TY - JOUR

T1 - Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

AU - Christensen, Gitte Lund

AU - Kelstrup, Christian

AU - Lyngsø, Christina

AU - Sarwar, Uzma

AU - Bøgebo, Rikke

AU - Sheikh, Søren P

AU - Gammeltoft, Steen

AU - Olsen, Jesper V

AU - Hansen, Jakob L

PY - 2010/7

Y1 - 2010/7

N2 - 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 (AT1R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists", with intrinsic "functional selectivity", that simultaneously blocks Gaq protein activity, and activitates G protein independent pathways of the AT1R, confer important perspectives in treatment of cardiovascular diseases.n this study we performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC cells by high-resolution mass spectrometry (LTQ Orbitrap MS) and compared the phosphoproteomes of the AT1R agonist Angiotensin II and the biased agonist SII Angiotensin II, which only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation site patterns displays a striking distinction between protein kinases activated by Gaq protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Gaq-dependent and independent AT1R signaling.his 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 Gaq 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.

AB - 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 (AT1R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists", with intrinsic "functional selectivity", that simultaneously blocks Gaq protein activity, and activitates G protein independent pathways of the AT1R, confer important perspectives in treatment of cardiovascular diseases.n this study we performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC cells by high-resolution mass spectrometry (LTQ Orbitrap MS) and compared the phosphoproteomes of the AT1R agonist Angiotensin II and the biased agonist SII Angiotensin II, which only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation site patterns displays a striking distinction between protein kinases activated by Gaq protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Gaq-dependent and independent AT1R signaling.his 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 Gaq 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.

U2 - 10.1074/mcp.M900550-MCP200

DO - 10.1074/mcp.M900550-MCP200

M3 - Journal article

C2 - 20363803

VL - 9

SP - 1540

EP - 1553

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 7

ER -