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.
Correa
Silvana A A
SA
Department of Biophysics, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Botucatu 862, 04023-060 São Paulo, Brazil.
Zalcberg
Heloisa
H
Han
Sang W
SW
Oliveira
Laerte
L
Costa-Neto
Claudio M
CM
Paiva
Antonio C M
AC
Shimuta
Suma I
SI
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005260:Female; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D000596:Amino Acids; D007295:Inositol Phosphates; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II