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Constitutive activity modulation of human metabotropic glutamate 5a receptors in HEK293 cells: a role for key amino‐terminal cysteine residues
Author(s) -
Mühlemann Andreas,
Diener Catherine,
Fischer Christophe,
Piussi Jenny,
Stucki Andy,
Porter Richard H
Publication year - 2005
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0706152
Subject(s) - metabotropic glutamate receptor , metabotropic glutamate receptor 6 , hek 293 cells , metabotropic glutamate receptor 2 , cysteine , metabotropic receptor , glutamate receptor , chemistry , metabotropic glutamate receptor 5 , terminal (telecommunication) , metabotropic glutamate receptor 3 , metabotropic glutamate receptor 4 , metabotropic glutamate receptor 7 , metabotropic glutamate receptor 1 , metabotropic glutamate receptor 8 , microbiology and biotechnology , receptor , biochemistry , biology , computer science , telecommunications , enzyme
1 Several combinations of cysteine to serine mutations at positions 57, 93, 99 and 129 in the extracellular N‐terminal domain of human metabotropic 5a (hmGlu5a) receptors were produced and expressed in HEK293 cells. Quisqualic acid‐induced intracellular calcium ([Ca 2+ ] i ) mobilization and inositol phosphates (IP) accumulation revealed an apparent increased efficacy and decreased potency for hmGlu5a mutants C57S, C99S and C57/99S, as well as a total loss of function for the mutant C57/93/99/129S. 2 [ 3 H]Quisqualate saturation analysis revealed mutants C57S, C99S, C57/99S and the tetramutant C57/93/99/129S to have unchanged K D but reduced B max values. [ 3 H]MPEP saturation analysis on the same membrane preparations revealed no difference in K D for any mutant, but a decrease in B max value for the C57/93/99/129S receptor. 3 Inverse agonism of MPEP at hmGlu5a receptors was partially reduced by mutation C57S, significantly reduced by C99S and C57/99S mutations and totally abolished in the tetramutant. 4 We confirmed the surface expression of all the mutated receptors using [ 3 H]MPEP binding analysis on whole cells. However, B max values were increased for mutant C57S, C99S, and C57/99S but decreased in the C57/93/99/129S receptor. 5 The 24 h preincubation of cells expressing hmGlu5a receptors with 1 μ M MPEP followed by extensive washing dramatically increased the wild‐type receptor efficacy to quisqualate, to the same levels seen with C57/99S receptors. MPEP preincubation did not affect C57/99S function. 6 We conclude that cysteines 57 and 99 are key residues necessary for modulating hmGlu5a receptor function.British Journal of Pharmacology (2005) 144 , 1118–1125. doi: 10.1038/sj.bjp.0706152