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Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.

Tsvetanova NG, von Zastrow M - Nat. Chem. Biol. (2014)

Bottom Line: G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization.Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response.Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of California-San Francisco, San Francisco, California, USA.

ABSTRACT
G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, we wondered whether the endosome-initiated signal encodes any discrete spatial information. Using the β2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signaling specificity based on endosome-mediated spatial encoding of intracellular second messenger production and 'location-aware' downstream transcriptional control.

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Endocytosis of β2-AR is required for cAMP accumulation and transcriptional response(a–b) Real-time cAMP levels were measured using the enzyme-based biosensor pGLO-20F (Promega) in response to bath application of (a) 1 μM or (b) 10 nM isoproterenol in cells pre-treated for 15 min with vehicle (DMSO) (blue) or 30 μM Dyngo (red). Data = average from n = 2–3 experiments. (c–e) Measurement by oligonucleotide microarrays of global gene expression in response to activation of β2-AR. Cells were pre-treated with either DMSO or Dyngo for 15 min as indicated. (c) Heat map showing the median-centered expression of core isoproterenol-responsive target genes (indicated on the y-axis; Supplementary Table 1) across all microarray experiments. (d–e) Scatter plots comparing expression levels for the target genes upon treatment of cells with (d) 1 μM or (e) 10 nM isoproterenol. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. In grey – isoproterenol targets, in red – endocytosis-dependent genes (Supplementary Table 3), dotted line has a slope of 1 (y = x). Arrows indicate RNA levels for PCK1 and DACT2. (f) Confirmation by qRT-PCR of PCK1 and DACT2 expression in response to block of receptor endocytosis. (g) Clathrin knockdown effects on β2-AR signalling in cells transfected with CHC17 or control siRNAs. RNA levels of PCK1 were analyzed by qRT-PCR. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. ND = no drug; Iso = isoproterenol. ** p < 0.005, * p < 0.05, p-values by unpaired t-test; error bars = ± s.e.m.
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Figure 1: Endocytosis of β2-AR is required for cAMP accumulation and transcriptional response(a–b) Real-time cAMP levels were measured using the enzyme-based biosensor pGLO-20F (Promega) in response to bath application of (a) 1 μM or (b) 10 nM isoproterenol in cells pre-treated for 15 min with vehicle (DMSO) (blue) or 30 μM Dyngo (red). Data = average from n = 2–3 experiments. (c–e) Measurement by oligonucleotide microarrays of global gene expression in response to activation of β2-AR. Cells were pre-treated with either DMSO or Dyngo for 15 min as indicated. (c) Heat map showing the median-centered expression of core isoproterenol-responsive target genes (indicated on the y-axis; Supplementary Table 1) across all microarray experiments. (d–e) Scatter plots comparing expression levels for the target genes upon treatment of cells with (d) 1 μM or (e) 10 nM isoproterenol. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. In grey – isoproterenol targets, in red – endocytosis-dependent genes (Supplementary Table 3), dotted line has a slope of 1 (y = x). Arrows indicate RNA levels for PCK1 and DACT2. (f) Confirmation by qRT-PCR of PCK1 and DACT2 expression in response to block of receptor endocytosis. (g) Clathrin knockdown effects on β2-AR signalling in cells transfected with CHC17 or control siRNAs. RNA levels of PCK1 were analyzed by qRT-PCR. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. ND = no drug; Iso = isoproterenol. ** p < 0.005, * p < 0.05, p-values by unpaired t-test; error bars = ± s.e.m.

Mentions: We began by assessing the effects of endosome signalling on the integrated β2-AR response. To do so, we profiled receptor-mediated regulation of cellular gene expression for > 20,000 human genes, and asked if endocytosis is important for this response. HEK293 cells endogenously express β2-ARs at low levels, making them a useful model for studying signalling effects without potential complications of receptor over-expression 6. We examined the endogenous HEK293 β2-AR-cAMP response elicited by the β2-AR agonist isoproterenol at two agonist concentrations: 1 μM, a saturating concentration, and 10 nM, a sub-saturating concentration that is close to the EC50 for stimulating acute cAMP accumulation. Both concentrations of isoproterenol promoted significant β2-AR internalization (Supplementary Results, Supplementary Figure 1a). To examine cAMP production in response to agonist stimulation, we measured real-time accumulation of the second messenger with a previously described luminescence-based cAMP biosensor that localizes diffusely throughout the cytoplasm 3,7,8. While the net cAMP produced in response to 1 μM isoproterenol was greater than that to 10 nM agonist (Figure 1a–b, blue plots), microarray analysis revealed a similar gene expression response elicited by both concentrations of isoproterenol. This indicates that even sub-saturating concentrations of agonist produce net amounts of cAMP capable of triggering efficient transcriptional signalling. We identified a core set of 55 isoproterenol-responsive genes (Supplementary Table 1) that were consistently induced over 1.5-fold in response to both concentrations of isoproterenol. This set is strongly enriched for cAMP response element-binding protein (CREB) target genes 9 (30/55, p < 1.0×10−19 by hypergeometric test) and spans a diverse range of biological processes based on gene ontology (GO) analysis (Supplementary Table 2). To investigate whether endocytosis impacts the β2-AR-mediated transcriptional response, we first took a pharmacological approach using Dyngo, a chemical inhibitor of dynamin that blocks regulated endocytosis of β2-ARs acutely 3,10. Pre-treatment of cells with Dyngo for 15 min was sufficient to strongly (> 90%) and significantly (p = 4.0×10−4 by t-test) inhibit isoproterenol-induced internalization of β2-ARs (Supplementary Figure 1b). Dyngo had little effect on basal cAMP levels as quantified biochemically and no effect whatsoever on cAMP detection by the biosensor (Supplementary Figure 1c–d). However, it markedly reduced the magnitude of isoproterenol-induced cytoplasmic cAMP accumulation at both saturating (Figure 1a) and sub-saturating (Figure 1b) concentrations, confirming previous reports that endosome-localized receptors contribute to the overall cytoplasmic cAMP signal 3.


Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.

Tsvetanova NG, von Zastrow M - Nat. Chem. Biol. (2014)

Endocytosis of β2-AR is required for cAMP accumulation and transcriptional response(a–b) Real-time cAMP levels were measured using the enzyme-based biosensor pGLO-20F (Promega) in response to bath application of (a) 1 μM or (b) 10 nM isoproterenol in cells pre-treated for 15 min with vehicle (DMSO) (blue) or 30 μM Dyngo (red). Data = average from n = 2–3 experiments. (c–e) Measurement by oligonucleotide microarrays of global gene expression in response to activation of β2-AR. Cells were pre-treated with either DMSO or Dyngo for 15 min as indicated. (c) Heat map showing the median-centered expression of core isoproterenol-responsive target genes (indicated on the y-axis; Supplementary Table 1) across all microarray experiments. (d–e) Scatter plots comparing expression levels for the target genes upon treatment of cells with (d) 1 μM or (e) 10 nM isoproterenol. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. In grey – isoproterenol targets, in red – endocytosis-dependent genes (Supplementary Table 3), dotted line has a slope of 1 (y = x). Arrows indicate RNA levels for PCK1 and DACT2. (f) Confirmation by qRT-PCR of PCK1 and DACT2 expression in response to block of receptor endocytosis. (g) Clathrin knockdown effects on β2-AR signalling in cells transfected with CHC17 or control siRNAs. RNA levels of PCK1 were analyzed by qRT-PCR. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. ND = no drug; Iso = isoproterenol. ** p < 0.005, * p < 0.05, p-values by unpaired t-test; error bars = ± s.e.m.
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Figure 1: Endocytosis of β2-AR is required for cAMP accumulation and transcriptional response(a–b) Real-time cAMP levels were measured using the enzyme-based biosensor pGLO-20F (Promega) in response to bath application of (a) 1 μM or (b) 10 nM isoproterenol in cells pre-treated for 15 min with vehicle (DMSO) (blue) or 30 μM Dyngo (red). Data = average from n = 2–3 experiments. (c–e) Measurement by oligonucleotide microarrays of global gene expression in response to activation of β2-AR. Cells were pre-treated with either DMSO or Dyngo for 15 min as indicated. (c) Heat map showing the median-centered expression of core isoproterenol-responsive target genes (indicated on the y-axis; Supplementary Table 1) across all microarray experiments. (d–e) Scatter plots comparing expression levels for the target genes upon treatment of cells with (d) 1 μM or (e) 10 nM isoproterenol. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. In grey – isoproterenol targets, in red – endocytosis-dependent genes (Supplementary Table 3), dotted line has a slope of 1 (y = x). Arrows indicate RNA levels for PCK1 and DACT2. (f) Confirmation by qRT-PCR of PCK1 and DACT2 expression in response to block of receptor endocytosis. (g) Clathrin knockdown effects on β2-AR signalling in cells transfected with CHC17 or control siRNAs. RNA levels of PCK1 were analyzed by qRT-PCR. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. ND = no drug; Iso = isoproterenol. ** p < 0.005, * p < 0.05, p-values by unpaired t-test; error bars = ± s.e.m.
Mentions: We began by assessing the effects of endosome signalling on the integrated β2-AR response. To do so, we profiled receptor-mediated regulation of cellular gene expression for > 20,000 human genes, and asked if endocytosis is important for this response. HEK293 cells endogenously express β2-ARs at low levels, making them a useful model for studying signalling effects without potential complications of receptor over-expression 6. We examined the endogenous HEK293 β2-AR-cAMP response elicited by the β2-AR agonist isoproterenol at two agonist concentrations: 1 μM, a saturating concentration, and 10 nM, a sub-saturating concentration that is close to the EC50 for stimulating acute cAMP accumulation. Both concentrations of isoproterenol promoted significant β2-AR internalization (Supplementary Results, Supplementary Figure 1a). To examine cAMP production in response to agonist stimulation, we measured real-time accumulation of the second messenger with a previously described luminescence-based cAMP biosensor that localizes diffusely throughout the cytoplasm 3,7,8. While the net cAMP produced in response to 1 μM isoproterenol was greater than that to 10 nM agonist (Figure 1a–b, blue plots), microarray analysis revealed a similar gene expression response elicited by both concentrations of isoproterenol. This indicates that even sub-saturating concentrations of agonist produce net amounts of cAMP capable of triggering efficient transcriptional signalling. We identified a core set of 55 isoproterenol-responsive genes (Supplementary Table 1) that were consistently induced over 1.5-fold in response to both concentrations of isoproterenol. This set is strongly enriched for cAMP response element-binding protein (CREB) target genes 9 (30/55, p < 1.0×10−19 by hypergeometric test) and spans a diverse range of biological processes based on gene ontology (GO) analysis (Supplementary Table 2). To investigate whether endocytosis impacts the β2-AR-mediated transcriptional response, we first took a pharmacological approach using Dyngo, a chemical inhibitor of dynamin that blocks regulated endocytosis of β2-ARs acutely 3,10. Pre-treatment of cells with Dyngo for 15 min was sufficient to strongly (> 90%) and significantly (p = 4.0×10−4 by t-test) inhibit isoproterenol-induced internalization of β2-ARs (Supplementary Figure 1b). Dyngo had little effect on basal cAMP levels as quantified biochemically and no effect whatsoever on cAMP detection by the biosensor (Supplementary Figure 1c–d). However, it markedly reduced the magnitude of isoproterenol-induced cytoplasmic cAMP accumulation at both saturating (Figure 1a) and sub-saturating (Figure 1b) concentrations, confirming previous reports that endosome-localized receptors contribute to the overall cytoplasmic cAMP signal 3.

Bottom Line: G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization.Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response.Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of California-San Francisco, San Francisco, California, USA.

ABSTRACT
G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, we wondered whether the endosome-initiated signal encodes any discrete spatial information. Using the β2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signaling specificity based on endosome-mediated spatial encoding of intracellular second messenger production and 'location-aware' downstream transcriptional control.

Show MeSH
Related in: MedlinePlus