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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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The α-MSH analogue MC4-NN2-0453 (NOVO) is a partial agonist of the MC4R in a murine colon mucosal assay of MC4R activityThe activation MC4R inhibits vectorial ion transport across colonic epithelium, measured as reductions in the short circuit current (Isc). a, kinetic response to a sub-maximal basolateral concentration of α-MSH or NOVO, showing more rapid achievement of maximal activity with α-MSH, *p<0.05, 1-way ANOVA with Dunnett’s post test. b, full concentration-response to MC4-NN2-0453 (NOVO), showing that the compound does not achieve the efficacy reached by a maximal dose of α-MSH (denoted by the dashed line. Full characterization of the MC4R mediated α-MSH response in colonic epithelium is presented elsewhere18.
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Figure 12: The α-MSH analogue MC4-NN2-0453 (NOVO) is a partial agonist of the MC4R in a murine colon mucosal assay of MC4R activityThe activation MC4R inhibits vectorial ion transport across colonic epithelium, measured as reductions in the short circuit current (Isc). a, kinetic response to a sub-maximal basolateral concentration of α-MSH or NOVO, showing more rapid achievement of maximal activity with α-MSH, *p<0.05, 1-way ANOVA with Dunnett’s post test. b, full concentration-response to MC4-NN2-0453 (NOVO), showing that the compound does not achieve the efficacy reached by a maximal dose of α-MSH (denoted by the dashed line. Full characterization of the MC4R mediated α-MSH response in colonic epithelium is presented elsewhere18.

Mentions: Additional data support a role for Kir7.1 in regulation of food intake by MC4R in vivo. The AgRP analogue (miniAgRP; AgRP87-120) retains normal affinity for MC4R, and potency in inhibition of MC4R coupling to Gαs (Fig. 4a), yet exhibits 70% reduction in its ability to stimulate food intake in rats5. In parallel with loss of orexigenic activity, this peptide has lost its ability to couple MC4R to Kir7.1 (Fig. 4b). Conversely, we have also identified an α-MSH analogue, MC4-NN2-045317 (Novo), that preferentially couples MC4R to Kir7.1 over GαS in cell culture, and potently depolarizes PVN MC4R neurons (Fig. 4c–f). MC4-NN2-0453 also exhibited biased actions at MC4R in vivo. In a cAMP-mediated response, the peptide was unable to induce intestinal PYY release in vivo18 (Fig. 4g), or ex vivo (Extended Data Fig. 8). In contrast, the peptide potently inhibited food intake at doses equimolar to other α-MSH analogues (Fig. 4h). Knockdown of Kir7.1 gene expression in WT and MC4R mutant larval zebrafish, but not Kir7.1 mutant jaguar zebrafish, produced a reduction in linear growth and upregulation of GHRH gene expression, responses reported previously for activation of the MC4R19, (Extended Data Fig. 9a–e), further supporting the argument that arguing that Kir7.1 acts downstream of MC4R.


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)

The α-MSH analogue MC4-NN2-0453 (NOVO) is a partial agonist of the MC4R in a murine colon mucosal assay of MC4R activityThe activation MC4R inhibits vectorial ion transport across colonic epithelium, measured as reductions in the short circuit current (Isc). a, kinetic response to a sub-maximal basolateral concentration of α-MSH or NOVO, showing more rapid achievement of maximal activity with α-MSH, *p<0.05, 1-way ANOVA with Dunnett’s post test. b, full concentration-response to MC4-NN2-0453 (NOVO), showing that the compound does not achieve the efficacy reached by a maximal dose of α-MSH (denoted by the dashed line. Full characterization of the MC4R mediated α-MSH response in colonic epithelium is presented elsewhere18.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: The α-MSH analogue MC4-NN2-0453 (NOVO) is a partial agonist of the MC4R in a murine colon mucosal assay of MC4R activityThe activation MC4R inhibits vectorial ion transport across colonic epithelium, measured as reductions in the short circuit current (Isc). a, kinetic response to a sub-maximal basolateral concentration of α-MSH or NOVO, showing more rapid achievement of maximal activity with α-MSH, *p<0.05, 1-way ANOVA with Dunnett’s post test. b, full concentration-response to MC4-NN2-0453 (NOVO), showing that the compound does not achieve the efficacy reached by a maximal dose of α-MSH (denoted by the dashed line. Full characterization of the MC4R mediated α-MSH response in colonic epithelium is presented elsewhere18.
Mentions: Additional data support a role for Kir7.1 in regulation of food intake by MC4R in vivo. The AgRP analogue (miniAgRP; AgRP87-120) retains normal affinity for MC4R, and potency in inhibition of MC4R coupling to Gαs (Fig. 4a), yet exhibits 70% reduction in its ability to stimulate food intake in rats5. In parallel with loss of orexigenic activity, this peptide has lost its ability to couple MC4R to Kir7.1 (Fig. 4b). Conversely, we have also identified an α-MSH analogue, MC4-NN2-045317 (Novo), that preferentially couples MC4R to Kir7.1 over GαS in cell culture, and potently depolarizes PVN MC4R neurons (Fig. 4c–f). MC4-NN2-0453 also exhibited biased actions at MC4R in vivo. In a cAMP-mediated response, the peptide was unable to induce intestinal PYY release in vivo18 (Fig. 4g), or ex vivo (Extended Data Fig. 8). In contrast, the peptide potently inhibited food intake at doses equimolar to other α-MSH analogues (Fig. 4h). Knockdown of Kir7.1 gene expression in WT and MC4R mutant larval zebrafish, but not Kir7.1 mutant jaguar zebrafish, produced a reduction in linear growth and upregulation of GHRH gene expression, responses reported previously for activation of the MC4R19, (Extended Data Fig. 9a–e), further supporting the argument that arguing that Kir7.1 acts downstream of MC4R.

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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