MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3.
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, 125I-NDPalpha-MSH, and the antagonist, 125I-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.
Cox
Alan
A
School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK. a.cox@leeds.ac.uk
Donnelly
Dan
D
Kaur
Manminder
M
Cheetham
Sharon C
SC
Cockcroft
Victor B
VB
Findlay
John B C
JB
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism
D008024:Ligands; D044105:Receptor, Melanocortin, Type 4; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D003545:Cysteine