Role of Arg182 in the second extracellular loop of angiotensin II receptor AT2 in ligand binding.
The phenolic side chain of Tyr(4) present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, (125)I-Ang II and [(125)I-Sar(1)-Ile(8)]Ang II, as well as the AT2 receptor-specific peptidic ligand (125)I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of (125)I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor.
Kurfis
J
J
Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403, USA.
Knowle
D
D
Pulakat
L
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D017931:DNA Primers; D005260:Female; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D017931:DNA Primers; D008024:Ligands; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D000804:Angiotensin II; D001120:Arginine