Abstract
The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn(2+) binding site in mGluR1b by mutating the residue Lys(260) to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC(50) value of 2 microm. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn(2+) to antagonize the agonist-induced response. Zn(2+) binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.
Highlights
The metabotropic glutamate receptors belong to family C of the G-protein-coupled receptor (GPCR) superfamily
Inositol Phosphate (IP) Assay—1 ϫ 106 tsA cells were split into a 10-cm tissue culture plate and transfected with 5 g of plasmid the following day using Superfect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany)
Effects of Zn2ϩ on wild type (WT) mGluR1b—Exposure of tsA cells transiently transfected with WT mGluR1b to Zn2ϩ in concentrations of 300 M and above led to an inhibition of agonistinduced IP accumulation with an IC50 value of the metal ion of ϳ1 mM (Fig. 1)
Summary
Glu, (S)-glutamic acid; mGluR, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; GABA, ␥-aminobutyric acid; GABABR, ␥-aminobutyric acid receptor type B; CaR, calcium-sensing receptor; ATD, amino-terminal domain; PBP, mitter in the central nervous system and plays an essential role in a wide range of functions, such as brain development, memory formation, and neurotoxicity [1]. We have previously applied the crystal structure of one of these PBPs, the leucine/isoleucine/valine-binding protein, as a template for the construction of a tertiary molecular model of the ATD of mGluR1, and similar models have been published for periplasmic binding protein; [3H]Quis, [3H]quisqualic acid; (S)-4CPG, (S)-4-Carboxyphenylglycine; CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylic acid ethyl ester; IP, inositol phosphate; HBSS, Hanks’ balanced saline solution; WT, wild type; PBS, phosphate-buffered saline solution; CHO, Chinese hamster ovary; TM, transmembrane region; 7TM, seven-transmembrane moiety. The results of the study provide interesting information regarding the ATD closure mechanism and the structural flexibility within each ATD lobe
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