title: Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation.
abstract: G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated β-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.
authors.affiliation: Departments of Pharmacology.
authors.forename: Brent M
authors.initials: BM
authors.lastname: Young
authors.affiliation: Departments of Pharmacology.
authors.forename: Elaine
authors.initials: E
authors.lastname: Nguyen
authors.affiliation: Departments of Pharmacology.
authors.forename: Matthew A J
authors.initials: MAJ
authors.lastname: Chedrawe
authors.affiliation: Biochemistry & Molecular Biology; Chemistry, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Rainey
authors.affiliation: Departments of Pharmacology. Electronic address: denis.dupre@dal.ca.
authors.forename: Denis J
authors.initials: DJ
authors.lastname: Dupré
mesh_terms: D000809:Angiotensins / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D055503:Protein Multimerization; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins / Q000378:metabolism
chemical_list: D000809:Angiotensins; D044140:Receptor, Angiotensin, Type 1; D000071557:beta-Arrestins
keywords: G protein-coupled receptor (GPCR); angiotensin II; angiotensin II type 1 receptor (AT1R); bioluminescence resonance energy transfer (BRET); dimerization; oligomerization; protein structure; transmembrane domain