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

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.


B2AR activates Gαs exclusively in ASRT endosomal microdomains. (A) Example images of B2AR, an ASRT marker; cortactin; and Nb80. Nb80 localizes to endosomes and tubules (arrowheads). Bars, 5 µm. (B) Example images of B2AR and SS>AA with cortactin and Nb80. Nb80 localizes to ASRT (arrowheads) and non-ASRT SS>AA tubules (arrows). Bars, 2 µm. (C) Percent tubules per cell with cortactin and Nb80, cortactin or Nb80 only, or neither. n = 15 (B2AR) and 21 (SS>AA) cells. (D) Example images of B2AR, cortactin, and Nb37. Nb37 localizes exclusively to the base of B2AR tubules (arrowheads) with cortactin (ASRT). Bars, 5 µm. (E) Example images of B2AR and SS>AA with cortactin and Nb37. Nb37 is not recruited to SS>AA constitutive tubules (arrows). Arrowheads show B2AR in ASRT domains. Bars, 2 µm. (F) Quantitation of the percentage of total tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither. n = 42 (B2AR) and 43 (SS>AA) cells.
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fig2: B2AR activates Gαs exclusively in ASRT endosomal microdomains. (A) Example images of B2AR, an ASRT marker; cortactin; and Nb80. Nb80 localizes to endosomes and tubules (arrowheads). Bars, 5 µm. (B) Example images of B2AR and SS>AA with cortactin and Nb80. Nb80 localizes to ASRT (arrowheads) and non-ASRT SS>AA tubules (arrows). Bars, 2 µm. (C) Percent tubules per cell with cortactin and Nb80, cortactin or Nb80 only, or neither. n = 15 (B2AR) and 21 (SS>AA) cells. (D) Example images of B2AR, cortactin, and Nb37. Nb37 localizes exclusively to the base of B2AR tubules (arrowheads) with cortactin (ASRT). Bars, 5 µm. (E) Example images of B2AR and SS>AA with cortactin and Nb37. Nb37 is not recruited to SS>AA constitutive tubules (arrows). Arrowheads show B2AR in ASRT domains. Bars, 2 µm. (F) Quantitation of the percentage of total tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither. n = 42 (B2AR) and 43 (SS>AA) cells.

Mentions: To test whether B2AR endosomal signaling could be spatially resolved in the context of endosomal microdomains, we used a GFP-tagged nanobody biosensor, Nb80, that recognizes the agonist-activated conformation of B2AR (Westfield et al., 2011; Irannejad et al., 2013). Nb80 was recruited to B2AR endosomes 5 min after iso stimulation (Fig. 2 A), consistent with previous data that B2ARs exist in an active conformation at endosomes (Irannejad et al., 2013). Nb80 localized to all domains of the endosome that contained B2AR, including the ASRT microdomains and constitutive tubules containing SS>AA (Fig. 2, B and C; and Fig. S2 A), suggesting that B2AR is in an active conformation in all regions of the endosome, irrespective of whether B2AR was sorted to sequence-dependent or constitutive recycling tubules. As a direct readout of where Gαs was active, we used GFP-tagged Nb37, a nanobody that recognizes the nucleotide-free form of Gαs (Irannejad et al., 2013). In contrast to Nb80, Nb37 localized to punctate regions on the endosome at the base of B2AR recycling tubules (Fig. 2, D and E). Nb37 localization was largely restricted to domains containing the ASRT domain marker cortactin (Fig. 2, E and F; and Fig. S2 B). Interestingly, Nb37 was still localized only to ASRT domains in SS>AA endosomes, even when active receptors were partitioned to both ASRT domains and constitutive tubules were not marked by ASRT components (Fig. 2 E). 50% of the SS>AA tubules were devoid of both Nb37 and cortactin markers (Fig. 2 F and Fig. S2 B). The distinct localization patterns of the nanobodies that detect active receptor and active Gαs indicate that Gαs activation is restricted to ASRT domains, even though B2AR may be in an active conformation in a broader region of the endosome.


Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling
B2AR activates Gαs exclusively in ASRT endosomal microdomains. (A) Example images of B2AR, an ASRT marker; cortactin; and Nb80. Nb80 localizes to endosomes and tubules (arrowheads). Bars, 5 µm. (B) Example images of B2AR and SS>AA with cortactin and Nb80. Nb80 localizes to ASRT (arrowheads) and non-ASRT SS>AA tubules (arrows). Bars, 2 µm. (C) Percent tubules per cell with cortactin and Nb80, cortactin or Nb80 only, or neither. n = 15 (B2AR) and 21 (SS>AA) cells. (D) Example images of B2AR, cortactin, and Nb37. Nb37 localizes exclusively to the base of B2AR tubules (arrowheads) with cortactin (ASRT). Bars, 5 µm. (E) Example images of B2AR and SS>AA with cortactin and Nb37. Nb37 is not recruited to SS>AA constitutive tubules (arrows). Arrowheads show B2AR in ASRT domains. Bars, 2 µm. (F) Quantitation of the percentage of total tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither. n = 42 (B2AR) and 43 (SS>AA) cells.
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Related In: Results  -  Collection

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fig2: B2AR activates Gαs exclusively in ASRT endosomal microdomains. (A) Example images of B2AR, an ASRT marker; cortactin; and Nb80. Nb80 localizes to endosomes and tubules (arrowheads). Bars, 5 µm. (B) Example images of B2AR and SS>AA with cortactin and Nb80. Nb80 localizes to ASRT (arrowheads) and non-ASRT SS>AA tubules (arrows). Bars, 2 µm. (C) Percent tubules per cell with cortactin and Nb80, cortactin or Nb80 only, or neither. n = 15 (B2AR) and 21 (SS>AA) cells. (D) Example images of B2AR, cortactin, and Nb37. Nb37 localizes exclusively to the base of B2AR tubules (arrowheads) with cortactin (ASRT). Bars, 5 µm. (E) Example images of B2AR and SS>AA with cortactin and Nb37. Nb37 is not recruited to SS>AA constitutive tubules (arrows). Arrowheads show B2AR in ASRT domains. Bars, 2 µm. (F) Quantitation of the percentage of total tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither. n = 42 (B2AR) and 43 (SS>AA) cells.
Mentions: To test whether B2AR endosomal signaling could be spatially resolved in the context of endosomal microdomains, we used a GFP-tagged nanobody biosensor, Nb80, that recognizes the agonist-activated conformation of B2AR (Westfield et al., 2011; Irannejad et al., 2013). Nb80 was recruited to B2AR endosomes 5 min after iso stimulation (Fig. 2 A), consistent with previous data that B2ARs exist in an active conformation at endosomes (Irannejad et al., 2013). Nb80 localized to all domains of the endosome that contained B2AR, including the ASRT microdomains and constitutive tubules containing SS>AA (Fig. 2, B and C; and Fig. S2 A), suggesting that B2AR is in an active conformation in all regions of the endosome, irrespective of whether B2AR was sorted to sequence-dependent or constitutive recycling tubules. As a direct readout of where Gαs was active, we used GFP-tagged Nb37, a nanobody that recognizes the nucleotide-free form of Gαs (Irannejad et al., 2013). In contrast to Nb80, Nb37 localized to punctate regions on the endosome at the base of B2AR recycling tubules (Fig. 2, D and E). Nb37 localization was largely restricted to domains containing the ASRT domain marker cortactin (Fig. 2, E and F; and Fig. S2 B). Interestingly, Nb37 was still localized only to ASRT domains in SS>AA endosomes, even when active receptors were partitioned to both ASRT domains and constitutive tubules were not marked by ASRT components (Fig. 2 E). 50% of the SS>AA tubules were devoid of both Nb37 and cortactin markers (Fig. 2 F and Fig. S2 B). The distinct localization patterns of the nanobodies that detect active receptor and active Gαs indicate that Gαs activation is restricted to ASRT domains, even though B2AR may be in an active conformation in a broader region of the endosome.

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.