Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT(1) receptor were designed to explore a possible role of a leucine at two positions, Leu(265) and Leu(268). Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu(265), located in the C-terminal region of transmembrane VI (TM-VI), and Leu(268), in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar(1)Leu(8)]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT(1) receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu(265), at the C-terminal portion of the AT(1)'s TM-VI, but not Leu(268), which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu(265) may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.