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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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AgRP-induced increase in thallium flux does not involve Gi signaling or β-arrestin recruitmenta & b, Subtracted Tl+ flux examining effects of 200 nM AgRP indicates that 8 hour pre incubation with pertussis toxin of MC4R and Kir7.1 expressing HEK293 cells fails to block AgRP -induced Kir7.1 regulation (mean +/−SEM, n=110). c, Addition of α-MSH stimulates β-arrestin recruitment to the MC4R in HEK cells stably expressing MC4R and β-arrestin fused to complementary fragments of β-galactosidase (DiscoverRx PathHunter assay, black line, LogEC50 = −7.69). In contrast, increasing concentrations of AgRP are without any effect using the same assay (red line). Individual points show mean +/− SEM.
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Figure 10: AgRP-induced increase in thallium flux does not involve Gi signaling or β-arrestin recruitmenta & b, Subtracted Tl+ flux examining effects of 200 nM AgRP indicates that 8 hour pre incubation with pertussis toxin of MC4R and Kir7.1 expressing HEK293 cells fails to block AgRP -induced Kir7.1 regulation (mean +/−SEM, n=110). c, Addition of α-MSH stimulates β-arrestin recruitment to the MC4R in HEK cells stably expressing MC4R and β-arrestin fused to complementary fragments of β-galactosidase (DiscoverRx PathHunter assay, black line, LogEC50 = −7.69). In contrast, increasing concentrations of AgRP are without any effect using the same assay (red line). Individual points show mean +/− SEM.

Mentions: By loading HEK293 cells with a Tl+-sensitive fluorescent dye, Thallos, the flux of Tl+ ions through open K+ channels can be measured15. The effect of receptor modulation on channel conductance can then be studied by subtracting flux intensity after experimental treatments from levels after vehicle. Using this system, dose-response analysis indicated that α-MSH mediates a MC4R-dependent closure of Kir7.1 channels with an IC50 of 10−7.5M (Fig. 3d–e). Conversely, AgRP mediated a MC4R-dependent increase in Tl+ flux through Kir7.1, with an EC50 of 10−8.6M (Fig. 3 f–g). AgRP did not appear to couple the MC4R to the cAMP inhibitory G protein, Gαi16, or to recruit β-arrestin to the receptor (Extended Data Fig. 6a–c). These data support a G-protein independent mechanism for the MC4R-mediated regulation of Kir7.1 by AgRP as well. To examine the selectivity of coupling between melanocortin receptors and Kir7.1, we created stable HEK293 cells expressing Kir7.1 M125R alone, or Kir7.1 M125R plus MC1R, MC3R, or MC4R. α-MSH (100 nM) significantly decreased Tl+ flux in Kir7.1 HEK293 cells expressing MC1R or MC4R, but not MC3R (Fig. 3h). α-MSH (100 nM) significantly decreased Tl+ flux in HEK293 cells transfected with MC4R plus Kir7.1 or Kir4.1, but not cells expressing Kir2.1 or Kir2.3 (Fig. 3i). The Kir7.1 specific channel blocker VU573 blocked the α-MSH effects on Kir7.1 function in the HEK293 cells (Fig. 3j), as in the slice (Fig. 2l). However, VU573 had no effect on α-MSH inhibition of Kir4.1 in HEK293 cells (Fig 3k), suggesting the conductance regulated by α-MSH in MC4R PVN neurons primarily involves Kir7.1. PKA activity was also not required for α-MSH induced closure of Kir7.1 in HEK293 cells (Extended Data Fig. 7a–b), and we also observed a synergistic effect of α-MSH and cAMP on Kir7.1 closure, in this system (Extended Data Fig. 7a–d).


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)

AgRP-induced increase in thallium flux does not involve Gi signaling or β-arrestin recruitmenta & b, Subtracted Tl+ flux examining effects of 200 nM AgRP indicates that 8 hour pre incubation with pertussis toxin of MC4R and Kir7.1 expressing HEK293 cells fails to block AgRP -induced Kir7.1 regulation (mean +/−SEM, n=110). c, Addition of α-MSH stimulates β-arrestin recruitment to the MC4R in HEK cells stably expressing MC4R and β-arrestin fused to complementary fragments of β-galactosidase (DiscoverRx PathHunter assay, black line, LogEC50 = −7.69). In contrast, increasing concentrations of AgRP are without any effect using the same assay (red line). Individual points show mean +/− SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383680&req=5

Figure 10: AgRP-induced increase in thallium flux does not involve Gi signaling or β-arrestin recruitmenta & b, Subtracted Tl+ flux examining effects of 200 nM AgRP indicates that 8 hour pre incubation with pertussis toxin of MC4R and Kir7.1 expressing HEK293 cells fails to block AgRP -induced Kir7.1 regulation (mean +/−SEM, n=110). c, Addition of α-MSH stimulates β-arrestin recruitment to the MC4R in HEK cells stably expressing MC4R and β-arrestin fused to complementary fragments of β-galactosidase (DiscoverRx PathHunter assay, black line, LogEC50 = −7.69). In contrast, increasing concentrations of AgRP are without any effect using the same assay (red line). Individual points show mean +/− SEM.
Mentions: By loading HEK293 cells with a Tl+-sensitive fluorescent dye, Thallos, the flux of Tl+ ions through open K+ channels can be measured15. The effect of receptor modulation on channel conductance can then be studied by subtracting flux intensity after experimental treatments from levels after vehicle. Using this system, dose-response analysis indicated that α-MSH mediates a MC4R-dependent closure of Kir7.1 channels with an IC50 of 10−7.5M (Fig. 3d–e). Conversely, AgRP mediated a MC4R-dependent increase in Tl+ flux through Kir7.1, with an EC50 of 10−8.6M (Fig. 3 f–g). AgRP did not appear to couple the MC4R to the cAMP inhibitory G protein, Gαi16, or to recruit β-arrestin to the receptor (Extended Data Fig. 6a–c). These data support a G-protein independent mechanism for the MC4R-mediated regulation of Kir7.1 by AgRP as well. To examine the selectivity of coupling between melanocortin receptors and Kir7.1, we created stable HEK293 cells expressing Kir7.1 M125R alone, or Kir7.1 M125R plus MC1R, MC3R, or MC4R. α-MSH (100 nM) significantly decreased Tl+ flux in Kir7.1 HEK293 cells expressing MC1R or MC4R, but not MC3R (Fig. 3h). α-MSH (100 nM) significantly decreased Tl+ flux in HEK293 cells transfected with MC4R plus Kir7.1 or Kir4.1, but not cells expressing Kir2.1 or Kir2.3 (Fig. 3i). The Kir7.1 specific channel blocker VU573 blocked the α-MSH effects on Kir7.1 function in the HEK293 cells (Fig. 3j), as in the slice (Fig. 2l). However, VU573 had no effect on α-MSH inhibition of Kir4.1 in HEK293 cells (Fig 3k), suggesting the conductance regulated by α-MSH in MC4R PVN neurons primarily involves Kir7.1. PKA activity was also not required for α-MSH induced closure of Kir7.1 in HEK293 cells (Extended Data Fig. 7a–b), and we also observed a synergistic effect of α-MSH and cAMP on Kir7.1 closure, in this system (Extended Data Fig. 7a–d).

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