Photolabeling Identifies Position 172 of the Human AT₁ Receptor as a Ligand Contact Point:  Receptor-Bound Angiotensin II Adopts an Extended Structure

Abstract

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.

Determination of a Binding Site of Cu, Ni, Mg, and Ca Metal Ions with Angiotensin II Peptide by Electrospray Tandem Mass Spectrometry

“…[9][10][11][12][13] It is evident that several reports are not consistent with each other and that no clear agreement exists regarding the biologically active structure of Ang II during the biological process.…”

“…1 Ang II is an octapeptide hormone (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8, DRVYIHPF) that has been studied for several decades in the structure-dependent activity of the biological pathway of the Ang II receptor. [2][3][4][5][6][7][8][9][10][11][12][13] Free Ang II molecular structures in solutions have been investigated with a variety of techniques. The results have been reported as β-turn, random coil, hair-pin and other structures.…”

Analysis of the Third Transmembrane Domain of the Human Type 1 Angiotensin II Receptor by Cysteine Scanning Mutagenesis

“…For this receptor, the binding site would thus be contained within a water-accessible crevice, the binding pocket, extending from the extracellular surface of the receptor to the transmembrane portion. Using a photoaffinity labeling approach, we directly identified ligand contact points within the second extracellular loop and the seventh TMD of the AT 1 receptor (12)(13)(14). Interestingly, numerous mutagenesis studies have provided the basis for a model in which an interaction between Asn 111 in TMD3 and Tyr 292 in TMD7 maintains the AT 1 receptor in the inactive conformation.…”

DFT Study of the [Cu-Angiotensin II]2+Complex Structure

“…[1][2][3][4] Metal ion influences on the biological activity of Ang II have also been investigated. [5][6][7][8][9][10][11][12][13][14] Blood pressure was observed to increase as a result of the influence of Li, Na, Mg, and Ca ions on the renin-angiotensin system.…”