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

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 Galpha(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 Galpha(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 Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(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 Galpha(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.