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Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

Jean-Alphonse F, Bowersox S, Chen S, Beard G, Puthenveedu MA, Hanyaloglu AC - J. Biol. Chem. (2013)

Bottom Line: Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling.Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment.We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, London W12 0NN, United Kingdom and.

ABSTRACT
Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

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The LHR and B2AR traffic to distinct compartments in the endocytic pathway.A, HEK 293 cells stably expressing FLAG-tagged LHR or B2AR were labeled with fluorescently tagged anti-FLAG antibodies and imaged live with confocal microscopy before and after agonist treatment. The LHR was stimulated with 10 nm LH, and the B2AR was stimulated with 10 μm isoproterenol. The frames shown were taken from a time-lapse series of supplemental Movies S1 and S2. B, the size of the LHR or B2AR containing endosomes was assessed by measuring the diameter of 10 endosomes at each time point stated across three to four movies. Data represent mean ± S.E. C, cells expressing either the LHR or B2AR were fed with anti-FLAG antibody and treated with the agonist for 10 min. Cells were fixed, permeabilized, and stained with an anti-EEA1 antibody and imaged with confocal microscopy. The images shown are representative of 16 cells. D, numbers of LHRs or B2ARs containing endosomes positive for EEA1 following either 10 or 30 min of agonist stimulation were quantified, and the percentage was calculated. Data are mean + S.E.; n = 16 cells, 298 and 295 endosomes respectively for each receptor. E and F, cells were treated as in C and D except that cells were transfected with the PI3P marker 2xFYVE-GFP F, and the number of receptor-containing endosomes positive for 2xFYVE-GFP was quantified (n = 24 and 22 cells, respectively; LHR and B2AR, ∼980 and 500 endosomes, respectively). The arrows represent examples of colocalization. Scale bars = 5 μm. ***, p < 0.001. See also supplemental Movies S1 and S2.
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Figure 1: The LHR and B2AR traffic to distinct compartments in the endocytic pathway.A, HEK 293 cells stably expressing FLAG-tagged LHR or B2AR were labeled with fluorescently tagged anti-FLAG antibodies and imaged live with confocal microscopy before and after agonist treatment. The LHR was stimulated with 10 nm LH, and the B2AR was stimulated with 10 μm isoproterenol. The frames shown were taken from a time-lapse series of supplemental Movies S1 and S2. B, the size of the LHR or B2AR containing endosomes was assessed by measuring the diameter of 10 endosomes at each time point stated across three to four movies. Data represent mean ± S.E. C, cells expressing either the LHR or B2AR were fed with anti-FLAG antibody and treated with the agonist for 10 min. Cells were fixed, permeabilized, and stained with an anti-EEA1 antibody and imaged with confocal microscopy. The images shown are representative of 16 cells. D, numbers of LHRs or B2ARs containing endosomes positive for EEA1 following either 10 or 30 min of agonist stimulation were quantified, and the percentage was calculated. Data are mean + S.E.; n = 16 cells, 298 and 295 endosomes respectively for each receptor. E and F, cells were treated as in C and D except that cells were transfected with the PI3P marker 2xFYVE-GFP F, and the number of receptor-containing endosomes positive for 2xFYVE-GFP was quantified (n = 24 and 22 cells, respectively; LHR and B2AR, ∼980 and 500 endosomes, respectively). The arrows represent examples of colocalization. Scale bars = 5 μm. ***, p < 0.001. See also supplemental Movies S1 and S2.

Mentions: We compared the trafficking and endosomal targeting of two physiologically relevant Gαs-coupled GPCRs for which membrane trafficking is critical for signaling, the luteinizing hormone receptor (LHR), and the β2-adrenergic receptor (B2AR) (12, 24–26). Upon activation, both the LHR and B2AR are known to bind β-arrestin, undergo clathrin-mediated endocytosis, and are sorted to a sequence-dependent/regulated recycling pathway (12, 18, 20, 27–29). To study the endosomal targeting of these two receptors, we first visualized the dynamics of receptor sorting in live HEK 293 cells stably expressing FLAG-tagged B2AR or LHR using confocal microscopy. Both receptors were observed on the cell surface before addition of the agonist (Fig. 1A), whereas after agonist addition, both the B2AR and the LHR internalized and appeared in endosomes within 5 min of agonist stimulation, although this was more evident for the B2AR than the LHR at this time point (Fig. 1A and supplemental movies S1 and S2). However, the most striking observation was the physical difference in the size of LHR endosomes compared with B2AR. The larger size of the B2AR endosomes enables visualization of the endosomal lumen and the receptor on the limiting membrane of the endosome, as observed previously in live cells (30, 31). The measurement of endosome size over time revealed that the B2AR enters an endosomal structure of 1200–1400 nm in diameter within 4 min of agonist treatment (Fig. 1B). This is a size consistent with prior observations of the recycling Fc receptor trafficking to EEA1-positive EEs in a distinct cell type (32) and, thus, is not a feature of transfection or HEK 293 cells. Although LHR endosome size also increases over time, it is enriched in a smaller endosome population of 400–500 nm where the limiting membrane and lumen are not visible at the resolution level of confocal microscopy. The receptor reaches this endosome population size within 3 min of agonist stimulation (Fig. 1B), although there was a delay in the appearance of visible LHR endosomes following 90 s of agonist treatment as compared with the B2AR (Fig. 1B).


Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

Jean-Alphonse F, Bowersox S, Chen S, Beard G, Puthenveedu MA, Hanyaloglu AC - J. Biol. Chem. (2013)

The LHR and B2AR traffic to distinct compartments in the endocytic pathway.A, HEK 293 cells stably expressing FLAG-tagged LHR or B2AR were labeled with fluorescently tagged anti-FLAG antibodies and imaged live with confocal microscopy before and after agonist treatment. The LHR was stimulated with 10 nm LH, and the B2AR was stimulated with 10 μm isoproterenol. The frames shown were taken from a time-lapse series of supplemental Movies S1 and S2. B, the size of the LHR or B2AR containing endosomes was assessed by measuring the diameter of 10 endosomes at each time point stated across three to four movies. Data represent mean ± S.E. C, cells expressing either the LHR or B2AR were fed with anti-FLAG antibody and treated with the agonist for 10 min. Cells were fixed, permeabilized, and stained with an anti-EEA1 antibody and imaged with confocal microscopy. The images shown are representative of 16 cells. D, numbers of LHRs or B2ARs containing endosomes positive for EEA1 following either 10 or 30 min of agonist stimulation were quantified, and the percentage was calculated. Data are mean + S.E.; n = 16 cells, 298 and 295 endosomes respectively for each receptor. E and F, cells were treated as in C and D except that cells were transfected with the PI3P marker 2xFYVE-GFP F, and the number of receptor-containing endosomes positive for 2xFYVE-GFP was quantified (n = 24 and 22 cells, respectively; LHR and B2AR, ∼980 and 500 endosomes, respectively). The arrows represent examples of colocalization. Scale bars = 5 μm. ***, p < 0.001. See also supplemental Movies S1 and S2.
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Figure 1: The LHR and B2AR traffic to distinct compartments in the endocytic pathway.A, HEK 293 cells stably expressing FLAG-tagged LHR or B2AR were labeled with fluorescently tagged anti-FLAG antibodies and imaged live with confocal microscopy before and after agonist treatment. The LHR was stimulated with 10 nm LH, and the B2AR was stimulated with 10 μm isoproterenol. The frames shown were taken from a time-lapse series of supplemental Movies S1 and S2. B, the size of the LHR or B2AR containing endosomes was assessed by measuring the diameter of 10 endosomes at each time point stated across three to four movies. Data represent mean ± S.E. C, cells expressing either the LHR or B2AR were fed with anti-FLAG antibody and treated with the agonist for 10 min. Cells were fixed, permeabilized, and stained with an anti-EEA1 antibody and imaged with confocal microscopy. The images shown are representative of 16 cells. D, numbers of LHRs or B2ARs containing endosomes positive for EEA1 following either 10 or 30 min of agonist stimulation were quantified, and the percentage was calculated. Data are mean + S.E.; n = 16 cells, 298 and 295 endosomes respectively for each receptor. E and F, cells were treated as in C and D except that cells were transfected with the PI3P marker 2xFYVE-GFP F, and the number of receptor-containing endosomes positive for 2xFYVE-GFP was quantified (n = 24 and 22 cells, respectively; LHR and B2AR, ∼980 and 500 endosomes, respectively). The arrows represent examples of colocalization. Scale bars = 5 μm. ***, p < 0.001. See also supplemental Movies S1 and S2.
Mentions: We compared the trafficking and endosomal targeting of two physiologically relevant Gαs-coupled GPCRs for which membrane trafficking is critical for signaling, the luteinizing hormone receptor (LHR), and the β2-adrenergic receptor (B2AR) (12, 24–26). Upon activation, both the LHR and B2AR are known to bind β-arrestin, undergo clathrin-mediated endocytosis, and are sorted to a sequence-dependent/regulated recycling pathway (12, 18, 20, 27–29). To study the endosomal targeting of these two receptors, we first visualized the dynamics of receptor sorting in live HEK 293 cells stably expressing FLAG-tagged B2AR or LHR using confocal microscopy. Both receptors were observed on the cell surface before addition of the agonist (Fig. 1A), whereas after agonist addition, both the B2AR and the LHR internalized and appeared in endosomes within 5 min of agonist stimulation, although this was more evident for the B2AR than the LHR at this time point (Fig. 1A and supplemental movies S1 and S2). However, the most striking observation was the physical difference in the size of LHR endosomes compared with B2AR. The larger size of the B2AR endosomes enables visualization of the endosomal lumen and the receptor on the limiting membrane of the endosome, as observed previously in live cells (30, 31). The measurement of endosome size over time revealed that the B2AR enters an endosomal structure of 1200–1400 nm in diameter within 4 min of agonist treatment (Fig. 1B). This is a size consistent with prior observations of the recycling Fc receptor trafficking to EEA1-positive EEs in a distinct cell type (32) and, thus, is not a feature of transfection or HEK 293 cells. Although LHR endosome size also increases over time, it is enriched in a smaller endosome population of 400–500 nm where the limiting membrane and lumen are not visible at the resolution level of confocal microscopy. The receptor reaches this endosome population size within 3 min of agonist stimulation (Fig. 1B), although there was a delay in the appearance of visible LHR endosomes following 90 s of agonist treatment as compared with the B2AR (Fig. 1B).

Bottom Line: Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling.Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment.We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, London W12 0NN, United Kingdom and.

ABSTRACT
Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

Show MeSH
Related in: MedlinePlus