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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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Targeting of LHR to pre-EEs requires the distal C-terminal tail.A, schematic of the LHR C-terminal tail. Residues highlighted in red have been described to be essential for receptor recycling (29, 40, 41). The arrow indicates the residue mutated to a stop codon to create truncation mutant LHR-683T. B and C, agonist-induced (LH, 10 nm; isoproterenol, 10 μm) internalization (B) and recycling (C) of the LHR, LHR-683T, and B2AR were quantitatively measured by flow cytometry. Data are mean ± S.E., n = 3. D, representative frame from a time-lapse movie of cells stably expressing either LHR or LHR-683T treated with an agonist, indicating the difference in size of endosomes to which each receptor internalizes. Scale bars = 5 μm and 1 μm (insets). E, average diameter of endosomes containing the LHR, LHR-683T, or B2AR following treatment with an agonist across the indicated times. For each time point, 10 endosomes are measured across three to four movies for each receptor. Data are mean ± S.E. F, representative confocal images of fixed cells stably expressing FLAG-LHR-683T following 30 min of agonist treatment and treated with anti-EEA1 antibody. The arrows indicate examples of colocalization of the receptor with EEA1. Scale bars = 5 μm. G, the percentage of receptor positive endosomes with EEA1 was quantified for the LHR, LHR 683T, and B2AR. Data are mean + S.E. (n = 15 cells, 220 endosomes for each receptor). ***, p < 0.001. See also supplemental Movie S3.
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Figure 3: Targeting of LHR to pre-EEs requires the distal C-terminal tail.A, schematic of the LHR C-terminal tail. Residues highlighted in red have been described to be essential for receptor recycling (29, 40, 41). The arrow indicates the residue mutated to a stop codon to create truncation mutant LHR-683T. B and C, agonist-induced (LH, 10 nm; isoproterenol, 10 μm) internalization (B) and recycling (C) of the LHR, LHR-683T, and B2AR were quantitatively measured by flow cytometry. Data are mean ± S.E., n = 3. D, representative frame from a time-lapse movie of cells stably expressing either LHR or LHR-683T treated with an agonist, indicating the difference in size of endosomes to which each receptor internalizes. Scale bars = 5 μm and 1 μm (insets). E, average diameter of endosomes containing the LHR, LHR-683T, or B2AR following treatment with an agonist across the indicated times. For each time point, 10 endosomes are measured across three to four movies for each receptor. Data are mean ± S.E. F, representative confocal images of fixed cells stably expressing FLAG-LHR-683T following 30 min of agonist treatment and treated with anti-EEA1 antibody. The arrows indicate examples of colocalization of the receptor with EEA1. Scale bars = 5 μm. G, the percentage of receptor positive endosomes with EEA1 was quantified for the LHR, LHR 683T, and B2AR. Data are mean + S.E. (n = 15 cells, 220 endosomes for each receptor). ***, p < 0.001. See also supplemental Movie S3.

Mentions: We next assessed whether the postendocytic sorting of the LHR to pre-EEs is a regulated, receptor-driven process. The distal region of the LHR C-terminal tail (C-tail) contains the sequence required for its plasma membrane recycling that also includes a known PDZ ligand (29, 40, 41) (Fig. 3A). The receptor was truncated to remove its recycling sequence and PDZ ligand (LHR-683T). Accordingly internalized LHR-683T is unable to recycle and thus exhibits greater internalization (Fig. 3B-C) as previously shown (40). However, upon live imaging of this LHR mutant, we observed that the receptor was able to internalize in to larger endosomes than the full-length LHR, with a similar size and kinetic profile to that of B2AR (Fig. 3D-E and Movie S3). Following 30 min of agonist treatment, a significantly greater number of LHR-683T endosomes were positive for EEA1, compared with LHR (Fig. 3F-G) indicating that the truncated receptor localized primarily to the EE compartment. These results demonstrate that the LHR distal C-tail is necessary for receptor targeting to small EEA1-negative 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)

Targeting of LHR to pre-EEs requires the distal C-terminal tail.A, schematic of the LHR C-terminal tail. Residues highlighted in red have been described to be essential for receptor recycling (29, 40, 41). The arrow indicates the residue mutated to a stop codon to create truncation mutant LHR-683T. B and C, agonist-induced (LH, 10 nm; isoproterenol, 10 μm) internalization (B) and recycling (C) of the LHR, LHR-683T, and B2AR were quantitatively measured by flow cytometry. Data are mean ± S.E., n = 3. D, representative frame from a time-lapse movie of cells stably expressing either LHR or LHR-683T treated with an agonist, indicating the difference in size of endosomes to which each receptor internalizes. Scale bars = 5 μm and 1 μm (insets). E, average diameter of endosomes containing the LHR, LHR-683T, or B2AR following treatment with an agonist across the indicated times. For each time point, 10 endosomes are measured across three to four movies for each receptor. Data are mean ± S.E. F, representative confocal images of fixed cells stably expressing FLAG-LHR-683T following 30 min of agonist treatment and treated with anti-EEA1 antibody. The arrows indicate examples of colocalization of the receptor with EEA1. Scale bars = 5 μm. G, the percentage of receptor positive endosomes with EEA1 was quantified for the LHR, LHR 683T, and B2AR. Data are mean + S.E. (n = 15 cells, 220 endosomes for each receptor). ***, p < 0.001. See also supplemental Movie S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Targeting of LHR to pre-EEs requires the distal C-terminal tail.A, schematic of the LHR C-terminal tail. Residues highlighted in red have been described to be essential for receptor recycling (29, 40, 41). The arrow indicates the residue mutated to a stop codon to create truncation mutant LHR-683T. B and C, agonist-induced (LH, 10 nm; isoproterenol, 10 μm) internalization (B) and recycling (C) of the LHR, LHR-683T, and B2AR were quantitatively measured by flow cytometry. Data are mean ± S.E., n = 3. D, representative frame from a time-lapse movie of cells stably expressing either LHR or LHR-683T treated with an agonist, indicating the difference in size of endosomes to which each receptor internalizes. Scale bars = 5 μm and 1 μm (insets). E, average diameter of endosomes containing the LHR, LHR-683T, or B2AR following treatment with an agonist across the indicated times. For each time point, 10 endosomes are measured across three to four movies for each receptor. Data are mean ± S.E. F, representative confocal images of fixed cells stably expressing FLAG-LHR-683T following 30 min of agonist treatment and treated with anti-EEA1 antibody. The arrows indicate examples of colocalization of the receptor with EEA1. Scale bars = 5 μm. G, the percentage of receptor positive endosomes with EEA1 was quantified for the LHR, LHR 683T, and B2AR. Data are mean + S.E. (n = 15 cells, 220 endosomes for each receptor). ***, p < 0.001. See also supplemental Movie S3.
Mentions: We next assessed whether the postendocytic sorting of the LHR to pre-EEs is a regulated, receptor-driven process. The distal region of the LHR C-terminal tail (C-tail) contains the sequence required for its plasma membrane recycling that also includes a known PDZ ligand (29, 40, 41) (Fig. 3A). The receptor was truncated to remove its recycling sequence and PDZ ligand (LHR-683T). Accordingly internalized LHR-683T is unable to recycle and thus exhibits greater internalization (Fig. 3B-C) as previously shown (40). However, upon live imaging of this LHR mutant, we observed that the receptor was able to internalize in to larger endosomes than the full-length LHR, with a similar size and kinetic profile to that of B2AR (Fig. 3D-E and Movie S3). Following 30 min of agonist treatment, a significantly greater number of LHR-683T endosomes were positive for EEA1, compared with LHR (Fig. 3F-G) indicating that the truncated receptor localized primarily to the EE compartment. These results demonstrate that the LHR distal C-tail is necessary for receptor targeting to small EEA1-negative 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