Constitutive activation of the angiotensin II type 1 receptor alters the spatial proximity of transmembrane 7 to the ligand-binding pocket.
Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TM) following binding of agonist. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which constitutes a water-accessible crevice surrounded by the seven TM helices. Using the substituted cysteine accessibility method, we initially identified those residues within the seventh transmembrane domain (TM7) of wild type angiotensin II type 1 (AT1) receptor that contribute to forming the binding site pocket. We have substituted successively TM7 residues ranging from Ile276 to Tyr302 to cysteine. Treatment of A277C, V280C, T282C, A283C, I286C, A291C, and F301C mutant receptors with the charged sulfhydryl-specific alkylating agent MTSEA significantly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was greatly reduced for TM7 reporter cysteines engineered in a constitutively active mutant of the AT1 receptor. Our data suggest that upon activation, TM7 of the AT1 receptor goes through a pattern of helical movements that results in its distancing from the binding pocket per se. These studies support accumulating evidence whereby elements of TM7 of class A GPCRs promote activation of the receptor through structural rearrangements.
Boucard
Antony A
AA
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Roy
Marise
M
Beaulieu
Marie-Eve
ME
Lavigne
Pierre
P
Escher
Emanuel
E
Guillemette
Gaetan
G
Leduc
Richard
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D014867:Water / Q000737:chemistry
D008024:Ligands; D008698:Mesylates; D009841:Oligonucleotides; D044140:Receptor, Angiotensin, Type 1; C093171:methanethiosulfonate ethylammonium; D007532:Isoleucine; D014867:Water; D014443:Tyrosine; C074502:methanethiosulfonate; D005020:Ethyl Methanesulfonate; D003545:Cysteine