Dissection of Aberrant GPCR Signaling in Tumorigenesis – A Systems Biology Approach

GPCR Signaling Pathways

All GPCRs have a common central core domain consisting of seven transmembrane helices connected by three intracellular loops and three extracellular loops. The length of the individual N-terminal and C-terminal domains are variable in different GPCRs (21). The basic signaling unit of a GPCR system comprises three parts: the receptor, the trimeric αβγ G protein, and an effector (22). Upon activation by extracellular ligands, GPCRs bind heterotrimeric GTP-binding proteins, which promote not only the release of GDP from the G protein α subunit and the exchange for GTP, but also dissociation of the GTP-bound α-subunit and βγ-dimer from the GPCR (23, 24). On one hand, dissociated Gα subunits can couple with an effector, such as adenylyl cyclase and phospholipase C β, or an ion channel (25). The G-protein-activated effectors in turn regulate multiple downstream signaling cascades that integrate at the level of glucose metabolism, visual excitation, cardiac contractility, development and cancer (26, 27). On the other hand, the dissociated Gβγ subunits stimulate effector molecules including adenylyl cyclases, phospholipase C, and phosphoinositide 3-kinases (PI3Ks) (1) and target a range of signaling pathways involved in desensitization, apoptosis, and ion channel activation (3, 25, 28).

About 20 mammalian G protein α subunits have been identified, which can be divided into four families based on their primary sequence similarity: G_s, G_i, G_q/11, and G_12/13 (29). The signals mediated by the four G protein α subunits are named respectively: G_s pathway, G_i pathway, G_q/11 pathway, and G_12/13 pathway (30-32), as shown in Figure 1. For example, G_s couples with many extracellular signals to activate adenylyl cyclases, 12-transmembrane glucoproteins which catalyze ATP to cAMP, thereby controlling the intracellular concentrations of adenosine 3’,5’-monophosphate (cAMP) (23). The cAMP produced is a second messenger in cellular metabolism and activates at least three known effectors: protein kinase A (PKA), guanine nucleotide exchange factor activated by cAMP (EPAC), and cyclic nucleotide-gated channels (33-35). PKA is an important enzyme that can regulate cell metabolism, cellular secretion, membrane permeability, and even specific gene expression (36). Activated EPAC activates the small GTPase RAP proteins, which are involved in cell growth and motility (35).

Members of the G_i family, including Gα_i1, Gα_i2, Gα_i3, Go, transducin (Gα_t) and gustducin (Gα_gust), activate a variety of phospholipases and phosphodiesterases, and promote the opening of several ion channels (37). Gi family members can inhibit a subset of these enzymes, thereby controlling the intracellular concentrations of cAMP. All isoforms of this family can be irreversibly uncoupled from their receptors by pertussis toxin (PTX). Inhibition of G_i by pretreatment with PTX causes strong impairment of lymphocyte migration in vitro (38), suggesting that signaling through the G_i is involved in cell motility processes.

The G_q/11 family can be divided into four subfamilies (G_q/11, G₁₁, G₁₄, G_15/16). G_q-Coupled receptors activate phospholipase Cβ, which converts phosphatidylinositol-4,5,-bisphosphate into inositol-1,4,5-trisphosphate and diacylglycerol (39). These in turn lead to an increase in the intracellular concentrations of free calcium and the activation of a number of protein kinases, including protein kinase C (PKC) which phosphorylates several downstream effectors such as calmodulin known to regulate calcium dependent signaling (40-43). G_q-Coupled receptors can stimulate mitogen-activated protein kinase in a PKC-dependent and Ras-independent manner depending on the cell type and the receptor expression levels (1). In addition, G_q can activate the PKC-glycogen synthase kinase-3β-β-catenin pathway, which induces cell proliferation and production of cytokines (44).

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Figure 1.

G-Protein-coupled receptor (GPCR)-mediated signaling pathways. Upon activation by extracellular ligands, GPCRs can regulate key biological functions, such as cell proliferation, metabolism, secretion, motility, tumor progression, invasion, and metastasis, through signalling pathway mediated by the four G protein α subunits (G_s, G_i, G_q/11, and G^12/13).

Interestingly, G_12/13 seem to be the most potent oncogenes because they comprise the only family for which overexpression of wild-type proteins has been found to be transforming (45, 46). Activation of the G_12/13 protein has been linked to the activation of small GTP-binding proteins of the RHO family, which contribute to diverse cellular processes involved in tumor progression. For example, lysophosphatidic acid-induced tumor cell migration was shown to require the activation of the G_12/13/RHOA/ROCK signaling pathway in SK-OV3 ovarian cancer cells (47). In addition, G_12/13 has been reported to interact with the cytoplasmic domain of cadherins, a family of integral membrane proteins involved in mediating cell–cell adhesion, altering their interaction with cytoplasmic proteins such as β-catenins, which suggests that G_12/13 protein mediates cancer invasion and metastasis through interaction with other cancer-related proteins (48, 49).

Classical GPCR-mediated signal transduction involves the agonist-dependent interaction of GPCRs with G proteins at the plasma membrane and the subsequent generation of soluble second messengers or ion currents by membrane localized effectors (22). However, many GPCRs directly interact with non-G protein signaling effectors, G protein-coupled receptor kinases (GRKs) and β-arrestins through specific protein–protein interaction domains (50). Active GPCRs are the target of GRKs that phosphorylate the agonist-activated form of GPCRs. This phosphorylation leads to the rapid recruitment and binding of cytosolic arrestins (known as arrestin-2 or β-arrestin-1, and arrestin-3 or β-arrestin-2) (51). Binding of β-arrestin proteins to the GPCRs not only uncouples the receptor from its cognate G protein, resulting in a decreased responsiveness of the signaling system to agonist (termed ‘desensitization’) but also initiates the process of receptor sequestration by targeting it to clathrin-coated vesicles for endocytosis (termed ‘internalization’) (52). Indeed, β-arrestins have been shown to act as multifunctional scaffolds and activators for a growing number of signaling proteins including ERK, p38, JNK, I-κB, AKT, and RHOA (53-55). Moreover, it has been reported that GRKs interact with a variety of proteins involved in signaling and trafficking such as Gα_q, Gβγ, PI3Kγ, clathrin, GPCR kinase-interacting protein (GIT), and caveolin (56). The characteristics discussed above highlight the complexity of the GPCR signaling pathways and the importance of considering their dynamic properties.

Aberrant GPCR Signaling in Tumorigenesis

Diverse GPCRs have been found to be overexpressed in primary and metastatic melanoma (57), human colon carcinoma (58), squamous cell carcinoma (SCC) of the lung (59), basal cell carcinoma (BCC) (60), hepatocellular carcinoma (HCC) (61), and glioblastoma multiforme (62). Some of the GPCRs and ligands reported to be involved in cancer are shown in Table III, together with their observed function. Recently, a large body of evidence has linked orphan receptors such as GPR18, GPR48/LGR4, GPR49, GPR56, GPR87, and CXCR7/CMKOR1 to the cancer phenotype. For example, Qin et al. found that GPR18, the most abundantly overexpressed orphan GPCR in melanoma metastasis, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival (57). The links between cancer and GPR48 are somewhat more cryptic. GPR48/LGR4 is widely expressed in multiple organs (63-64), playing a vital role in development and adult physiological functions. Interestingly, Gao et al. found previously that up-regulation of GPR48 contributed to human colon carcinoma cell invasion and metastasis (58). Moreover, Lgr4 knockout leads to reduced viability and retarded growth in the mouse (65). Similarly, de Lau et al. discovered that conditional deletion of both Lgr4 and Lgr5 genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts (66), which suggested that LGR4 may be a potential target for therapy of intestinal cancer (67). GPR49 has been reported to be a novel gene marker of follicular and other tissue stem cells, overexpression of which was frequently observed in HCC with mutations in β-catenin exon 3 (61). Aberrant expression of some GPCRs also plays a role in tumor cell biology. For example, GPR56, an orphan GPCR of the secretin family, is overexpressed in gliomas and functions in tumor cell adhesion by activating the nuclear factor-kappa B, plasminogen activator inhibitor-1, and transcriptional response elements (62). Gugger and colleagues, using laser capture microdissection and GPCR-focused Affymetrix microarrays, identified that GPR87, and CXCR7/CMKOR1 are overexpressed GPCRs in SCC of the lung (59), and could be explored as novel therapeutic targets for cancer treatment and prevention.

Recently, a series of studies showed that overexpression of some novel genes including those for GPCR-PCa, PSGR2, CaSR, GPR30, and GPR39 were associated with tumorigenesis or metastasis in diverse types of cancer tissues. In human prostate cancer, GPCR-PCa (68), which belongs to the subfamily of odorant-like orphan GPCRs, and PSGR2 (69), are selectively overexpressed and may be useful as tissue markers and molecular targets for the early detection and treatment of human prostate cancer. A previous clinical study had demonstrated that CaSR was expressed at higher levels in breast cancer cells from patients with bone metastases (70, 71). Recently, CaSR has been shown to be involved in the progression and spread of a variety of cancer types such as colorectal, breast and parathyroid, and is likely to be the focus of much research to further elucidate its precise role (72). GPR30, a seven membrane-spanning estrogen receptor, is linked to estrogen binding and heparin-bound epidermal growth factor release and induces proliferation and migration of breast cancer cells through connective tissue growth factor (73, 74). Xie et al. found that GPR39 was frequently overexpressed in primary esophageal SCC at both the mRNA level and protein level, which was significantly associated with lymph node metastasis and advanced TNM stage (75). In addition, many GPCR ligands, including those for sphingosine-1-phosphate (76), LPA (77-80), platelet-activating factor (81, 82), thrombin (83), interleukin-8 (84), growth regulated oncogene α-γ (1), monocyte chemoattractant protein 1 (85), and stromal cell-derived factor, have also been shown to play a key role in tumor growth, metastasis, vasculogenesis and tumor-induced angiogenesis (86). Taken together, these results suggest that interfering with these receptors and their downstream targets might provide an opportunity for the development of new strategies for cancer diagnosis, prevention and treatment (15).

High-throughput Screening (HTS) for Discovery of GPCRs Drug Targets

HTS plays a crucial role in the preclinical discovery process of many pharmaceutical companies (87). Given the importance of GPCRs as drug targets, the development of HTS for identifying target GPCRs has become a major focus in the pharmaceutical industry. Classical HTS approaches for screening GPCRs including GTP-binding assays, cAMP assays, intracellular calcium assays, inositol phosphate accumulation assays, and reporter gene assays (88). Despite the success of drug discovery aimed at GPCRs over the past decade, there remains a need to identify GPCR-targeted drugs with greater selectivity and to effectively develop screening assays for identifying lead agonists or antagonists for orphan receptors and validate these targets. In recent years, new experimental approaches, including green fluorescent protein (GFP), fluorescence resonance energy transfer (FRET), protein complementation assays (PCAs), and GPCR microarrays, have been used to analyse activation, localization, trafficking, ligand-screening assays and protein–protein interactions for GPCRs.

Tagging of GPCRs with GFP has allowed for their direct visualization of localization and real-time trafficking in living cells, which have provided crucial insight into the mechanisms involved in controlling GPCR function (89). It has been shown that GFP mutants can exhibit enhanced fluorescence properties, opening up possibilities for the development of ligand screening assays for GPCRs based on cell imaging. For instance, mutation of the tyrosine residue at position 66 to histidine generated a protein with altered spectral properties and blue fluorescence emission (90). Proof of principle following expression of a form of the β2-adrenoceptor, in which GFP was appended to the C-terminal tail of the GPCR (91) initiated interest in this process as a direct screening strategy. Ambrosio et al. have recently reviewed the use of different strategies for inserting fluorescent labels into purified, reconstituted receptors, or into receptors in intact cells to sense receptor activation via changes in fluorescence using modern spectroscopic and crystallographic techniques (92).

Fluorescence resonance energy transfer, a most promising approach, relies upon the dissociation of G proteins into separate α- and β/γ-subunits stimulated by GPCR activation (90). In order to study the characteristics and kinetics of GPCR activation in living cells, Milligan et al. have developed a strategy based on the use of FRET between donor and acceptor fluorophores attached to the receptor sequence (90). Vilardaga et al. initially reported this approach in 2003 (93). The principle of this assay is as follows: placement of the donor fluorescent protein CFP (27 kDa) into the third intracellular loop of the receptor and the acceptor fluorescent protein YFP (27 kDa) at the C-terminus results in a sensor for which conformational switches performed by the receptor after stimulation with an agonist cause an increase in the distance between the two fluorescent probes that leads to a concomitant decrease of the FRET signal. Using this strategy, parathyroid hormone and α 2 A-the adrenaline can receptor construct were generated with preserved ligand binding and signaling properties (92). Fluorescent protein-fragment complementation assay, also termed bimolecular fluorescence complementation, allow for visualization of either single or dual protein–protein interactions at the subcellular level and only require basic experimental setups (94). More recently, multicolor PCAs has been applied to quantitatively measure drug-induced changes in GPCR interactions. For example, Przybyla and Watts used multicolor PCAs to study ligand-induced regulation and localization of cannabinoid CB1 and dopamine D2L receptor heterodimers (95). In addition, the application of PCAs-FRET combined techniques demonstrated that higher-order A2AR oligomers accumulate at the plasmamembrane in a neuronal cell model, providing insight into drug-mediated effects on GPCR signaling and oligomerization (96). With recent advances in instrumentation and the understanding of cellular mechanisms underlying the signals measured, multiplexed assays have become important tools for measuring ligand-induced receptor activation in the pharmaceutical industry. Multiplexed assays, combining reverse transfection in a 96-well plate format with a calcium flux readout, can quantitatively measure receptor activation and inhibition and permit the determination of compound potency and selectivity for entire families of GPCRs in parallel (97). However, arraying of membrane-bound proteins is complicated because they typically need to be embedded in membranes to maintain their correctly folded conformations (98). To address this problem, GPCR microarrays that require the co-immobilization of lipid molecules and the probe receptors of interest have been fabricated, using conventional robotic printing technologies (99). Using multiplexed assays by high-content imaging, Ross and colleagues identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both, which indicates that the imaging assays can be used as tools to study GPCR activation and internalization (100).

Systems Biology-based Annotation for GPCR Signaling

Recently, the multidisciplinary field of systems biology has emerged as a holistic approach to interpreting the complexity and dynamics of cellular signaling (101-105). In contrast to the earlier reductionist approaches which focus on the manipulation of one gene or protein (e.g., tumor suppressor or oncogene) to understand a complex entity (e.g., a cell, an organ or a disease) (106), systems biology attempts to integrate global information into a comprehensive map which can be used to predict the behavior of the total system and understand how it regulates specific cells or tissues (107). Nowadays, integrative systems biology approaches have been increasingly applied to the identification of novel GPCRs and related proteins, to investigate the function of GPCRs in various disease states and to unravel GPCR complexes and signal transduction networks. Such studies should speed up the discovery of new specific drugs in cancer prevention and treatment (20). To conduct a systems-level analysis, a comprehensive set of quantitative data from ‘omic’ approaches is required. ‘Omics’ refers to the biological sciences that study the genes (genomics), their initial products (RNA transcripts) (transcriptomics), their final products (proteins) (proteomics) and the metabolites produced in the processes in which these proteins are involved (metabolomics) (108). In this review, we focus on omics-based systems biology approaches used to study GPCRs.

Genomic Profiling of Gene Expression of GPCRs

Genomics is a discipline in genetics which interrogates the molecular organization of DNA and its physical mapping, and which encompasses structural genomics and functional genomics. Advances in genomic technologies such as DNA microarrays (109, 110), and DNA chips (111) now provide an unparalleled opportunity to perform large–scale global analyses of the variability of gene expression. DNA arrays can be used for many different purposes, most prominently to measure levels of gene expression (messenger RNA abundance) for tens of thousands of genes across different tissues or cells simultaneously (109). Several groups have now taken advantage of this technology to perform global analyses of the variability of gene expression between normal tissue and abnormal tissue, especially in tumor tissue. For example, Ye et al. used 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least 1 out of 15 of the independent microarrays, to analyze samples from normal adrenals, aldosterone-producing adenomas and cortisol-producing adenomas (112). The array results indicated that certain GPCRs exhibit elevated expression in the former. These data were further confirmed using real-time RT-PCR (qPCR), which suggested a potential role for elevated expression of GPCR in many cases of primary hyperaldosteronism and provided candidate GPCRs for further clinical study. Lockhart and Winzeler used high-resolution oligonucleotide comparative genomic hybridization (CGH) arrays to match gene expression array data and identified dysregulated genes that were able to classify breast cancer according to gene copy number anomalies (109). This study first showed that CGH arrays were a robust technology for assessing gene copy number anomalies and provided a new strategy to screen novel therapeutic targets for molecular subsets of cancer.

An understanding of mutations and alterations in the expression of various genes resulting in carcinogenesis combined with the development of microarray technology has enabled the identification of comprehensive gene expression alterations during oncogenesis (113-116). In recent years, many studies have applied this technology for BCC (60), HCC (113) and other type of cancer and have identified a number of candidate genes with potential as biomarkers in cancer staging, prediction of recurrence and prognosis, and treatment selection. Interestingly, Tanese et al. screened a DNA microarray database of human BCC cases to identify genes responsible for tumor formation in BCC and found up-regulation of the orphan GPCR GPR49 in all BCC cases compared with controls, which was confirmed by real-time qPCR analysis and in situ hybridization (60). These data suggested that GPR49 may play a vital role in tumor formation. Similarly, Weigle et al. screened a DNA Chip-based expression database and identified an expressed sequence tag (EST) originally sequenced in a lung cancer cDNA library for the identification of target structures specifically expressed in prostate tissue (68). They identified GPCR-PCa as a novel putative GPCR that was overexpressed in prostate cancer.

Transcriptomics Profiling of GPCR Expression

Transciptomics (or expression profiling) describes the complete set of RNA transcripts present in a particular cell including messenger RNA (mRNA), micro-RNA (miRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) (117). Unlike the genome, which is fixed for a given cell type (excluding mutations), the transcriptome can be modulated by external environmental conditions. Nowadays, a number of techniques are available for studying transcriptomics, including cDNA hybridization microarrays, serial analysis of gene expression (SAGE), conventional RT-PCR and qPCR.

Using qPCR, Maurel et al. identified a number of GPCRs that had not been detected previously in cerebellar granule neurons and neuroblasts during postnatal development (118). These GPCRs represent novel candidates in the development and survival of cerebellar granule neurons. Using cDNA microarray analysis, Xie and co-workers identified a GPCR, GPR39, which is significantly up-regulated in esophageal SCC. They then further analyzed the mRNA expression level of GPR39 in 9 esophageal SCC cell lines and 50 primary esophageal SCC tumors using semi-quantitative RT-PCR (75). Interestingly, functional studies in vitro and in vivo showed that GPR39 plays an important tumorigenic role in the development and progression of esophageal SCC. Atwood et al. using microarray analysis revealed expression of GPCRs and related proteins in HEK293, AtT20, BV2, and N18 cell lines, providing a better understanding of the potential interactions between GPCRs and these signaling proteins (119). In addition to mRNA, ESTs can also give valuable information about expression patterns. Fredriksson and Schioth identified more than 20,000 sequences that match GPCRs through searching EST databases (120). GPCRs receptors are encoded by low abundance mRNAs and can be fully functional at levels of 1×10⁴ protein molecules per cell (121). It can therefore be difficult to determine the expression profiles of GPCRs in cell lines or in tissues using conventional cDNA or oligonucleotide arrays. Thus there is a need to develop new methodologies to apply to the GPCR genome. The use of microarray technology allows for a large sampling of receptor families with low abundance mRNAs to be performed and also allows monitoring of the message levels of thousands of genes simultaneously in a given sample (122, 123). Hansen et al. developed a new method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression using as little as 1 μg of total RNA. Using this method, they profiled three human bone marrow stromal cell lines (124). Hakak and co-workers used a custom high-density oligonucleotide microarray containing probes designed to measure the gene expression levels of over 700 human GPCRs, along with a number of molecules involved in GPCR signaling and regulation (125). These studies revealed complex signaling networks in many cell types.

Evaluation of the transcriptional levels for these genes across a large panel of tissues would thus provide a global view of GPCR expression and function in the human body (125). Yamamoto and colleagues have investigated the differences in mRNA expression in several HCC cell lines by using mRNA differential display polymerase chain reaction (mRNADD-PCR) (61). This study demonstrated that GPR49, which belongs to the glycoprotein hormone receptor subfamily, is markedly up-regulated in HCCs carrying β-catenin mutations. To better understand the functions of GPCRs in vivo, Regard et al., by analyzing the pattern of GPCR mRNA expression across tissues and the relative abundance for each of 353 nonodorant GPCRs in 41 tissues from adult mice, developed a dataset that provides a useful resource for finding previously unidentified roles for GPCRs with known ligands, and importantly provides clues regarding the function of orphan GPCRs and the source of their ligands (126). Additionally, they demonstrated that the GPR91, a receptor for the citric acid cycle intermediate succinate, can regulate lipolysis in white adipose tissue, suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis.

Proteomics Identification of Novel GPCRs and Their Interactive Proteins

Proteomics is the large-scale analysis of proteins to profile a whole proteome or sub-proteome in a single experiment, so that the protein alterations corresponding to a pathological or biochemical condition at a given time can be annotated in an integrated way (127). Proteomics can be divided into three main areas: protein micro-characterization for large-scale identification of proteins and their post-translational modifications; ‘differential display’ proteomics for comparison of protein levels with potential application in a wide range of diseases; and studies of protein–protein interactions using techniques such as mass spectrometry (MS) or the yeast two-hybrid system to give information on signaling pathways (74). Nowadays, proteomics strategies encompass many analytical techniques including high resolution two-dimensional electrophoresis (2-DE), multidimensional separation protocols for the purification of trace components (128), two-dimensional difference gel electrophoresis (2D-DIGE), antibody/protein arrays, metabolic or chemical labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), gel-free systems such as isotope-coded affinity tagging (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), proteolytic ¹⁸O labeling, coupled with advances in MS such as multidimensional protein identification technology (MudPIT) and multiple-reaction monitoring (MRM) (129), which combine to provide the large–scale and unbiased platforms required to determine the dynamic profiles of GPCR proteomes.

Proteomics offers specific advantages for the analysis of GPCR complexes and signal transduction networks. Because these regulatory mechanisms are not necessarily dependent on de novo synthesis, genomic and transcriptomic approaches might fail to identify these processes (130). During the past decade, MS analysis has gained prominence for revealing detailed information on the individual GPCR class, including characterization of the GPCR binding pocket and specific post-translational modifications. For example, using MALDI-TOF and/or LC-ion trap MS, Kamonchanok et al. were able to directly determine >80% of the primary amino acid composition of the histamine H1 receptor covering five of the transmembrane domain regions after baculovirus-driven and in vitro cell-free expression (131).

GPCRs are associated with large protein networks organised by protein–protein interactions involving multidomain proteins. The combination of 2-DE or high-resolution chromatography and MS allows more direct observations to be made based on the study of molecular interaction networks. Using the C-terminal tail of the 5-hydroxytryptamine 2C (5-HT2C) receptor as an example, Bécamel and co-workers identified at least 15 proteins that interact with the C-terminal tail of the 5-HT2C receptor using a proteomics approach based on peptide affinity chromatography followed by MS and immunoblotting. These studies indicated that the 5-HT2C receptor is associated with protein networks that are important for synaptic localization and coupling to the signaling machinery (132). β-Arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by GPCR kinases. Xiao and co-workers performed a global proteomics analysis of β-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1α receptor, in an attempt to assess the full range of functions of these versatile molecules. In this study, they combined gel-based and non-gel-based proteomics methods in order to enhance the coverage and reliability of the analysis and identified 337 nonredundant proteins interacting with β-arrestin 1 and 2 using nano-LC MS/MS (133). These proteins, which were ubiquitously distributed in the cell, had multiple functions, including receptor desensitization, endocytosis, signal transduction, regulation of gene expression, protein synthesis, cellular reorganization, chemotaxis, and apoptosis.

Protein phosphorylation is a general and important mechanism of cellular regulation that involves at least two interacting protein partners (134, 135). In recent years, global and site-specific analysis of in vivo phosphorylation sites by quantitative MS has emerged as the method of choice to investigate cell signaling pathways in an unbiased fashion (136). For instance, using SILAC in combination with specific phosphopeptide enrichment using TiO2 chromatography (135) and high performance MS using a LTQ-Orbitrap MS, Christensen et al. compared the phosphoproteomes of the AT1R agonist angiotensin II and the biased agonist [Sar¹, Ile⁴, Ile⁸] angiotensin II (SII angiotensin II). As a result, they quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analog SII angiotensin II (137).