Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1: noncompetitive antagonism originating from the amino-terminal domain of a family C G-protein-coupled receptor

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Anders A. Jensen, P O Sheppard, L B Jensen, P J O'Hara, H Bräuner-Osborne

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.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind276
Udgave nummer13
Sider (fra-til)10110-8
Antal sider9
ISSN0021-9258
DOI
StatusUdgivet - 2001

ID: 38485322