The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors.
We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [(3)H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [(3)H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.
Novartis Pharma AG, Nervous System Research, Klybeckstrasse 141, CH-4057 Basel, Switzerland.
A
A
Pagano
D
D
Ruegg
S
S
Litschig
N
N
Stoehr
C
C
Stierlin
M
M
Heinrich
P
P
Floersheim
L
L
Prezèau
F
F
Carroll
J P
JP
Pin
A
A
Cambria
I
I
Vranesic
P J
PJ
Flor
F
F
Gasparini
R
R
Kuhn
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002867:Chromones / Q000378:metabolism*; D006224:Cricetinae; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
C117789:7-(hydroxyimino)cyclopropan(b)chromen-1a-carbxoylic acid ethyl ester; D002867:Chromones; D018691:Excitatory Amino Acid Antagonists; D011725:Pyridines; D018094:Receptors, Metabotropic Glutamate; C121465:6-methyl-2-(phenylethynyl)pyridine