Disulfide bridges in extracellular domains of angiotensin II receptor type IA.
Angiotensin II receptor type IA (AT1A) has a cysteine (Cys) residue in each of four extracellular domains, and these Cys residues are believed to form two disulfide bridges. However, the question as to which pairs of Cys residues form disulfide bridges have not been experimentally determined. We constructed four mutants of rat AT1A, in which extracellular Cys residues were individually replaced by glycine (mutant C-1, C-2, C-3 and C-4). Further, we constructed two double mutants, in which two extracellular Cys residues were simultaneously substituted for by glycine. The binding affinity for angiotensin II in a double mutant C-1 + 4 (Cys18,274Gly) was similar to that in individually substituted mutants (C-1, C-2, C-3 and C-4) whereas the ligand binding of a double mutant C-2 + 4 (Cys101,274Gly) was completely abolished. The bindings of the non-peptide AT1A antagonist [125I]EXP-985 to mutants C-1, C-4 and C-1 + 4 were only slightly reduced whereas in mutant C-2, C-3 and C-2 + 4 the specific binding for [125I]EXP-985 was completely abolished. These results suggest that disulfide bridges in AT1A are formed between Cys18 and Cys274, and between Cys101 and Cys180, and the latter disulfide bond is essential for the binding of the non-peptidic antagonists [125I]EXP-985 or losartan.
Ohyama
K
K
Department of Pediatrics, Yamanashi Medical University, Japan.
Yamano
Y
Y
Sano
T
T
Nakagomi
Y
Y
Hamakubo
T
T
Morishima
I
I
Inagami
T
T
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001713:Biphenyl Compounds / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry; D019808:Losartan; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013777:Tetrazoles / Q000378:metabolism / Q000494:pharmacology
D001713:Biphenyl Compounds; D004220:Disulfides; D007093:Imidazoles; D011945:Receptors, Angiotensin; D013777:Tetrazoles; D000804:Angiotensin II; C078212:EXP 985; D019808:Losartan; D004229:Dithiothreitol