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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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LHR endosomes are upstream of cargo trafficking to the EE and contain APPL1.A, cells stably expressing FLAG-LHR were fed with anti-FLAG antibody and then treated with the agonist LH (10 nm) for 15 min. AF555-labeled Tf was added to LH-stimulated cells for the final 2, 5, or 10 min of LH treatment. For the 15-min Tf treatment, Tf was added simultaneously with LH. Cells were then washed with PBS/0.04% EDTA (see “Experimental Procedures”) and then fixed and permeabilized for treatment with AF488-labeled secondary antibody. Representative confocal microscopy images from three independent experiments are shown. The arrows represent examples of FLAG-LHR and Tf colocalization. Scale bar = 5 μm. B, numbers of FLAG-LHR endosomes positive for Tf following 2, 5, 10, or 15 min of Tf treatment were quantified, and the percentage was calculated. Data are mean + S.E.; n = 8 cells, ∼240 endosomes per condition. ***, p < 0.001. C, cells stably expressing FLAG-LHR were transfected with APPL1-GFP and fed with AF555-labeled FLAG antibody (15min) prior to agonist stimulation (LH, 10 nm). Shown is a representative frame from live imaging of cells via confocal microscopy following 20 min of agonist stimulation. Scale bar = 5 μm.
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Figure 2: LHR endosomes are upstream of cargo trafficking to the EE and contain APPL1.A, cells stably expressing FLAG-LHR were fed with anti-FLAG antibody and then treated with the agonist LH (10 nm) for 15 min. AF555-labeled Tf was added to LH-stimulated cells for the final 2, 5, or 10 min of LH treatment. For the 15-min Tf treatment, Tf was added simultaneously with LH. Cells were then washed with PBS/0.04% EDTA (see “Experimental Procedures”) and then fixed and permeabilized for treatment with AF488-labeled secondary antibody. Representative confocal microscopy images from three independent experiments are shown. The arrows represent examples of FLAG-LHR and Tf colocalization. Scale bar = 5 μm. B, numbers of FLAG-LHR endosomes positive for Tf following 2, 5, 10, or 15 min of Tf treatment were quantified, and the percentage was calculated. Data are mean + S.E.; n = 8 cells, ∼240 endosomes per condition. ***, p < 0.001. C, cells stably expressing FLAG-LHR were transfected with APPL1-GFP and fed with AF555-labeled FLAG antibody (15min) prior to agonist stimulation (LH, 10 nm). Shown is a representative frame from live imaging of cells via confocal microscopy following 20 min of agonist stimulation. Scale bar = 5 μm.

Mentions: To determine whether LHR endosomes represent a compartment that is either upstream or a distinct pathway from cargo trafficking to EEs, we employed fluorescently labeled transferrin to monitor the internalization of transferrin (Tf) and its receptor (TfR) following agonist-induced LHR internalization. Because we have shown previously that the LHR heterodimerizes with the B2AR (37), this precluded the coexpression of this receptor with the LHR. Tf/TfR, a classic marker of clathrin-mediated endocytosis and EEs, rapidly internalizes to a common EE pool, like the B2AR, prior to its sorting to the default plasma membrane recycling pathway (30). LHR-expressing cells were treated with labeled FLAG antibody prior to stimulation with LH for 15 min to induce receptor endocytosis. Cells were then treated with labeled Tf for the last 2, 5, or 10 min of LH treatment (Fig. 2, A and B). For cells treated with labeled Tf for 15 min, the labeled Tf was added simultaneously with LH (Fig. 2, A and B, right column and right bar, respectively). Therefore, for all Tf time points, all cells were stimulated with LH for 15 min only. Following 2 min of Tf treatment, many of the internalized labeled Tf colocalized with LHR-positive endosomes, with ∼43% of LHR endosomes positive for Tf (Fig. 2B). This colocalization decreased significantly following a 5 min treatment with labeled Tf, with only ∼10% of LHR endosomes positive for Tf. This is consistent with the rapid internalization of Tf to EEs within 5 min (38, 39). Because Tf transiently colocalizes with the LHR early on in the trafficking of Tf, this suggests that Tf rapidly internalizes through LHR endosomes.


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)

LHR endosomes are upstream of cargo trafficking to the EE and contain APPL1.A, cells stably expressing FLAG-LHR were fed with anti-FLAG antibody and then treated with the agonist LH (10 nm) for 15 min. AF555-labeled Tf was added to LH-stimulated cells for the final 2, 5, or 10 min of LH treatment. For the 15-min Tf treatment, Tf was added simultaneously with LH. Cells were then washed with PBS/0.04% EDTA (see “Experimental Procedures”) and then fixed and permeabilized for treatment with AF488-labeled secondary antibody. Representative confocal microscopy images from three independent experiments are shown. The arrows represent examples of FLAG-LHR and Tf colocalization. Scale bar = 5 μm. B, numbers of FLAG-LHR endosomes positive for Tf following 2, 5, 10, or 15 min of Tf treatment were quantified, and the percentage was calculated. Data are mean + S.E.; n = 8 cells, ∼240 endosomes per condition. ***, p < 0.001. C, cells stably expressing FLAG-LHR were transfected with APPL1-GFP and fed with AF555-labeled FLAG antibody (15min) prior to agonist stimulation (LH, 10 nm). Shown is a representative frame from live imaging of cells via confocal microscopy following 20 min of agonist stimulation. Scale bar = 5 μm.
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Figure 2: LHR endosomes are upstream of cargo trafficking to the EE and contain APPL1.A, cells stably expressing FLAG-LHR were fed with anti-FLAG antibody and then treated with the agonist LH (10 nm) for 15 min. AF555-labeled Tf was added to LH-stimulated cells for the final 2, 5, or 10 min of LH treatment. For the 15-min Tf treatment, Tf was added simultaneously with LH. Cells were then washed with PBS/0.04% EDTA (see “Experimental Procedures”) and then fixed and permeabilized for treatment with AF488-labeled secondary antibody. Representative confocal microscopy images from three independent experiments are shown. The arrows represent examples of FLAG-LHR and Tf colocalization. Scale bar = 5 μm. B, numbers of FLAG-LHR endosomes positive for Tf following 2, 5, 10, or 15 min of Tf treatment were quantified, and the percentage was calculated. Data are mean + S.E.; n = 8 cells, ∼240 endosomes per condition. ***, p < 0.001. C, cells stably expressing FLAG-LHR were transfected with APPL1-GFP and fed with AF555-labeled FLAG antibody (15min) prior to agonist stimulation (LH, 10 nm). Shown is a representative frame from live imaging of cells via confocal microscopy following 20 min of agonist stimulation. Scale bar = 5 μm.
Mentions: To determine whether LHR endosomes represent a compartment that is either upstream or a distinct pathway from cargo trafficking to EEs, we employed fluorescently labeled transferrin to monitor the internalization of transferrin (Tf) and its receptor (TfR) following agonist-induced LHR internalization. Because we have shown previously that the LHR heterodimerizes with the B2AR (37), this precluded the coexpression of this receptor with the LHR. Tf/TfR, a classic marker of clathrin-mediated endocytosis and EEs, rapidly internalizes to a common EE pool, like the B2AR, prior to its sorting to the default plasma membrane recycling pathway (30). LHR-expressing cells were treated with labeled FLAG antibody prior to stimulation with LH for 15 min to induce receptor endocytosis. Cells were then treated with labeled Tf for the last 2, 5, or 10 min of LH treatment (Fig. 2, A and B). For cells treated with labeled Tf for 15 min, the labeled Tf was added simultaneously with LH (Fig. 2, A and B, right column and right bar, respectively). Therefore, for all Tf time points, all cells were stimulated with LH for 15 min only. Following 2 min of Tf treatment, many of the internalized labeled Tf colocalized with LHR-positive endosomes, with ∼43% of LHR endosomes positive for Tf (Fig. 2B). This colocalization decreased significantly following a 5 min treatment with labeled Tf, with only ∼10% of LHR endosomes positive for Tf. This is consistent with the rapid internalization of Tf to EEs within 5 min (38, 39). Because Tf transiently colocalizes with the LHR early on in the trafficking of Tf, this suggests that Tf rapidly internalizes through LHR endosomes.

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