title: Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists.
abstract: 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.
authors.affiliation: Laboratory for Molecular Cardiology, Department of Biomedical Sciences, Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.
authors.forename: Gitte L
authors.initials: GL
authors.lastname: Christensen
authors.forename: Christian D
authors.initials: CD
authors.lastname: Kelstrup
authors.forename: Christina
authors.initials: C
authors.lastname: Lyngsø
authors.forename: Uzma
authors.initials: U
authors.lastname: Sarwar
authors.forename: Rikke
authors.initials: R
authors.lastname: Bøgebo
authors.forename: Søren P
authors.initials: SP
authors.lastname: Sheikh
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.forename: Jakob L
authors.initials: JL
authors.lastname: Hansen
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D044140:Receptor, Angiotensin, Type 1 / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D044140:Receptor, Angiotensin, Type 1; D043802:GTP-Binding Protein alpha Subunits, Gq-G11