GABAB Receptor Subunit GB1 at the Cell Surface Independently Activates ERK1/2 through IGF-1R Transactivation

BACKGROUND: Functional GABA(B) receptor is believed to require hetero-dimerization between GABA(B1) (GB1) and GABA(B2) (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 GABA(B) 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 GABA(B) 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

In the absence of GB2, GB1asa induction of ERK1/2 phosphorylation is greater than induction by wild type GB1.

<p>(A) Detection of the cell surface expression of GB1 or GB1asa (upper panel) and total expression by Western blots with anti-HA and anti-β-actin (lower panel). (B) Time course of the endogenous ERK1/2 phosphorylation induced by GABA (100 µM) 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.</p

GB1asa can induce ERK1/2 phosphorylation independent of GB2.

<p>(A) Effects of GABA (100 µM) and Baclofen (100 µM) 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 µM; 20 min) is incubated before treatment with GABA (100 µM; 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 µM) 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 ± SEM).</p