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Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling

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ABSTRACT

GPCRs can activate different programs of gene expression from the plasma membrane and the endosome. Bowman et al. show that signaling by endosomal β-2 adrenergic receptors occurs at the microdomains that GPCRs use for sequence-dependent recycling.

No MeSH data available.


B2AR phosphorylation at S345 and S346 restricts B2AR to ASRT endosomal microdomains. (A) Images of iso-induced B2AR endocytosis and PKA inhibition. Before iso, B2ARs localize to the cell surface. 5 min after iso, B2AR redistributes to endosomes and is sorted into tubules (arrowheads). KT was added 5 min after iso. Bars: (main images) 5 µm; (insets) 2 µm. (B) Example images of B2AR and coronin (ASRT marker) with iso and KT. KT causes B2AR to enter non-ASRT domains. (C) Quantitation of percent B2AR in ASRT and non-ASRT tubules. n = 31 (−KT, iso) and 14 (KT) cells. (D) Example images of B2AR and SNX1 (ASRT marker) with iso and KT. (B and D) Arrowheads show B2AR in ASRT domains, and arrows show B2AR in non-ASRT domains. Bars, 2 µm. (E) Quantitation of the percent B2AR in ASRT and non-ASRT tubules. n = 11 (−KT, iso only) and 18 (+KT, PKA inhibited) cells. (F) 5 min after iso, SS>AA also localized to endosomes and tubules. Bars, 5 µm. (G) Example images of B2AR and SS>AA with coronin. SS>AA localizes to both ASRT domains (arrowheads) and constitutive tubules (arrows). Bars, 2 µm. (H) Quantitation of percent B2AR and SS>AA ASRT tubules. SS>AA localizes to both ASRT and non-ASRT tubules. n = 13 (B2AR) and 14 (SS>AA) cells. Error bars in all graphs are SEM across cells.
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fig1: B2AR phosphorylation at S345 and S346 restricts B2AR to ASRT endosomal microdomains. (A) Images of iso-induced B2AR endocytosis and PKA inhibition. Before iso, B2ARs localize to the cell surface. 5 min after iso, B2AR redistributes to endosomes and is sorted into tubules (arrowheads). KT was added 5 min after iso. Bars: (main images) 5 µm; (insets) 2 µm. (B) Example images of B2AR and coronin (ASRT marker) with iso and KT. KT causes B2AR to enter non-ASRT domains. (C) Quantitation of percent B2AR in ASRT and non-ASRT tubules. n = 31 (−KT, iso) and 14 (KT) cells. (D) Example images of B2AR and SNX1 (ASRT marker) with iso and KT. (B and D) Arrowheads show B2AR in ASRT domains, and arrows show B2AR in non-ASRT domains. Bars, 2 µm. (E) Quantitation of the percent B2AR in ASRT and non-ASRT tubules. n = 11 (−KT, iso only) and 18 (+KT, PKA inhibited) cells. (F) 5 min after iso, SS>AA also localized to endosomes and tubules. Bars, 5 µm. (G) Example images of B2AR and SS>AA with coronin. SS>AA localizes to both ASRT domains (arrowheads) and constitutive tubules (arrows). Bars, 2 µm. (H) Quantitation of percent B2AR and SS>AA ASRT tubules. SS>AA localizes to both ASRT and non-ASRT tubules. n = 13 (B2AR) and 14 (SS>AA) cells. Error bars in all graphs are SEM across cells.

Mentions: We first asked whether B2AR endosomal signaling was restricted to functional tubular domains. We directly quantitated agonist-induced redistribution of B2AR between endosomal microdomains in living cells in real time using live-cell confocal fluorescence microscopy in HEK 293 cells stably expressing fluorescently tagged B2AR. We and others have confirmed that this system accurately reflects the trafficking and signaling of B2AR (Kobilka, 1995; Seachrist et al., 2000; Yudowski et al., 2009; Puthenveedu et al., 2010; Han et al., 2012; Vistein and Puthenveedu, 2013). B2AR localized to the cell surface before addition of the B2AR agonist, isoproterenol (iso; Fig. 1 A). 5-min incubation with iso caused endocytosis and redistributed B2ARs to endosomes (Fig. 1 A). Within endosomal membranes, B2AR localized to tubular structures—previously characterized as ASRT domains that mediate sequence-dependent recycling—that are biochemically distinct from tubules that mediate constitutive recycling (Puthenveedu et al., 2010; Vistein and Puthenveedu, 2013). PKA inhibition increased the percentage of B2AR endosomes with more than one B2AR tubule (Fig. 1 A and Fig. S1 A), consistent with B2AR sorting into both ASRT domains and constitutive tubules (Puthenveedu et al., 2010; Vistein and Puthenveedu, 2013).


Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling
B2AR phosphorylation at S345 and S346 restricts B2AR to ASRT endosomal microdomains. (A) Images of iso-induced B2AR endocytosis and PKA inhibition. Before iso, B2ARs localize to the cell surface. 5 min after iso, B2AR redistributes to endosomes and is sorted into tubules (arrowheads). KT was added 5 min after iso. Bars: (main images) 5 µm; (insets) 2 µm. (B) Example images of B2AR and coronin (ASRT marker) with iso and KT. KT causes B2AR to enter non-ASRT domains. (C) Quantitation of percent B2AR in ASRT and non-ASRT tubules. n = 31 (−KT, iso) and 14 (KT) cells. (D) Example images of B2AR and SNX1 (ASRT marker) with iso and KT. (B and D) Arrowheads show B2AR in ASRT domains, and arrows show B2AR in non-ASRT domains. Bars, 2 µm. (E) Quantitation of the percent B2AR in ASRT and non-ASRT tubules. n = 11 (−KT, iso only) and 18 (+KT, PKA inhibited) cells. (F) 5 min after iso, SS>AA also localized to endosomes and tubules. Bars, 5 µm. (G) Example images of B2AR and SS>AA with coronin. SS>AA localizes to both ASRT domains (arrowheads) and constitutive tubules (arrows). Bars, 2 µm. (H) Quantitation of percent B2AR and SS>AA ASRT tubules. SS>AA localizes to both ASRT and non-ASRT tubules. n = 13 (B2AR) and 14 (SS>AA) cells. Error bars in all graphs are SEM across cells.
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Related In: Results  -  Collection

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fig1: B2AR phosphorylation at S345 and S346 restricts B2AR to ASRT endosomal microdomains. (A) Images of iso-induced B2AR endocytosis and PKA inhibition. Before iso, B2ARs localize to the cell surface. 5 min after iso, B2AR redistributes to endosomes and is sorted into tubules (arrowheads). KT was added 5 min after iso. Bars: (main images) 5 µm; (insets) 2 µm. (B) Example images of B2AR and coronin (ASRT marker) with iso and KT. KT causes B2AR to enter non-ASRT domains. (C) Quantitation of percent B2AR in ASRT and non-ASRT tubules. n = 31 (−KT, iso) and 14 (KT) cells. (D) Example images of B2AR and SNX1 (ASRT marker) with iso and KT. (B and D) Arrowheads show B2AR in ASRT domains, and arrows show B2AR in non-ASRT domains. Bars, 2 µm. (E) Quantitation of the percent B2AR in ASRT and non-ASRT tubules. n = 11 (−KT, iso only) and 18 (+KT, PKA inhibited) cells. (F) 5 min after iso, SS>AA also localized to endosomes and tubules. Bars, 5 µm. (G) Example images of B2AR and SS>AA with coronin. SS>AA localizes to both ASRT domains (arrowheads) and constitutive tubules (arrows). Bars, 2 µm. (H) Quantitation of percent B2AR and SS>AA ASRT tubules. SS>AA localizes to both ASRT and non-ASRT tubules. n = 13 (B2AR) and 14 (SS>AA) cells. Error bars in all graphs are SEM across cells.
Mentions: We first asked whether B2AR endosomal signaling was restricted to functional tubular domains. We directly quantitated agonist-induced redistribution of B2AR between endosomal microdomains in living cells in real time using live-cell confocal fluorescence microscopy in HEK 293 cells stably expressing fluorescently tagged B2AR. We and others have confirmed that this system accurately reflects the trafficking and signaling of B2AR (Kobilka, 1995; Seachrist et al., 2000; Yudowski et al., 2009; Puthenveedu et al., 2010; Han et al., 2012; Vistein and Puthenveedu, 2013). B2AR localized to the cell surface before addition of the B2AR agonist, isoproterenol (iso; Fig. 1 A). 5-min incubation with iso caused endocytosis and redistributed B2ARs to endosomes (Fig. 1 A). Within endosomal membranes, B2AR localized to tubular structures—previously characterized as ASRT domains that mediate sequence-dependent recycling—that are biochemically distinct from tubules that mediate constitutive recycling (Puthenveedu et al., 2010; Vistein and Puthenveedu, 2013). PKA inhibition increased the percentage of B2AR endosomes with more than one B2AR tubule (Fig. 1 A and Fig. S1 A), consistent with B2AR sorting into both ASRT domains and constitutive tubules (Puthenveedu et al., 2010; Vistein and Puthenveedu, 2013).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

GPCRs can activate different programs of gene expression from the plasma membrane and the endosome. Bowman et al. show that signaling by endosomal β-2 adrenergic receptors occurs at the microdomains that GPCRs use for sequence-dependent recycling.

No MeSH data available.