The N-Terminal Domain of γ-Aminobutyric Acid_BReceptors Is Sufficient to Specify Agonist and Antagonist Binding

Abstract

The recently identified gamma-aminobutyric acid type B receptors (GABA(B)Rs) share low sequence similarity with the metabotropic glutamate (mGlu) receptors. Like the mGlu receptors, the N-terminal extracellular domain (NTED) of GABA(B)Rs is proposed to be related to bacterial periplasmic binding proteins (PBPs). However, in contrast to the mGlu receptors, the GABA(B)Rs lack a cysteine-rich region that links the PBP-like domain to the first transmembrane domain. This cysteine-rich region is necessary for the PBP-like domain of mGlu receptors to bind glutamate. To delimit the ligand-binding domain of GABA(B)Rs, we constructed a series of chimeric GABA(B)R1/mGluR1 and truncated GABA(B)R1 receptor mutants. We provide evidence that despite the lack of a cysteine-rich region, the NTED of GABA(B)Rs contains all of the structural information that is necessary and sufficient for ligand binding. Moreover, a soluble protein corresponding to the NTED of GABA(B)Rs reproduces the binding pharmacology of wild-type receptors. This demonstrates that the ligand-binding domain of the GABA(B)Rs can correctly fold when dissociated from the transmembrane domains.

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

“…Accordingly, a direct interaction between HD and VFT in GB1 is likely responsible for the low agonist affinity state. Such an interaction has already been proposed by others (22,41), but has never been demonstrated. Such a negative effect of HD on agonist affinity in VFT has also been reported for other class III GPCRs, the mGlu4 and mGlu8 receptors, using a similar approach (42,43).…”

“…The ligand-binding site of GB1 has been extensively studied (17)(18)(19)(20)(21)(22). Modeling and site-directed mutagenesis studies indicate that ligands bind in the cleft that separates both lobes of GB1 VFT, as observed for ligand binding in many similar protein modules (23), including mGlu1 VFT (24,25).…”

Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator

“…Substituted by Alanine-Although there is strong evidence that the VFT domain of the ECD of family 3 GPCRs, including the CaR, is responsible for agonist binding (7)(8)(9)(10), the ability of Rho-C-hCaR to respond to calcium upon addition of NPS R-568 or by imposing the F788C mutation indicates the existence of calcium-binding site(s) in the 7TM domain of the hCaR as well. Acidic residues in the VFT have been speculated to be involved in calcium binding to and activation of the CaR.…”

“…One possible role for these amino acids would be their involvement in directly binding Ca 2ϩ . Although considerable evidence points to the VFT as the primary site of Ca 2ϩ binding (7)(8)(9)(10), evidence for positive cooperativity in Ca 2ϩ response is consistent with multiple sites for binding on the CaR, including the possibility of lower affinity sites within the 7TM domain (31). Our data showing that mutation of both Asp 758 and Glu 759 or of Glu 767 to alanine facilitates activation of both full-length and ECD-deleted receptors rather than diminishing Ca 2ϩ response argues against a role for these residues in directly binding Ca 2ϩ .…”

Sushi domains confer distinct trafficking profiles on GABA B receptors

“…These receptors are increasingly considered as potential therapeutic targets for a range of diseases, including epilepsy, schizophrenia, anxiety, depression, and substance abuse (1,5). Functional GABA B receptors are heteromers formed from GABA B R1 subunits containing the agonist binding domain (6) and R2 subunits that link to G protein signaling (7,8).…”