Relevant role of Leu²⁶⁵in helix VI of the angiotensin AT₁receptor in agonist binding and activity

Abstract

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 AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, 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 [Sar1Leu8]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 AT1 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 Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 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.

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

“…We previously reported that photoreactive AngII analogues substituted at positions 1, 2, 3, and 5 with either Bpa or aryldiazirine-based photoprobes all labeled exactly the same domains of the AT1R-binding site with only a single exception for analogues substituted in position 2, thus enabling the actual detailed MPA analysis of AT 1 R (20). Interestingly, an exhaustive literature survey on previously available mutagenesis data appears to corroborate these findings because nearly half of the actual ligand/receptor contact residues that we found in the NT (36), ECL1 (36 -44), ECL2 (36,41,(45)(46)(47), and ECL3 (36,43,44,(47)(48)(49) were postulated to be involved in ligand binding and receptor functions or were adjacent to such residues. The present study suggests that those mutated residues do not have an indirect influence on the ligand-binding site but instead may directly contact the bound ligand.…”

Association between the angiotensin-converting enzyme (insertion/deletion) and angiotensin II type 1 receptor (A1166C) polymorphisms and breast cancer among Brazilian women

“…[28][29][30] These interesting findings showed that this component of the RAS could be involved in control of cell growth mechanisms.…”

“…Moreover, in a previous study, we demonstrated that expression of L262D and L265D mutants of the AT 1 -receptor caused morphological changes and inhibited the proliferation in CHO cells. [28][29][30] These interesting findings showed that this component of the RAS could be involved in control of cell growth mechanisms.…”

Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor

“…Mutation of Leu265 decreased ligand-binding affinity and ability to stimulate IP 3 formation. This led authors (Correa et al 2002) to suggest that the aliphatic side chain of Leu265 may be involved in the formation of the ligand-binding sites through allosteric effects, which stabilizes the receptor structure around ligand-binding site for full-agonist activity. As noted previously, a charge pair interaction of Arg 2 of Ang II with residues in the ECL3 (278 and 281) is essential for initial ligand positioning, opening of the ligand-binding pocket, and full-agonistic activity.…”