Aliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity
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2002-06-15
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Corrêa, Silvana Aparecida Alves 
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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 All and for non-peptide (losartan) and peptide ([Sar(1)Leu(8)]All) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of All 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. (C) 2002 Elsevier Science B.V. All rights reserved.
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Regulatory Peptides. Amsterdam: Elsevier B.V., v. 106, n. 1-3, p. 33-38, 2002.