Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT 1 R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks G␣ q protein activity and activates G protein-independent pathways of the AT 1 R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT 1 R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT 1 R agonist angiotensin II and the biased agonist [Sar 1 ,Ile 4 ,Ile 8 ]angiotensin II (SII angiotensin II), which only activates the G␣ q protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT 1 R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by G␣ q protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both G␣ q -dependent and -independent AT 1 R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of G␣ q protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future. Molecular & Cellular Proteomics 9:1540 -1553, 2010. 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 ␤-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 o...