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G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors - PubMed

  • ️Thu Jan 01 2009

Review

G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors

J-P Pin et al. J Physiol. 2009.

Abstract

G-protein-coupled receptors (GPCRs) are key players in the precise tuning of intercellullar communication. In the brain, both major neurotransmitters, glutamate and GABA, act on specific GPCRs [the metabotropic glutamate (mGlu) and GABA(B) receptors] to modulate synaptic transmission. These receptors are encoded by the largest gene family, and have been found to associate into both homo- and hetero-oligomers, which increases the complexity of this cell communication system. Here we show that dimerization is required for mGlu and GABA(B) receptors to function, since the activation process requires a relative movement between the subunits to occur. We will also show that, in contrast to the mGlu receptors, which form strict dimers, the GABA(B) receptors assemble into larger complexes, both in transfected cells and in the brain, resulting in a decreased G-protein coupling efficacy. We propose that GABA(B) receptor oligomerization offers a way to increase the possibility of modulating receptor signalling and activity, allowing the same receptor protein to have specific properties in neurons at different locations.

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Figures

Figure 1
Figure 1. A model for the activation mechanism of the heterodimeric GABAB receptor based on the solved three-dimensional structure of the dimeric extracellular domain of mGlu1

GABAB1 is in blue, GABAB2 in yellow. Binding of GABA in the cleft of GABAB1 Venus Flytrap domain (VFT) leads to domain closure and a reorientation of both VFTs in the dimer. This is expected to induce a relative movement of the seven transmembrane (7TM) domains, allowing activation of GABAB2 7TM. A similar activation mechanism is proposed for the mGlu receptors even though these have an additional cystein-rich domain that links their VFT to their 7TM.

Figure 2
Figure 2. Insertion of an N-glycan at the lobe 2 interface of GABAB1 prevents GABAB receptor activation

The illustrated models depict the resting conformation with both VFTs open (Roo) and the active conformation with one VFT closed and the other open (Aco), as based on the solved structure of the mGlu1 VFT dimer without (Roo) and with bound agonist (Aco). Intracellular Ca signals geberated by different concentrations of GABA were measured in cells expressing the wild-type GABAB receptor (GB2-WT), or that containg the GB2 subunit carrying an additional N-glycosylation site at position 209 (GB2-N209), or a variant not glycosylated (GB2-Q209). Note that replacement of the Asn residue in the glycosylation site by a Gln residue that is not glycosylated restores function. Adapted from Rondard et al. (2008).

Figure 4
Figure 4. GABAB receptors organize into larger complexes

GABAB1 (blue), GABAB2 (yellow) or both subunits were fused to a snap-tag (ST) at their N-termini, expressed at various densities in transfected cells, and labelled with benzylguanine derivatives carrying either Eu-crytate (TR-FRET donor) or d2-crytate (TR-FRET acceptor). The TR-FRET measurements revealed a close proximity between GABAB1 and GABAB2 subunits, but also a similar FRET between two GABAB1 subunits. In contrast, very low FRET was measured between two GABAB2 subunits. Note that the FRET emission is proportional to the amount of receptors at the cell surface (represented here as the amount of ST that can be labelled on intact cells) and is clearly detectable for densities similar to those reported in brain membranes or in cultured cortical neurons (arrows in the graph). In the graph, TR-FRET efficacy is represented by the slope of the curve and is then constant over the receptor densities analysed. Adapted from Maurel et al. (2008).

Figure 3
Figure 3. Metabotropic glutamate receptors form strict dimers at the cell surface

mGlu5 subunits carrying the C-tail of GABAB1 (mG5c1 in orange, not reaching the cell surface alone) or GABAB2 (mG5c2, in green, allowing targeting to the cell surface of the C1-C2 combination), and taged with a myc and HA epitope, respectively, were labelled with FRET-compatible anti-HA antibodies only, or with a combination of anti-HA and anti-myc antibodies. Experiments were performed with cells expressing different amounts of receptors at the cells surface as quantified by ELISA. Note that a large TR-FRET emission, proportional to the amount of cell surface receptors, is observed when both subunits of the dimer are labelled. In contrast, no TR-FRET can be measured if only one subunit per dimer is labelled. Adapted from Kniazeff et al. (2004a).

Figure 5
Figure 5. Schematic view of the oligomeric assembly of mGlu and GABAB receptors

While the mGlu receptors are strict dimers, with a single subunit at a time activating G-proteins (Goudet et al. 2005; Hlavackova et al. 2005), the GABAB receptors assemble into dimers of dimers via interaction of the GABAB1 subunits (blue), with possibly a single receptor entity activating a G-protein at a time.

Figure 6
Figure 6. Disruption of GABAB oligomers increases G-protein coupling efficacy

Left graph: FRET signals between the ST-labelled GB1 subunits co-expressed with a GB2 subunits carrying an ER retention signal (GB2-KKXX), as a function of the cell surface expression of the truncated form of GB1 (HA-GB1-HD). Right panel, Ca-mediated responses generated by increasing concentration of GABA in cells expresing GB1+GB2-KKXX alone (Back squares), or in the presence of a excess HA-CD4 (blue triangles), or the truncated form of GB1 (HA-GB1-HD) (inverted red triangles). By overexpressing the 7TM domain of GABAB1, deleted of both the VFT and the coiled coil domain, a disruption of the large GABAB receptor complexes is observed, as revealed by the large decrease in the TR-FRET signal between snap-tag (ST) GABAB1. In parallel, a twofold increase in the G-protein activation is observed, even though the amount of wild-type GABAB receptor heterodimers at the cell surface remained constant. Adapted from Maurel et al. (2008).

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