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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.


The localization of B2AR to ASRT or bulk recycling domains does not influence global cAMP levels. (A) Example images of Epac and B2AR. 1–2 min after iso, the CFP/FRET ratio increases as cAMP is produced and FRET decreases. Bar, 10 µm. (B) CFP/FRET ratio across multiple B2AR cells. Error bars are SEM across cells. n = 10 cells. (C) The total cAMP produced at 20 min increased significantly for both B2AR and SS>AA. Data are Tukey box plots from 13 (B2AR) and 10 (SS>AA) cells.
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fig4: The localization of B2AR to ASRT or bulk recycling domains does not influence global cAMP levels. (A) Example images of Epac and B2AR. 1–2 min after iso, the CFP/FRET ratio increases as cAMP is produced and FRET decreases. Bar, 10 µm. (B) CFP/FRET ratio across multiple B2AR cells. Error bars are SEM across cells. n = 10 cells. (C) The total cAMP produced at 20 min increased significantly for both B2AR and SS>AA. Data are Tukey box plots from 13 (B2AR) and 10 (SS>AA) cells.

Mentions: Emerging data indicate that production of cAMP signaling at the endosome and the cell surface have distinct downstream effects, as they activate the transcription of different sets of downstream genes (Tsvetanova and von Zastrow, 2014). Based on our data that Nb37 was not recruited to constitutive recycling tubules (Fig. 2, D–F), we hypothesized that the strength of endosomal G protein signaling could be controlled by the dynamic relocation of B2AR to constitutive recycling tubules. To test this, we measured total cAMP response in conditions where B2AR localized exclusively to ASRT domains or to bulk recycling domains. We used a Forster resonance energy transfer (FRET)–based sensor (DiPilato et al., 2004; Ponsioen et al., 2004), which measures cAMP-induced conformational changes in exchange proteins activated by cAMP (Epac’s), to resolve cAMP production over time. When cells expressing this sensor and either B2AR or SS>AA were exposed to iso, cAMP levels increased within 2 min. This increase was sustained over 25 min, and B2AR endocytosis was visible within 2 min of iso stimulation in the same cells (Fig. 4, A and B). Total cAMP increased significantly for both B2AR and SS>AA to a similar degree 20 min after iso (Fig. 4 C), indicating that the SS>AA was fully competent to signal through cAMP, comparable to B2AR.


Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling
The localization of B2AR to ASRT or bulk recycling domains does not influence global cAMP levels. (A) Example images of Epac and B2AR. 1–2 min after iso, the CFP/FRET ratio increases as cAMP is produced and FRET decreases. Bar, 10 µm. (B) CFP/FRET ratio across multiple B2AR cells. Error bars are SEM across cells. n = 10 cells. (C) The total cAMP produced at 20 min increased significantly for both B2AR and SS>AA. Data are Tukey box plots from 13 (B2AR) and 10 (SS>AA) cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037407&req=5

fig4: The localization of B2AR to ASRT or bulk recycling domains does not influence global cAMP levels. (A) Example images of Epac and B2AR. 1–2 min after iso, the CFP/FRET ratio increases as cAMP is produced and FRET decreases. Bar, 10 µm. (B) CFP/FRET ratio across multiple B2AR cells. Error bars are SEM across cells. n = 10 cells. (C) The total cAMP produced at 20 min increased significantly for both B2AR and SS>AA. Data are Tukey box plots from 13 (B2AR) and 10 (SS>AA) cells.
Mentions: Emerging data indicate that production of cAMP signaling at the endosome and the cell surface have distinct downstream effects, as they activate the transcription of different sets of downstream genes (Tsvetanova and von Zastrow, 2014). Based on our data that Nb37 was not recruited to constitutive recycling tubules (Fig. 2, D–F), we hypothesized that the strength of endosomal G protein signaling could be controlled by the dynamic relocation of B2AR to constitutive recycling tubules. To test this, we measured total cAMP response in conditions where B2AR localized exclusively to ASRT domains or to bulk recycling domains. We used a Forster resonance energy transfer (FRET)–based sensor (DiPilato et al., 2004; Ponsioen et al., 2004), which measures cAMP-induced conformational changes in exchange proteins activated by cAMP (Epac’s), to resolve cAMP production over time. When cells expressing this sensor and either B2AR or SS>AA were exposed to iso, cAMP levels increased within 2 min. This increase was sustained over 25 min, and B2AR endocytosis was visible within 2 min of iso stimulation in the same cells (Fig. 4, A and B). Total cAMP increased significantly for both B2AR and SS>AA to a similar degree 20 min after iso (Fig. 4 C), indicating that the SS>AA was fully competent to signal through cAMP, comparable to B2AR.

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.