GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation

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Abstract

Background: Functional GABAB receptor is believed to require hetero-dimerization between GABAB1 (GB1) and GABAB2 (GB2) subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABAB receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. Methodology/Principal Findings: Here, by using cells overexpressing a GB1 mutant (GB1asa) with the ability to translocate to the cell surface in the absence of GB2, we show that GABAB receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. Conclusions/Significance: Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2. © 2012 Baloucoune et al.

Figures

  • Figure 1. GB1asa can induce ERK1/2 phosphorylation independent of GB2. (A) Effects of GABA (100 mM) and Baclofen (100 mM) on ERK1/2 phosphorylation in cells overexpressing GB1asa over the indicated time course. (B) Effects of CGP54626 on GABA-induced ERK1/2 phosphorylation. CGP54626 (10 mM; 20 min) is incubated before treatment with GABA (100 mM; 3 min). (C) Detection of expression of HAGB1asa alone or HAGB1 in the presence of FlagGB2 by ELISA (upper panel) and Western blots (lower panel). (D) Time course of the ERK1/2 phosphorylation induced by GABA (100 mM) in the HEK293 cells transfected with both GB1 and GB2 or GB1asa alone. The representative western blots are shown under the quantified data of ERK1/2 phosphorylation analyzed from at least three separate experiments (mean 6 SEM). doi:10.1371/journal.pone.0039698.g001
  • Figure 2. GB1asa-mediated ERK1/2 activation is independent of GABAB2. (A) Endogenous expression of GB2 is undetectable in HEK293 cells. Western blots of cell lysates performed with anti-HA or anti-GB1 antibodies (upper panel), anti-Flag or anti-GB2 antibodies (middle panel) and anti-Src antibody (bottom panel). Cells were transfected without or with HAGB1, FlagGB2, HAGB1with FlagGB2 and HAGB1asa. (B) No GB2 mRNA can be detected in HEK293 cells. HEK293 cells transfected with human GB1 or human GB1 with human GB2 are used as negative and positive control respectively. Images are representative of at least three independent RT-PCR analyses. (C) Time course of the ERK1/2 phosphorylation induced by GABA (100 mM) in the absence or presence of CGP7930 (25 mM) in the HEK293 cells transfected with both GB1 and GB2 (left panel) or GB1asa alone (right panel). All western blots shown here are representative of at least three separate experiments. doi:10.1371/journal.pone.0039698.g002
  • Figure 3. GB1asa-mediated ERK1/2 phosphorylation requires IGF-1R transactivation through Gi/o proteins and PLC pathway. (A) Effects of GABA (100 mM) and Baclofen (100 mM) on IGF-1R phosphorylation in cells overexpressing GB1asa for the indicated time course. (B) Effect of AG1024 (upper panel) and shRNA of IGF-1R (3476) (lower panel) on GABA-stimulated ERK1/2 phosphorylation. AG1024 (0.1 mM; 60 min) is incubated before treatment with GABA (100 mM; 5 min) in HEK293 cells overexpressing GB1asa. The shRNA knock-down assay is performed in MEF cells overexpressing GB1asa. (C) Effect of PTX on GABA-stimulated IGF-1R and ERK1/2 phosphorylation. PTX (200 ng/ml; 16 hrs) is incubated before and during treatment with GABA (100 mM; 5 min). (D) Effect of U73122 and U73343 on GABA-stimulated ERK1/2 phosphorylation. U73122 (5 mM; 60 min) or U73343 (5 mM; 60 min) are incubated before treatment with GABA (100 mM; 5 min). The western blots shown are representative of at least three separate experiments. doi:10.1371/journal.pone.0039698.g003
  • Figure 4. In the absence of GB2, GB1asa induction of ERK1/2 phosphorylation is greater than induction by wild type GB1. (A) Detection of the cell surface expression of GB1 or GB1asa (upper panel) and total expression by Western blots with anti-HA and anti-b-actin (lower panel). (B) Time course of the endogenous ERK1/2 phosphorylation induced by GABA (100 mM) in the HEK293 cells transfected with GB1asa or GB1 alone. (C) Schematic representation of the signaling pathway mediated by GB1asa at the cell surface. Activation of ERK1/2 phosphorylation by GB1asa requires Gi/o proteins to activate PLC pathway, which in turn transactivates IGF-IR. doi:10.1371/journal.pone.0039698.g004

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Baloucoune, G. A., Chun, L., Zhang, W., Xu, C., Huang, S., Sun, Q., … Liu, J. (2012). GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation. PLoS ONE, 7(6). https://doi.org/10.1371/journal.pone.0039698

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