Mutational analysis of the angiotensin type 2 receptor: contribution of conserved amino acids in the region of the sixth transmembrane domain.
Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding. In contrast, relatively little is known of the binding mechanism of the AT2 receptor. It has been hypothesized that residues that are conserved between the two subtypes that have been shown to be important in the AT1 receptor may also contribute to AngII binding in the AT2 receptor as well. To test this hypothesis, the role of two conserved residues in the sixth transmembrane domain of the AT2 receptor in ligand binding were investigated: tryptophan 269 and aspartate 279. In contrast to the AT1 receptor, mutation of Trp269 in the AT2 receptor to an alanine had no effect on AngII binding, while mutation of Asp279 to alanine similarly impaired AngII binding in both receptors. However, the more sterically conservative substitution of Asp279 to asparagine in the AT2 receptor showed near wild type affinity. Based on this finding, we mutated Asp263 in the AT1 receptor to asparagine. Subsequent studies indicated that this more conservative mutation had no effect on AngII binding to the AT1 receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT1 and AT2 AngII receptors, there are also clear differences.
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
J N
JN
Heerding
D K
DK
Yee
M Z
MZ
Krichavsky
S J
SJ
Fluharty
D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001665:Binding Sites; D019556:COS Cells; D017124:Conserved Sequence; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D000596:Amino Acids; D008024:Ligands; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D001224:Aspartic Acid; D014364:Tryptophan