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Mechanism of attenuation of leptin signaling under chronic ligand stimulation.

Knobelspies H, Zeidler J, Hekerman P, Bamberg-Lemper S, Becker W - BMC Biochem. (2010)

Bottom Line: Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance).In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077.The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.

ABSTRACT

Background: Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.

Results: Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.

Conclusions: Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

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Time course of STAT3 activation by LEPRb tyrosine/phenylalanine mutants. A, Schematic representation of the LEPRb point mutants used in this study. The membrane-proximal box1/2 region binds the JAK family kinase and Tyr1138 (box3) mediates the activation of STAT3. B, RINm5F cells expressing WT or mutant LEPRb (FFY, FYY, YFY) were treated with leptin (20 ng/ml) for the times indicated (0 min, 15 min, 45 min, 75 min, 135 min, 255 min). Total cellular lysates were subjected to Western blot analysis and immunodetection with the indicated antibodies. These results are representative of 2 (YFY) or at least 3 (WT, FFY, FYY) independent experiments.
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Figure 1: Time course of STAT3 activation by LEPRb tyrosine/phenylalanine mutants. A, Schematic representation of the LEPRb point mutants used in this study. The membrane-proximal box1/2 region binds the JAK family kinase and Tyr1138 (box3) mediates the activation of STAT3. B, RINm5F cells expressing WT or mutant LEPRb (FFY, FYY, YFY) were treated with leptin (20 ng/ml) for the times indicated (0 min, 15 min, 45 min, 75 min, 135 min, 255 min). Total cellular lysates were subjected to Western blot analysis and immunodetection with the indicated antibodies. These results are representative of 2 (YFY) or at least 3 (WT, FFY, FYY) independent experiments.

Mentions: To examine the role of Tyr985 and Tyr1077 in the attenuation of LEPRb signaling during prolonged ligand stimulation, we determined the kinetics of leptin-induced STAT3 tyrosine phosphorylation in RINm5F cells. We have previously established the insulinoma cell line RINm5F as a model system to define the differential effects of the intracellular tyrosines in LEPRb signal transduction and their effects on gene expression [43,44]. We used RINm5F subclones expressing wild type (WT) LEPRb or mutant receptor construct in which one or both of these tyrosines were mutated to phenylalanine. The presence of Tyr1138 in the mutant receptors allowed us to monitor STAT3 tyrosine phosphorylation as the principle downstream signaling event. As shown in Figure 1, continuous treatment of RINm5F-LEPRb-WT cells with leptin resulted in a maximal tyrosine phosphorylation of STAT3 from 15 - 75 min, after which the level of the phosphorylated STAT3 declined sharply but remained detectable at reduced levels for 3 h. Mutation of neither Tyr985 nor Tyr1077 in LEPRb-FYY and LEPRb-YFY abrogated the attenuation of LEPRb signaling. STAT3 activation by the LEPRb double mutant lacking both Tyr985 and Tyr1077 (LEPRb-FFY) did not decline during the 4-h treatment with leptin. Similar time courses of STAT3 phosphorylation were observed in HIT-T15 cells transiently expressing wild type and mutant LEPRb constructs (see additional file 1: supplementary Figure S1).


Mechanism of attenuation of leptin signaling under chronic ligand stimulation.

Knobelspies H, Zeidler J, Hekerman P, Bamberg-Lemper S, Becker W - BMC Biochem. (2010)

Time course of STAT3 activation by LEPRb tyrosine/phenylalanine mutants. A, Schematic representation of the LEPRb point mutants used in this study. The membrane-proximal box1/2 region binds the JAK family kinase and Tyr1138 (box3) mediates the activation of STAT3. B, RINm5F cells expressing WT or mutant LEPRb (FFY, FYY, YFY) were treated with leptin (20 ng/ml) for the times indicated (0 min, 15 min, 45 min, 75 min, 135 min, 255 min). Total cellular lysates were subjected to Western blot analysis and immunodetection with the indicated antibodies. These results are representative of 2 (YFY) or at least 3 (WT, FFY, FYY) independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821298&req=5

Figure 1: Time course of STAT3 activation by LEPRb tyrosine/phenylalanine mutants. A, Schematic representation of the LEPRb point mutants used in this study. The membrane-proximal box1/2 region binds the JAK family kinase and Tyr1138 (box3) mediates the activation of STAT3. B, RINm5F cells expressing WT or mutant LEPRb (FFY, FYY, YFY) were treated with leptin (20 ng/ml) for the times indicated (0 min, 15 min, 45 min, 75 min, 135 min, 255 min). Total cellular lysates were subjected to Western blot analysis and immunodetection with the indicated antibodies. These results are representative of 2 (YFY) or at least 3 (WT, FFY, FYY) independent experiments.
Mentions: To examine the role of Tyr985 and Tyr1077 in the attenuation of LEPRb signaling during prolonged ligand stimulation, we determined the kinetics of leptin-induced STAT3 tyrosine phosphorylation in RINm5F cells. We have previously established the insulinoma cell line RINm5F as a model system to define the differential effects of the intracellular tyrosines in LEPRb signal transduction and their effects on gene expression [43,44]. We used RINm5F subclones expressing wild type (WT) LEPRb or mutant receptor construct in which one or both of these tyrosines were mutated to phenylalanine. The presence of Tyr1138 in the mutant receptors allowed us to monitor STAT3 tyrosine phosphorylation as the principle downstream signaling event. As shown in Figure 1, continuous treatment of RINm5F-LEPRb-WT cells with leptin resulted in a maximal tyrosine phosphorylation of STAT3 from 15 - 75 min, after which the level of the phosphorylated STAT3 declined sharply but remained detectable at reduced levels for 3 h. Mutation of neither Tyr985 nor Tyr1077 in LEPRb-FYY and LEPRb-YFY abrogated the attenuation of LEPRb signaling. STAT3 activation by the LEPRb double mutant lacking both Tyr985 and Tyr1077 (LEPRb-FFY) did not decline during the 4-h treatment with leptin. Similar time courses of STAT3 phosphorylation were observed in HIT-T15 cells transiently expressing wild type and mutant LEPRb constructs (see additional file 1: supplementary Figure S1).

Bottom Line: Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance).In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077.The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.

ABSTRACT

Background: Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.

Results: Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.

Conclusions: Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

Show MeSH
Related in: MedlinePlus