title: Determination of key amino acids implicated in the actions of allosteric modulation by 3,3'-difluorobenzaldazine on rat mGlu5 receptors.
abstract: Several mutations in the seven-transmembrane region of rat metabotropic glutamate 5 (rmGlu5) receptors were produced by site-directed mutagenesis and expressed in CHO cells. Using functional intracellular calcium ([Ca(2+)](i)) mobilisation, we identified amino acids implicated in the positive allosteric modulation of quisqualate-induced response by 3,3'-difluorobenzaldazine (DFB). Human and rat mGlu5 receptors displayed a higher potency and a higher efficacy in the presence of DFB. Mutant receptors S657(3.39)C, T780(6.44)A and M801(7.39)T disrupted the DFB-mediated increase in functional response. DFB-induced increase in potency was abolished in mutant receptors N733(45.51)A, Y791(6.55)A, A809(7.47)V, P654(3.36)S/S657(3.39)C and P654(3.36)S/S657(3.39)C/L743(5.47)V without affecting the enhancement of efficacy observed in wild type receptors. Mutations at positions Leu-743(5.47) and Trp-784(6.48) resulted in significantly larger DFB-induced potentiation of EC(50) and E(max) values than in wild type receptors. DFB-mediated increase of efficacy was abolished and EC(50) values were right-shifted in mutant receptor F787A, resulting in DFB acting as a weak partial antagonist at this mutant receptor. Based on these findings, we constructed a homology model concluding that six key residues in transmembranes 3, 5, 6 and 7 are necessary for the allosteric modulation of rmGlu5a receptor by DFB. The model confirms an overlapping but distinct binding site to 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and in particular emphasises the key role of W784 in transmembrane (TM) 6 for controlling the receptor's activation state.
authors.affiliation: F. Hoffmann-La Roche Ltd., Pharma Division, CNS Research, CH-4070 Basel, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mühlemann
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Ward
authors.forename: Nicole
authors.initials: N
authors.lastname: Kratochwil
authors.forename: Catherine
authors.initials: C
authors.lastname: Diener
authors.forename: Christophe
authors.initials: C
authors.lastname: Fischer
authors.forename: Andy
authors.initials: A
authors.lastname: Stucki
authors.forename: Georg
authors.initials: G
authors.lastname: Jaeschke
authors.forename: Pari
authors.initials: P
authors.lastname: Malherbe
authors.forename: Richard H P
authors.initials: RH
authors.lastname: Porter
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006834:Hydrazines / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: C481447:3,3'-difluorobenzaldazine; D006834:Hydrazines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D002118:Calcium