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G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

Ghamari-Langroudi M, Digby GJ, Sebag JA, Millhauser GL, Palomino R, Matthews R, Gillyard T, Panaro BL, Tough IR, Cox HM, Denton JS, Cone RD - Nature (2015)

Bottom Line: Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding.Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN.Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology &Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

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MC4R and Kir7.1 coimmunoprecipitate from transfected HEK293 cellsa, Cells transfected with the indicated genetically flagged proteins were incubated with the reversible crosslinker dithiobismaleimidoethane (DTME) prior to lysis. Lysates were immunoprecipitated using the indicated antibody (F=Flag, HA=Hemaglutinin, X= no antibody), crosslinking was reversed with 100mM DL-dithiothreitol (DTT), and samples separated by SDS-PAGE. The membrane was blotted with the M2 anti-Flag antibody to detect Kir7.1. b, Relative quantitation of protein immunoprecipitation. Densitometry analysis to measure the amount of immunoreactive Kir7.1 material was performed using Adobe Photoshop. Amount of material immunoprecipitated with the Kir7.1-3X-Flag was set at 100%. Data shows relative amount of Kir7.1 immunoprecipitated using an antibody against the 3HA-MC4R protein; bars indicate range of data from 2 independent lanes. The protein molecular weight of Kir7.1 is calculated at 40kd, and the two larger bands represent glycosylated forms of the protein that are absent when the N-linked glycosylation site at position 93 is mutated (data not shown).
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Figure 9: MC4R and Kir7.1 coimmunoprecipitate from transfected HEK293 cellsa, Cells transfected with the indicated genetically flagged proteins were incubated with the reversible crosslinker dithiobismaleimidoethane (DTME) prior to lysis. Lysates were immunoprecipitated using the indicated antibody (F=Flag, HA=Hemaglutinin, X= no antibody), crosslinking was reversed with 100mM DL-dithiothreitol (DTT), and samples separated by SDS-PAGE. The membrane was blotted with the M2 anti-Flag antibody to detect Kir7.1. b, Relative quantitation of protein immunoprecipitation. Densitometry analysis to measure the amount of immunoreactive Kir7.1 material was performed using Adobe Photoshop. Amount of material immunoprecipitated with the Kir7.1-3X-Flag was set at 100%. Data shows relative amount of Kir7.1 immunoprecipitated using an antibody against the 3HA-MC4R protein; bars indicate range of data from 2 independent lanes. The protein molecular weight of Kir7.1 is calculated at 40kd, and the two larger bands represent glycosylated forms of the protein that are absent when the N-linked glycosylation site at position 93 is mutated (data not shown).

Mentions: To confirm that MC4R signaling is capable of modulating Kir7.1 function, we transfected MC4R and Kir7.1 channels into HEK293 cells, using an M125R variant of Kir7.1 that exhibits higher unitary conductance than the native channel, previously demonstrated to allow detection of the channel in cell lines14. Whole cell recordings were performed 24–48 h after cotransfecting cells with MC4R, Kir7.1 M125R and GFP expression plasmids to examine effects of α-MSH on membrane current by examining the I–V analysis of α-MSH response. α-MSH significantly decreased the amplitude (>50%) and slope of current responses indicating closure of Kir7.1 channels (Fig. 3a–c). This effect was reversible, and the current amplitude was reduced to less than 25% of its own control by 2 mM Ba2+, consistent with the increased sensitivity of the Kir7.1 M125R variant to Ba2+14. Use of HEK293 cells allowed for a more direct assessment of kinetics of the α-MSH-induced current, indicating a rapid activation time course with an average τrise of 32 seconds. Cotransfection of tagged MC4R and Kir7.1 genes, followed by immunoprecipitation and western blot analysis showed a quantitative association of the two proteins in this system (Extended Data Fig. 5).


G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

Ghamari-Langroudi M, Digby GJ, Sebag JA, Millhauser GL, Palomino R, Matthews R, Gillyard T, Panaro BL, Tough IR, Cox HM, Denton JS, Cone RD - Nature (2015)

MC4R and Kir7.1 coimmunoprecipitate from transfected HEK293 cellsa, Cells transfected with the indicated genetically flagged proteins were incubated with the reversible crosslinker dithiobismaleimidoethane (DTME) prior to lysis. Lysates were immunoprecipitated using the indicated antibody (F=Flag, HA=Hemaglutinin, X= no antibody), crosslinking was reversed with 100mM DL-dithiothreitol (DTT), and samples separated by SDS-PAGE. The membrane was blotted with the M2 anti-Flag antibody to detect Kir7.1. b, Relative quantitation of protein immunoprecipitation. Densitometry analysis to measure the amount of immunoreactive Kir7.1 material was performed using Adobe Photoshop. Amount of material immunoprecipitated with the Kir7.1-3X-Flag was set at 100%. Data shows relative amount of Kir7.1 immunoprecipitated using an antibody against the 3HA-MC4R protein; bars indicate range of data from 2 independent lanes. The protein molecular weight of Kir7.1 is calculated at 40kd, and the two larger bands represent glycosylated forms of the protein that are absent when the N-linked glycosylation site at position 93 is mutated (data not shown).
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Related In: Results  -  Collection

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Figure 9: MC4R and Kir7.1 coimmunoprecipitate from transfected HEK293 cellsa, Cells transfected with the indicated genetically flagged proteins were incubated with the reversible crosslinker dithiobismaleimidoethane (DTME) prior to lysis. Lysates were immunoprecipitated using the indicated antibody (F=Flag, HA=Hemaglutinin, X= no antibody), crosslinking was reversed with 100mM DL-dithiothreitol (DTT), and samples separated by SDS-PAGE. The membrane was blotted with the M2 anti-Flag antibody to detect Kir7.1. b, Relative quantitation of protein immunoprecipitation. Densitometry analysis to measure the amount of immunoreactive Kir7.1 material was performed using Adobe Photoshop. Amount of material immunoprecipitated with the Kir7.1-3X-Flag was set at 100%. Data shows relative amount of Kir7.1 immunoprecipitated using an antibody against the 3HA-MC4R protein; bars indicate range of data from 2 independent lanes. The protein molecular weight of Kir7.1 is calculated at 40kd, and the two larger bands represent glycosylated forms of the protein that are absent when the N-linked glycosylation site at position 93 is mutated (data not shown).
Mentions: To confirm that MC4R signaling is capable of modulating Kir7.1 function, we transfected MC4R and Kir7.1 channels into HEK293 cells, using an M125R variant of Kir7.1 that exhibits higher unitary conductance than the native channel, previously demonstrated to allow detection of the channel in cell lines14. Whole cell recordings were performed 24–48 h after cotransfecting cells with MC4R, Kir7.1 M125R and GFP expression plasmids to examine effects of α-MSH on membrane current by examining the I–V analysis of α-MSH response. α-MSH significantly decreased the amplitude (>50%) and slope of current responses indicating closure of Kir7.1 channels (Fig. 3a–c). This effect was reversible, and the current amplitude was reduced to less than 25% of its own control by 2 mM Ba2+, consistent with the increased sensitivity of the Kir7.1 M125R variant to Ba2+14. Use of HEK293 cells allowed for a more direct assessment of kinetics of the α-MSH-induced current, indicating a rapid activation time course with an average τrise of 32 seconds. Cotransfection of tagged MC4R and Kir7.1 genes, followed by immunoprecipitation and western blot analysis showed a quantitative association of the two proteins in this system (Extended Data Fig. 5).

Bottom Line: Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding.Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN.Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology &Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Show MeSH
Related in: MedlinePlus