Aliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity.

Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.

Importância de resíduos hidrofóbicos da alça extracelular III do receptor AT1 da angiotensina II na interação com seu ligante / Importance of the hydrophobic residues of loop extracellular III of the AT1 receptor of angiotensin II in the interaction with its ligands

The role of the external third loop (EC-3) of the angiotensin II (AII) AT1, receptor for the interaction with its ligand was investigated by performing triple simuitaneous mutation of L262, L265 and L268 for Asp ([L3D3]AT1), in addition to two single mutantions of Leu265 [L265A]AT1, and L268, [L268R]AT1. The mutants were permanently transfected to Chinese hamster ovary cells (CHO). Binding assays showed that [L3D3]AT1, had very low affinity to Ali and DuP753 whereas its binding to [Sar1,Leu8-AII was not detectable. However no great changes were observed towards [L265A]AT1 and to [L268R]AT1 which produced lC5O values similar to that obtained with wild type receptor.(control) The inositol phosphate responses to AII was much drastically reduced in [L3D3]AT1. Therefore the ED50 value and the maximal effect were significantiy lower that those of wild type. It was also observed that the expression of [L3D3]AT1 was drasticaily reduced. However [L268R]AT1 and [L265A]AT1 were able to stimulate inositol phosphate formation and its maximal effect not significantly different from the controls. These effects could be only detected when multiple but not single mutations were performed. Moreover the hydrophobic residues located in this N-terminal region of EC-3 should be substituted for residues bearing charges. From these results and from the literature it is suggested that AII was less affected than [Sar1,Leu8]-AII due to its strong interactions with transmembrane residues of the receptor while the extracellular residues seem to be more important to the interactions of [Sar1,Leu8]-AII with the receptor. This could explain why [Sar1,Leu8]-AII was more affected by the mutations of [L3D3]AT1 than AII. On the other hand as DuP753 partially share some residues such as Lysl99 and His256 for interaction with AII this could explain the similar effect exerted by [L3D3]AT1 on both ligands. These resuits suggest that the hydrophobic residues located in the EC-3, close to heiix VI are critical to maintain the stability of ligand binding