Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.
The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.
Heerding
J N
JN
Department of Animal Biology, University of Pennsylvania, Philadelphia, PA 19104-6046, USA.
Hines
J
J
Fluharty
S J
SJ
Yee
D K
DK
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D019556:COS Cells; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D004220:Disulfides; D010446:Peptide Fragments; D020033:Protein Isoforms; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D004229:Dithiothreitol