Identification of angiotensin II-binding domains in the rat AT2 receptor with photolabile angiotensin analogs.
To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT2), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of 125I-[Bpa1]AngII.AT2 receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of 125I-[Bpa1]AngII within amino acids 3-30 of AT2 receptor. Treatment of 125I-[Bpa8]AngII.AT2 receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT2 receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of 125I-[Bpa8]AngII within residues 129-138 of AT2 receptor. Our results indicate that the amino-terminal tail of the AT2 receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT2 receptor interacts with the carboxyl-terminal end of AngII.
Servant
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Département de Pharmacologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Laporte
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SA
Leduc
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Escher
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Guillemette
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D003488:Cyanogen Bromide / Q000378:metabolism; D007610:Kallikreins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D020840:Tissue Kallikreins; D014662:Vasoconstrictor Agents / Q000378:metabolism
D000345:Affinity Labels; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D010649:Phenylalanine; C053764:benzoylphenylalanine; D007610:Kallikreins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D020840:Tissue Kallikreins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide