Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor.
The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.
Pérodin
Jacqueline
J
Département de pharmacologie, Faculté de médecine, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada.
Deraët
Maud
M
Auger-Messier
Mannix
M
Boucard
Antony A
AA
Rihakova
Lenka
L
Beaulieu
Marie-Eve
ME
Lavigne
Pierre
P
Parent
Jean-Luc
JL
Guillemette
Gaétan
G
Leduc
Richard
R
Escher
Emanuel
E
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D003488:Cyanogen Bromide / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000596:Amino Acids; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D010649:Phenylalanine; D001216:Asparagine; D008715:Methionine; D003488:Cyanogen Bromide