Mutational analysis of the angiotensin II type 2 receptor: contribution of conserved extracellular amino acids.
While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view. To determine the commonalities in ligand binding of the AT1 and AT2 receptors, we have chosen several conserved extracellular amino acids which have been shown to be important in AngII binding [1,2] to the AT1 receptor for mutational studies of the AT2 receptor. Specifically, we have mutated tyrosine108 in extracellular loop 1 (ECL1), arginine182 in ECL2, and aspartate297 in ECL3 of the AT2 receptor in order to determine their contribution to AngII binding. In the AT2 receptor, mutation of tyrosine108 to an alanine resulted in a receptor with wild-type binding for AngII, while mutation of either arginine182 or aspartate297 drastically impaired AngII binding ( > 100 nM). These results demonstrate both similarities as well as clear differences between receptor subtypes in the contributions to AngII binding of several conserved extracellular amino acid residues.
Heerding
J N
JN
Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Yee
D K
DK
Jacobs
S L
SL
Fluharty
S J
SJ
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001667:Binding, Competitive; D019556:COS Cells; D005110:Extracellular Space / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000235:genetics
D000596:Amino Acids; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D001224:Aspartic Acid; D014443:Tyrosine; D001120:Arginine; D000409:Alanine