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MTSEA prevents ligand binding to the human melanocortin‐4 receptor by modification of cysteine 130 in transmembrane helix 3
Author(s) -
Cox Alan,
Donnelly Dan,
Kaur Manminder,
Cheetham Sharon C.,
Cockcroft Victor B.,
Findlay John B.C.
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.11.087
Subject(s) - agonist , ligand (biochemistry) , cysteine , chemistry , receptor , alanine , binding site , transmembrane domain , antagonist , stereochemistry , biophysics , biochemistry , amino acid , biology , enzyme
We have investigated the effect of the sulfhydryl‐reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin‐4 (MC4) receptor stably expressed in HEK‐293 cells. MTSEA inhibited binding of the agonist, 125 I‐NDPα‐MSH, and the antagonist, 125 I‐SHU9119, in a concentration‐dependent manner. Pre‐incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl‐reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close‐by negative charge, most likely on Asp126.