Quantitative Phosphoproteomics Dissection of Seven-transmembrane Receptor Signaling Using Full and Biased Agonists

Seven-transmembrane receptors (7TMRs)1 constitute the largest family of plasma membrane receptors. These receptors regulate a variety of biological processes and are currently the most prominent therapeutic targets, highlighted by an abundance of clinically available drugs (1–5). Their cellular responses were until recently thought to depend primarily on G protein activation and generation of second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and cAMP, reflected by the term G protein-coupled receptors. However, the 7TMR structure also confers activation of G protein-independent signaling initiated by direct interaction with β-arrestin or tyrosine kinases (1–3, 6, 7). The discovery of alternative signaling pathways to the G protein-initiated pathways has also led to the discovery of regulatory peptides that can have agonistic effects on one pathway while simultaneously antagonizing another. Such pharmacological separation of signaling pathways has an enormous clinical potential, which is described by pharmacological concepts such as “functional selectivity” or “biased agonism” (3, 8, 9). A number of antagonists have only been evaluated by G protein-dependent readouts and may therefore turn out to be biased agonists. An example of such biased agonism is the retrospective discovery that the widely used inverse β2-adrenergic receptor agonist carvedilol, which has been superior to other β-blockers in the prevention of heart failure, functions as a biased agonist. The reason for the increased survival of patients treated with carvedilol is unclear, but this drug acts as a biased agonist by activating the Erk1/2 mitogen-activated protein kinases while inhibiting Gαs-mediated effects (10). This could be the explanation behind its clinical success because the G protein-independent signaling could confer the effects that improve survival (10). Similar mechanisms are likely to underlie the molecular mechanism behind other successful 7TMR drugs. Comparable observations that G protein-independent signaling may provide beneficial effects have been made in model systems for angiotensin II (Ang II) signaling via the Ang II type 1 receptor (AT1R). The AT1R is commonly used as a model system to study Gαq-dependent versus -independent signaling because of the availability of excellent and well defined biological tools for this receptor. The AT1R serves as a key regulator of cardiovascular physiology, maintaining salt and fluid homeostasis and blood pressure (11, 12). The AT1R is also involved in a number of medical conditions including endothelial dysfunction and atheroma, cardiac hypertrophy and failure, atrial fibrillation, nephropathy, insulin resistance, and cancer (13) and is therefore a prominent drug target in cardiovascular diseases, reflected by the common use of AT1R blockers such as losartan and angiotensin-converting enzyme inhibitors, for example ramipril. AT1R signaling is particularly interesting with respect to functional selectivity because selective activation of Gαq protein-independent signaling has proven relevant in primary cells, in isolated organs, and in vivo (3, 14, 15). AT1R signaling pathways encompass a complex network of signaling molecules including heterotrimeric G proteins, protein kinases, and scaffold proteins (11, 12). The biased agonist [Sar1,Ile4,Ile8]Ang II (SII Ang II) is a well established AT1R ligand that provides the opportunity to study Gαq-independent effects (16). SII Ang II selectively inhibits the Gαq protein signaling branch while still activating the remaining network that is activated from the AT1R such as tyrosine kinase signaling (6, 7), β-arrestin signaling (16), and the possibly minor contribution from other trimeric G proteins such as G12/13, Gi, and Go (17–19) that collectively is called Gαq-independent signaling (Fig. 1A). Activation of Erk1/2 has been thoroughly investigated with respect to Gαq protein-dependent versus β-arrestin-dependent signaling (20–22). These studies have established that Erk1/2 is activated by both pathways and furthermore that there is a striking pathway-dependent difference in the function and localization of activated Erk1/2 (20). Gαq-dependent activation of Erk1/2 results in nuclear translocation and gene transcription, whereas Gαq-independent activation leads to a tight association with β-arrestin, which results in cytosolic sequestration of Erk1/2 (20–22). This difference in Erk1/2 localization has been related to several important phenotypic differences. The β-arrestin part of Gαq-independent signaling has been linked to cellular survival and renewal as well as regulation of cell migration (23, 24). Conversely, the Gαq protein-mediated component of AT1R signaling has been associated with cellular death and fibrosis leading to cardiac hypertrophy and progression to heart failure (3). These divergent roles of the two signaling pathways indicate that full therapeutic blockade of the AT1R may have drawbacks in some situations due to blocking desirable physiological functions. Biased agonism provides a novel pharmacological potential as the use of biased agonists may allow selective blocking of undesired effects while maintaining desired effects (2, 3). Fig. 1. Experimental setup. A, illustration showing the principle signaling pathways from the AT1R. The AT1R associates with heterotrimeric G proteins but can also signal through G protein-independent mechanisms, which include direct binding to tyrosine kinases ... Global and site-specific analysis of in vivo phosphorylation sites by quantitative mass spectrometry has emerged as the method of choice to investigate cell signaling pathways in an unbiased fashion (25). In this study, we characterized the AT1R signaling network by high resolution quantitative phosphoproteomics and thereby delineated the differences between the full agonist Ang II and the biased agonist SII Ang II. To characterize the AT1R signaling network, we used stable isotope labeling by amino acids in cell culture (SILAC) in combination with phosphopeptide enrichment and high performance mass spectrometry (26). This approach has become increasingly powerful in the analysis of signaling pathways because it allows for a high throughput and quantitative mapping of changes in cellular protein phosphorylation on a systems-wide scale. The majority of the identified proteins in our study have not previously been associated with AT1R signaling, and we found Gαq protein-independent signaling to play an unexpected large role in AT1R signaling.