ACE2 Activation by Tripeptide IRW (Ile-Arg-Trp) Depends on the G Protein-Coupled Receptor 30 Signaling Cascade

This study aimed to understand how specific cell-boundreceptorsinfluence ACE2 activation by IRW. Our results showed that G protein-coupledreceptor 30 (GPR30), a 7-transmembrane domain protein, was involvedin IRW-mediated ACE2 increase. IRW treatment (50 mu M) significantlyincreased the GPR30 pool levels (3.2 +/- 0.5 folds) (p < 0.001). IRW treatment also boosted the consecutive GEF (guaninenucleotide exchange factor) activity (2.2 +/- 0.2 folds) (p < 0.001), and GNB1 levels (2.0 +/- 0.5 folds) (p < 0.05), associated with the functional subunits ofG proteins, in cells. These results were translated in hypertensiveanimal studies as well (p < 0.05), indicated byan increase in the aortal levels of GPR30 (p <0.01); further experiments showed an increase in downstream PIP3/PI3K/Aktpathway activation following IRW treatment. The blockade of GPR30by an antagonist and siRNA in cells abolished the ACE2-activatingability of IRW, as shown by the depleted levels of ACE2 mRNA (p < 0.001), protein levels in whole cells and membrane,angiotensin (1-7) (p < 0.01), and ACE2promoter HNF1 alpha (p < 0.05). Finally, theGPR30 blockade in ACE2-overexpressing cells using the antagonist (p < 0.01) and siRNA (p < 0.05) significantlydepleted the innate cellular pool of ACE2, thus confirming the relationshipbetween the membrane-bound GPR30 and ACE2. Overall, these resultsshowed that the vasodilatory peptide IRW could activate ACE2 via themembrane-bound receptor GPR30.