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.

Background/Aims: CXCL12, acting via one of its G protein-coupled receptors, CXCR4, is a chemoattractant for a broad range of cell types, including several types of cancer cells. Elevated expression of CXCR4, and its ligand CXCL12, play important roles in promoting cancer metastasis. Cancer cells have the potential for rapid and unlimited growth in an area that may have restricted blood supply, as oxidative stress is a common feature of solid tumors. Recent studies have reported that enhanced expression of cytosolic superoxide dismutase (SOD1), a critical enzyme responsible for regulation of superoxide radicals, may increase the aggressive and invasive potential of malignant cells in some cancers. Methods: We used a variety of biochemical approaches and a prostate cancer cell line to study the effects of SOD1 on CXCR4 signaling. Results: Here, we report a direct interaction between SOD1 and CXCR4. We showed that SOD1 interacts directly with the first intracellular loop (ICL1) of CXCR4 and that the CXCL12/CXCR4-mediated regulation of AKT activation, apoptosis and cell migration in prostate cancer (PCa) cells is differentially modulated under normal versus hypoxic conditions when SOD1 is present. Conclusions: This study highlights a potential new regulatory mechanism by which a sensor of the oxidative environment could directly regulate signal transduction of a receptor involved in cancer cell survival and migration.

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CXCL12, acting via its G protein‐coupled receptor CXCR4, is a chemoattractant for a broad range of cell types, including several types of cancer cells. Indeed, elevated expression of CXCR4, and its ligand CXCL12 play important roles in promoting cancer metastasis. Cancer cells have the potential for rapid and unlimited growth in a restricted blood supply; oxidative stress is a common feature of several tumors. Superoxide dismutase (SOD) is a critical enzyme responsible for regulation of superoxide radicals. Recent studies have reported enhanced expression of SOD may increase the aggressive and invasive potential of malignant cells in some cancers. Here, we report a direct interaction between SOD1 and CXCR4. Given the important roles of these two proteins in cancer progression and metastasis, we characterized the effects of SOD1 on CXCR4 signal transduction. We showed that SOD1 interacts directly with the first intracellular loop of CXCR4. We demonstrated that CXCL12/CXCR4‐mediated modulation of ERK, AKT, apoptosis and cell migration in prostate cancer cells is different under normal versus hypoxic conditions when SOD1 is present. Finally, we identified a potential regulatory mechanism by which SOD1 directly interferes with CXCR4 function, regulating G protein coupling to CXCR4, and promoting a relative switch in G protein coupling from Gi to Gq depending on environmental conditions. This study highlights a potential new regulatory mechanism by which a sensor of the oxidative environment can directly regulate signal transduction of a receptor involved in cancer cell survival and migration. Supported the Dalhousie Medical Research Foundation and Natural Sciences and Engineering Research Council of Canada.