Influence of the Accessory Protein SET on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling

Simon, Violaine; Öner, Şükrü Sadik; Cohen‐Tannoudji, Joëlle; Tobin, Andrew B.; Lanier, Stephen M.

doi:10.1124/mol.111.075523

Cited by 7 publications

(7 citation statements)

References 40 publications

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“…A similar inhibitory action of SET on M3-MR coupling to calcium signaling has been reported (18). Interestingly, a recent study addressing some of the mechanisms involved in SET action on M3-MR signaling showed that SET decreased G q protein engagement by the M3-MR, possibly through a competition for binding to the receptor, given the close proximity of the two protein binding sites within ICL3 (31) (40,41), suggesting that SET may also inhibit G q protein binding to GnRHR in gonadotrope ␣T3-1 cells. SET has been described as an inhibitor of PP2A activity (22).…”

Section: Discussionsupporting

confidence: 67%

“…Using site-directed mutagenesis, we delineated SET binding sites to basic amino acid clusters contained within the ICL1 and ICL3 of GnRHR. Importantly, these sites were absent from the ICL2 of GnRHR, which does not bind SET, and their mutation in the ICL3 of M3-MR precluded SET binding (31). Interestingly, in silico analyses revealed the presence of similar basic amino acid clusters within intracellular domains of receptors known to interact with SET, such as the muscarinic (ICL3 and carboxyl-terminal domain) and the ␤ 1 -adrenergic receptors (ICL3), but also within other GPCR, such as rhodopsin and melatonin receptors.…”

Section: Discussionmentioning

confidence: 99%

“…There is very recent evidence showing that SET may regulate GPCR signaling by influencing the phosphorylation of proteins involved in GPCR signaling cascades through the inhibition of PP2A. For example, SET inhibits dephosphorylation of the M3-MR as well as of the ␤ 1 -and ␤ 2 -adrenergic receptors (31,38). Inhibition of ␤ 1 -adrenergic receptor dephosphorylation contributes to the attenuation of its signaling, probably by preventing receptor resensitization (38).…”

Section: Discussionmentioning

confidence: 99%

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SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

Avet

Garrel

Denoyelle

et al. 2013

Journal of Biological Chemistry

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Background: Mechanisms regulating signaling of mammalian GnRH receptor (GnRHR), which exhibits an atypical structure, are poorly known. Results: SET interacts with GnRHR, differentially impacts GnRHR coupling to calcium and cAMP signaling, and enhances GnRH stimulation of Gnrhr promoter activity. Conclusion:This represents the first identification of a GnRHR interacting partner that enhances its coupling to the cAMP pathway. Significance: SET is a novel regulator of GnRHR signaling.In mammals, the receptor of the neuropeptide gonadotropinreleasing hormone (GnRHR) is unique among the G proteincoupled receptor (GPCR) family because it lacks the carboxylterminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids 66 KRKK 69 and 246 RK 247 , located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHRmediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in ␣T3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway. G protein-coupled receptors (GPCR)3 represent the largest family of membranous receptors with more than 1000 members identified so far (1). GPCR process signals from a great diversity of endogenous and exogenous stimuli, including biogenic amines, peptides, glycoproteins, lipids, nucleotides, ions, proteases, photons, odors, and tastes. Because of their importance in a wide range of physiological and pathophysiological processes and the fact that they represent one of the most important drug targets, understanding the mechanisms regulating the efficacy and specificity of GPCR is a current challenge. GPCR-interacting proteins (GIP) have been shown to influence GPCR function (2). They are cytoplasmic proteins that bind to intracellular domains of GPCR and participate in the assembly of receptors into signal transduction complexes or "receptosomes." GIP influence signal transfer from the receptor to G ...

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

Avet

Garrel

Denoyelle

et al. 2013

Journal of Biological Chemistry

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“…Earlier work showed that the i3 loop of M3R interacts with several proteins, including Gq, Gβγ, calmodulin, and SET, and contains a number of phosphorylation sites. 19 , 53 − 56 Our analyses of the region of residues 304–345 did not reveal homology to other proteins or particular structural features, but Gβγ docking and GRK phosphorylation have been mapped to this region. 20 These results suggest that the Gβ5-RGS7 complex may attenuate M3R-stimulated Ca 2+ signaling by hindering Gβγ docking.…”

Section: Discussionmentioning

confidence: 72%

Helix 8 and the i3 Loop of the Muscarinic M3 Receptor Are Crucial Sites for Its Regulation by the Gβ5-RGS7 Complex

et al. 2015

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The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca2+ from intracellular stores or enhance the influx of Ca2+ across the plasma membrane. Here we show that deletion of amino acids 304–345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548–567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3RTP/LP) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.

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“…In the present study, BPA showed down regulation of SET and it was consistently confirmed by 2D-gel, western blot and realtime PCR analyses. SET was first described as part of the SET-CAN fusion gene, a putative oncogene associated with acute undifferentiated leukaemia (von Lindern et al, 1992): SE in SET refers to the patient with leukemia containing SET translocation and the T in SET refers to translocation (Simon et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Set, a Putative Oncogene, As a Biomarker for Prenatal Exposure to Bisphenol A

Lee

Pyo²,

Yang³

2012

Asian Pacific Journal of Cancer Prevention

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Background: Bisphenol A (BPA), an endocrine disrupting chemical, has been suspected to pose carcinogenic risks. However, likely mechanisms are obscure and there are difficulties to estimating its real significance for cancer development. Methods: We therefore studied BPA-induced proteomic alterations in immune organs of ICR mice offspring that were prenatally exposed to BPA (15 and 300 mg/L of drinking water). We performed 2D-gel analyses of samples, considering differences in spleen, exposure levels, sex, and ages. Results: From proteomic analyses, we found various proteins were up-or down-regulated by BPA. Among them, SET, a putative oncogene and inhibitor of phosphatase 2A, was significantly downregulated in a BPA dose-dependent manner. We also confirmed down-regulation of SET in western blot and real time PCR analyses. From gene network analysis, SET is predicted to communicate with other genes including CYP17, which is involved in biosynthesis and metabolism of sex-hormones. Conclusions: This study provided evidence that SET can be applied as a new biomarker for prenatal BPA exposure and suggests a potential new mechanism of action in that BPA may disrupt CYP17 via SET.

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Influence of the Accessory Protein SET on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling

Cited by 7 publications

References 40 publications

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

Helix 8 and the i3 Loop of the Muscarinic M3 Receptor Are Crucial Sites for Its Regulation by the Gβ5-RGS7 Complex

Set, a Putative Oncogene, As a Biomarker for Prenatal Exposure to Bisphenol A

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