Transmembrane Domains IV and V Form an Interface in Human Angiotensin II Type 1 Receptor Homomers

Of the 750 GPCRs said to be expressed in humans, only approximately 20 individual high-resolution crystal structures are available for human GPCRs. While GPCR oligomerization is a common occurrence, very few groups have had success in crystallizing these structures. Given the fact that the pharmacology of these entities has been shown to diverge from that of their monomeric constituents, there has been a push for biophysical characterization of GPCR oligomers. Here, we sought to characterize the structure of human angiotensin II type 1 receptor (AT1R) homomers with a bioluminescence resonance energy transfer (BRET)-based approach. An initial alignment was performed, and we developed a three-dimensional homology model of the AT1R using the GPCR Online MOdeling and DOcking server. The δ-opioid receptor (DOR) was selected as the best template for AT1R homology modeling using two scoring functions. This model received a normalized Discrete Optimized Protein Energy (DOPE) score of 0.03223 and a GA341 score of 0 (Fig.1). Accessible surface area was determined for each side chain in the modelled AT1R transmembrane (TM) region. This data, as well as published data surrounding the DOR,allowed us to focus our efforts on discrete AT1R TM regions. Site-directed mutagenesis and BRET experiments identified an AT1R homomer interface at TM domains IV and V (Fig.2). This was validated using GPCR Heteromer Identification Technology. These results contribute to our understanding of AT1R dimerization and may serve as the foundation for future drug discovery efforts that target GPCR oligomers. Supported by the Natural Sciences and Engineering Research Council of Canada.