Role of Lys215 located in the fifth transmembrane domain of the AT2 receptor in ligand-receptor interaction.

Studies on ligand-receptor interaction of Angiotensin II (Ang II) receptor type 1 have shown that for peptidic ligands to bind this receptor they must interact via their C-terminal carboxylate group to the positively charged side chain of the Lysine residue 199 located in the fifth transmembrane domain of this receptor. In the Ang II receptor type AT2, this Lysine residue is conserved at position 215 in the fifth transmembrane domain. To determine the specific mechanism of ligand binding to the Angiotensin II receptor type AT2, mutated AT2 receptors were generated in which the Lys215 was replaced with glutamic acid, glutamine, alanine and arginine. The ability of these mutated receptors to bind peptidic ligands 125I-[Sar1-Ile8]Ang II (non-specific for AT2 receptor type), 125I-CGP42112A (AT2 receptor specific) and the non-peptidic ligand PD123319 (AT2 receptor specific) was evaluated by expressing these receptors in Xenopus oocytes and performing binding assays. The Lys215Glu and Lys215Gln mutants of AT2 receptor lost their affinity to 125I-[Sar1-Ile8]Ang II, but retained their affinity to 125I-CGP42112A and PD123319. In contrast, Lys215Arg mutant retained its affinity to 125I-[Sar1-Ile8]Ang II, but exhibited lower affinity to 125I-CGP42112A. The Lys215Ala mutant lost its affinity to both 125I-[Sar1-Ile8]Ang II and 125I-CGP42112A. These results suggest that the binding mechanism of 125I-[Sar1-Ile8]Ang II to AT2 receptor is similar to that of AT1 receptor since an amino acid with positively charged side chain (Lys or Arg) located in the fifth transmembrane domain is required for this ligand to bind AT2 receptor. In contrast, although CGP42112A is a peptidic ligand, it does not require an interaction between its C-terminal carboxylate group and the positively charged side-chain of an amino acid in the fifth transmembrane domain for its binding to AT2 receptor.