The heteromeric GABA-B receptor recognizes G-protein α subunit C-termini

Franěk, Miloslav; Pagano, Adriana; Kaupmann, Klemens; Bettler, Bernhard; Pin, Jean‐Philippe; Blahoš, Jaroslav

doi:10.1016/s0028-3908(99)00135-5

Cited by 63 publications

(65 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, the GABA B receptor can activate efficiently phospholipase C when co-expressed with the chimeric G-protein Gqi9 (47). As noticed previously, the IP assay is much more sensitive than the [␥- 35 S]GTP assay as indicated by the 100-fold lower EC 50 value measured for GABA compare with the [␥- 35 S]GTP assay.…”

Section: Cgp7930 Is a Positive Allostericmentioning

confidence: 79%

“…Cell Culture and Transfection-Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected by electroporation as described elsewhere (47,48). Unless stated otherwise, 10 ϫ 10 6 cells were transfected with plasmid DNA containing the coding sequence of the receptor subunits and the amount of plasmid DNA was adjusted to 10 g with the empty plasmid pRK 6 .…”

Section: Methodsmentioning

confidence: 99%

“…Unless stated otherwise, 10 ϫ 10 6 cells were transfected with plasmid DNA containing the coding sequence of the receptor subunits and the amount of plasmid DNA was adjusted to 10 g with the empty plasmid pRK 6 . For determination of inositol phosphate accumulation, the cells also were transfected with the chimeric G␣qi9 G-protein, which allows the coupling of the recombinant heteromeric GABA B receptor to phospholipase C (47).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Heptahelical Domain of GABAB2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABAB Receptor

Binet

Brajon

Corre

et al. 2004

Journal of Biological Chemistry

194

138

View full text Add to dashboard Cite

The ␥-aminobutyric acid, type B (GABA B ) receptor is well recognized as being composed of two subunits, GABA B1 and GABA B2 . Both subunits share structural homology with other class-III G-protein-coupled receptors. They are composed of two main domains: a heptahelical domain (HD) typical of all G-protein-coupled receptors and a large extracellular domain (ECD). Although GABA B1 binds GABA, GABA B2 is required for GABA B1 to reach the cell surface. However, it is still not demonstrated whether the association of these two subunits is always required for function in the brain. Indeed, GABA B2 plays a major role in the coupling of the heteromer to G-proteins, such that it is possible that GABA B2 can transmit a signal in the absence of GABA B1 . Today only ligands interacting with GABA B1 ECD have been identified. Thus, the compounds acting exclusively on the GABA B2 subunit will be helpful in analyzing the specific role of this subunit in the brain. Here, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA B receptor. We showed that it activates the wild type GABA B receptor but with a low efficacy. The GABA B2 HD is necessary for this effect, although one cannot exclude that CGP7930 could also bind to GABA B1 . Of interest, CGP7930 could activate GABA B2 expressed alone and is the first described agonist of GABA B2 . Finally, we show that CGP7930 retains its agonist activity on a GABA B2 subunit deleted of its ECD. This demonstrates that the HD of GABA B2 behaves similar to a rhodopsin-like receptor, because it can reach the cell surface alone, can couple to G-protein, and be activated by agonists. These data open new strategies for studying the mechanism of activation of GABA B receptor and examine any possible role of homomeric GABA B2 receptors.

show abstract

Section: Cgp7930 Is a Positive Allostericmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Heptahelical Domain of GABAB2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABAB Receptor

Binet

Brajon

Corre

et al. 2004

Journal of Biological Chemistry

194

138

View full text Add to dashboard Cite

show abstract

“…Cell Culture and Transfection-Human embryonic kidney (HEK) 293 and COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected by electroporation as described (35). Cells (10 ϫ 10 6 ) were transfected with plasmid DNA containing GB1 ASA (2 g), GB2 (2 g), ⌬GB1 (4 g), ⌬GB2 (4 g), GB1 GPI (4 g), GB2 GPI (4 g), ⌬mG5 (4 g), ⌬GB1 S246A (4 g), or the V2 vasopressin receptor (165 ng) and completed to a total amount of 10 g of plasmid DNA with the pRK5 empty vector.…”

Section: Methodsmentioning

confidence: 99%

Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit

Liu

Maurel

Etzol

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The ␥-aminobutyric acid type B (GABA B ) receptor is an allosteric complex made of two subunits, GABA B1 (GB1) and GABA B2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF® technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.

show abstract

“…To allow detection of the recombinant receptors on cell surface the N-terminal 16 residues of rat GB1a and N-terminal 41 residues of rat GB2 were replaced by 36 residues encoding the mGluR5 signal peptide, and HA epitope. The adaptor Gqi9 protein, based on sequence of Gq with last 9 residues replaced by extreme C terminal 9 residues from Gi sequence was described (Conklin et al, 1993) and used previously in our studies (Franek et al, 1999;Galvez et al, 2001;Havlickova et al, 2003). All constructs were sequenced using Big Dye Terminater v. 3,1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).…”

Section: 1mutagenesismentioning

confidence: 99%

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

et al. 2008

View full text Add to dashboard Cite

Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show, that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 γ-amino butyric acid b (GABAb) receptor subunits does not exclude their heterodimerization.

show abstract

The heteromeric GABA-B receptor recognizes G-protein α subunit C-termini

Cited by 63 publications

References 48 publications

The Heptahelical Domain of GABAB2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABAB Receptor

The Heptahelical Domain of GABAB2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABAB Receptor

Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

Contact Info

Product

Resources

About