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G protein-independent effects of the Angiotensin II type 1 receptor

 

Gitte Lund Christensen

Summary

The angiotensin II type 1 receptor (AT1R) is an important drug target in cardiovascular medicineand a widely used model seven transmembrane receptor (7TMR), for studyingG protein-independent signal transduction. Classically the AT1R has been pictured as primarilysignaling through heterotrimeric G proteins. Besides G protein activation it can also signal byG protein-independent mechanisms of which the impact and complexity is less understood.“Biased agonists” blocking Gαq protein activity, while activating G protein-independentpathways have been applied to describe the two branches of AT1R signaling. The studiesprovided here indicate that biased agonists have potential to improve cardiovascular drug therapy.In this PhD thesis we have proved the relevance of biased signaling from the AT1R in primarycells and hearts and shown that we can separate signaling leading to maladaptive cardiachypertrophy from pathways involved in cytoprotective and proliferative effects. The focus hasbeen on evaluating the importance of G protein-independent effects mediated by AT1R activation,applying the biased agonist SII Angiotensin II (SII Ang II) and global analysis such as massspectrometry and expression microarrays. Using an unbiased mass spectrometry approach weprovide substantial novel insight into Angiotensin II (Ang II) signal transduction and dissect thedifferences between a full agonist and a biased agonist on AT1R signaling on a systems-widescale. The quantitative mass spectrometry reveals a previously unappreciated diversity andquantity of Gαq protein-independent signaling and uncovers novel signaling pathways. Withstandard molecular biology techniques we look further into the activation mechanisms behind themost central G protein-independently regulated kinase, PKD.Although we find the biased agonist SII Ang II to stimulate substantial signal transduction in themass spectrometric study, it was not critically involved in the regulation of gene expression. Westudied regulation of both mRNA and microRNA expression by microarray technology, andconclude that the majority of the Ang II mediated gene regulation is dependent on G proteinactivation. Despite the inability of SII Ang II to regulate mRNA and microRNA expression, thebiased agonist vastly potentiated gene expression induced by isoproterenol, indicating amodulatory role of G protein-independent signaling.Overall the studies included in this thesis have proved the relevance of biased agonism in nativecellular settings, described the involved players in, and scaled the relative contribution of, Gprotein-independent effects in AT1R signaling. Moreover, we find that G protein-independentsignaling does not autonomously regulate gene expression, but that this novel signalingopportunity may possess a function in modulation of G protein-dependent mediated geneexpression.