, Western blot analysis of cleared mHypoE-42 hypothalamic neurons lysates with the indicated antibodies. C, The intensity of the AMPKp and AMPK bands were measured as described in Materials and Methods, and data were expressed as the percentage of the untreated control. Averages and SDs were derived from 3 independent experiments.
Figure 7.
Figure 7.
Persistent cAMP signaling is a property of some synthetic MC4R agonists. A, Intracellular cAMP in N2AHA-MC4R-GFP cells was measured by ELISA in response to no additions, or addition of α-MSH and MTII (200nM), THIQ (1μM), BIM-22511 and LY2112688 (100nM). B–F, N2AHA-MC4R-GFP cells were transfected with TEpacVV, transferred to a heated stage at 37°C for confocal microscopy. Changes in intracellular cAMP were monitored by real-time FRET assay as in Figure 2. In all experiments, N2AHA-MC4R-GFP cells were treated with different agonists, as in A. Cells were washed approximately 10 minutes after addition of the agonists and then rechallenged by using 500nM α-MSH. The experiments in C–F were carried out after an initial experiment where the same population of transfected cells was treated with α-MSH followed by agonist wash-off, as in A. Changes in corrected FRET ratio were monitored as in Figure 2C, normalized to that observed in response to the initial exposure to the agonist (Materials and Methods), and shown in the graphs as averages and SDs (n cells per condition >/= 5).
Figure 8.
Figure 8.
The extent by which HA-MC4R-GFP redistributes to the intracellular localization in response to prolonged treatment with synthetic agonist is specific to the type of agonist being used. A, Live N2AHA-MC4R-GFP cells were treated with no additions or exposed to α-MSH and MTII (200nM), THIQ (1μM), BIM-22511 and LY2112688 (100nM), and to POD-conjugated anti-HA antibodies at 4°C. Cells were washed and transferred at 37°C in the presence of agonist to measure internalization of the pool of HA-MC4R-GFP by monitoring the abundance of agonist-free and agonist-bound receptor at the cell surface as outlined in Materials and Methods. B, N2AHA-MC4R-GFP cells are treated at 37°C for 4 hours with no additions or with α-MSH (1μM) and MTII (500nM) as shown in the schematic. C, Confocal images of live N2AHA-MC4R-GFP cells treated as in B and transferred to a heated stage at 37°C for confocal microscopy. Scale bar, 10 μm; blue tracing, ROI of intracellular GFP fluorescence; red tracing, ROI of GFP fluorescence within the cell perimeter. D, Experiments as outlined in B were carried out exposing cells to α-MSH (1μM), MTII (500nM), THIQ (1μM), and BIM-22511 and LY2112688 (500nM) for 4 hours. Changes in the cellular distribution of HA-MC4R-GFP upon prolonged agonist exposure were monitored by measuring the ratio intracellular GFP fluorescence intensity to total GFP fluorescence intensity as outlined in Materials and Methods. Each symbol corresponds to a single cell (∼60 cells per condition). E, Schematic of experiments where N2AHA-MC4R-GFP cells were pretreated at 37°C with no additions or with α-MSH, MTII, THIQ, BIM-22511, and LY2112688 for 4 hours as in Figure 6F. F, HA-MC4R-GFP desensitization is monitored by the cAMP levels in N2AHA-MC4R-GFP cells pretreated with the indicated agonists expressed as a percentage of that of control cells pretreated in the absence of MC4R ligands.

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