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...

Background: Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer.

The pre-analytical and analytical conditions of SELDI-TOF mass spectrometry of serum have a significant impact on the protein peaks, with the number of peaks low and the assay variation high. Consequently, SELDI-TOF mass spectrometry of serum needs further evaluation before application in clinical diagnostics.

Critical verification did not justify advancing the identified CRC serum protein pattern into clinical validation without improvement. We believe that proteomics biomarker research could benefit if the presented, or a similar, verification scheme was more commonly employed in explorative biomarker studies.

Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.