Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II.
To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.
Laporte
S A
SA
Department of Pharmacology, Medical School, Université de Sherbrooke, Québec, Canada.
Boucard
A A
AA
Servant
G
G
Guillemette
G
G
Leduc
R
R
Escher
E
E
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells / Q000378:metabolism; D002478:Cells, Cultured / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry*; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012521:Sarcosine / Q000737:chemistry
D007457:Iodine Radioisotopes; D019920:Photoaffinity Labels; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D000804:Angiotensin II; D010649:Phenylalanine; C053764:benzoylphenylalanine; D012521:Sarcosine