The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.
The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.
Domazet
Ivana
I
Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
Martin
Stéphane S
SS
Holleran
Brian J
BJ
Morin
Marie-Eve
ME
Lacasse
Patrick
P
Lavigne
Pierre
P
Escher
Emanuel
E
Leduc
Richard
R
Guillemette
Gaétan
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D007202:Indicators and Reagents; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D014662:Vasoconstrictor Agents; C093171:methanethiosulfonate ethylammonium; D000804:Angiotensin II; D005020:Ethyl Methanesulfonate; D010738:Type C Phospholipases